WO2026028165A1 - Méganucléases modifiées ayant une spécificité pour les séquences de reconnaissance dans le gène c9orf72 - Google Patents

Méganucléases modifiées ayant une spécificité pour les séquences de reconnaissance dans le gène c9orf72

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WO2026028165A1
WO2026028165A1 PCT/IB2025/057836 IB2025057836W WO2026028165A1 WO 2026028165 A1 WO2026028165 A1 WO 2026028165A1 IB 2025057836 W IB2025057836 W IB 2025057836W WO 2026028165 A1 WO2026028165 A1 WO 2026028165A1
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seq
engineered meganuclease
acid sequence
residues
amino acid
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Spencer MCKINSTRY
Hye Ran Lee
John Morris
James Jefferson Smith
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Precision Biosciences Inc
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Precision Biosciences Inc
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    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the application relates to the field of engineered meganucleases, molecular biology and recombinant nucleic acid technology.
  • the invention relates to engineered meganucleases useful for the removal of non-coding nucleotide sequences from the C90rf72 gene and for the treatment of subjects having a neurological disorder such as amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • ALS Amyotrophic lateral sclerosis
  • ALS is a fatal neurodegenerative disease characterized clinically by progressive paralysis leading to death from respiratory failure, typically within two to three years of symptom onset (Rowland and Shneider, N. Engl. J. Med., 2001, 344, 1688-1700).
  • ALS is the third most common neurodegenerative disease in the Western world (Hintz et al., Neurology, 2007, 68, 326-337), and there are currently no effective therapies. Approximately 10% of cases are familial in nature, whereas the bulk of patients diagnosed with the disease are classified as sporadic as they appear to occur randomly throughout the population (Chio et al., Neurology, 2008, 70, 533-537).
  • ALS and frontotemporal dementia represent an overlapping continuum of disease, characterized pathologically by the presence of TDP-43 positive inclusions throughout the central nervous system (Lillo and Hodges, J. Clin. Neurosci., 2009, 16, 1131-1135; Neumann et al., Science, 2006, 314, 130-133).
  • the ALS-FTD causing mutation is a large hexanucleotide (GGGGCC) repeat expansion in the first intron of the C90rf72 gene (Renton et al., Neuron, 2011, 72, 257-268; De Jesus-Hernandez et al., Neuron, 2011, 72, 245-256).
  • GGGGCC hexanucleotide repeat expansion in the first intron of the C90rf72 gene
  • This locus on chromosome 9p21 accounts for nearly half of familial ALS and nearly one-quarter of all ALS cases in a cohort of 405 Finnish patients (Laaksovirta et al, Lancet Neurol., 2010, 9, 978-985).
  • a founder haplotype, covering the C90rf72 gene, is present in the majority of cases linked to this region.
  • the present disclosure provides compositions and methods for treatment of neurodegenerative disease, such as ALS and FTD.
  • the invention is a permanent treatment for ALS and FTD that involves the excision of specific non-coding sequence (i.e., hexanucleotide repeat region) located 5’ to the C90rf72 coding sequence using a pair of engineered, site-specific homing endonucleases, often referred to as meganucleases.
  • a pair of such endonucleases By targeting a pair of such endonucleases to the hexanucleotide repeat region in the C90rf72 gene, it is possible to permanently remove the intervening fragment from the genome.
  • the resulting cell, and its progeny, will express a modified C90rf72 RNA in which a portion of the non-coding sequence comprising the sequence susceptible to mutation (i.e., hexanucleotide repeat expansion) that is associated with disease is removed.
  • a portion of the non-coding sequence comprising the sequence susceptible to mutation (i.e., hexanucleotide repeat expansion) that is associated with disease is removed.
  • Homing endonucleases are a group of naturally-occurring nucleases that recognize 15-40 base-pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double- stranded break in the chromosome, which recruits the cellular DNA-repair machinery (Stoddard (2006) Q. Rev. Biophys. 38:49-95).
  • LAGLID ADG Homing endonucleases are commonly grouped into four families: the LAGLID ADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLID ADG family are characterized by having either one or two copies of the conserved LAGLID ADG motif (see, Chevalier et al. (2001) Nucleic Acids Res. 29:3757-74). The LAGLID ADG homing endonucleases with a single copy of the LAGLID ADG motif form homodimers, whereas members with two copies of the LAGLID ADG motif are found as monomers.
  • I-Crel (SEQ ID NO: 1) is a member of the LAGLID ADG family of homing endonucleases that recognizes and cuts a 22 basepair recognition sequence in the chloroplast chromosome of the algae Chlamydomonas reinhardtii . Genetic selection techniques have been used to modify the wild-type I-Crel cleavage site preference (Sussman et al. (2004) J. Mol. Biol. 342:31-41; Chames et al. (2005) Nucleic Acids Res. 33:el78; Seligman et al. (2002) Nucleic Acids Res. 30:3870-79, Amould et al. (2006) J. Mol. Biol. 355:443-58).
  • I-Crel and its engineered derivatives are normally dimeric but can be fused into a single polypeptide using a short peptide linker that joins the C- terminus of a first subunit to the N-terminus of a second subunit (Li et al. (2009) Nucleic Acids Res. 37: 1650-62; Grizot et a/. (2009) Nucleic Acids Res. 37:5405-19).
  • a functional “single-chain” meganuclease can be expressed from a single transcript.
  • the present disclosure provides engineered meganucleases that bind and cleave recognition sequences in a C90rf72 gene, as well as compositions comprising such engineered meganucleases and methods of their use.
  • the recognition sequences targeted by the disclosed engineered meganucleases are selected to have identical four base pair center sequences, such that the first and second cleavage sites will have complementary four base pair 3 ’ overhangs that can perfectly ligase to one another (i.e., each base pair of one overhang pairs with its complement on the other overhang).
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7- 18. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7-18.
  • the HVR1 region comprises a residue corresponding to residue 29 of any one of SEQ ID NOs: 7-11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 41 of any one of SEQ ID NOs: 7-11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of any one of SEQ ID NOs: 7- 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of any one of SEQ ID NOs: 7-11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of any one of SEQ ID NOs: 7-11 or 9-17.
  • the HVR1 region comprises a residue corresponding to residue 72 of any one of SEQ ID NOs: 7-11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of any one of SEQ ID NOs: 7-11.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of any one of SEQ ID NOs: 7-18. In some embodiments, the HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 7-18.
  • the HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 7-18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 7-18.
  • the first subunit comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of any one of SEQ ID NOs: 7-18.
  • the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of any one of SEQ ID NOs: 7-18.
  • the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6- 153 of any one of SEQ ID NOs: 7-18.
  • the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of any one of SEQ ID NOs: 7-18.
  • the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-153 of any one of SEQ ID NOs: 7-18.
  • the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-153 of any one of SEQ ID NOs: 7-18.
  • the first subunit is an N-terminal subunit and the residue at position 1 of any one of SEQ ID NOs: 7-18 is modified from M to another amino acid. In some embodiments, the residue at position 1 is modified from M to A.
  • the first subunit is an N-terminal subunits that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of any one of SEQ ID NOs: 7-18.
  • the first subunit comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 to SEQ ID NO: 195.
  • the first subunit comprises a residue corresponding to residue 19 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of any one of SEQ ID NOs: 8-12, 14, or 1418. In some embodiments, the first subunit comprises a residue corresponding to residue 139 of SEQ ID NO: 18. In some embodiments, the first subunit comprises a residue corresponding to residue 142 of SEQ ID NO: 9. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of any one of SEQ ID NOs: 7-18.
  • the first subunit is an N-terminal subunit that comprises residues 7- subunit that comprises residues 6-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 5-153 of any one of SEQ ID NOs: 7- 18. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 4-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 3-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 2-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 1-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit comprises residues 1-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first sub
  • the first subunit comprises residues 7-153 of any one of SEQ ID NOs: 7-18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 1-153 of any one of SEQ ID NOs: 7-18.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 7-18.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 7-18. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 7-18.
  • the HVR2 region comprises a residue corresponding to residue 241 of any one of SEQ ID NOs: 7-18. In some embodiments, the HVR2 region comprises a residue corresponding to residue 258 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of any one of SEQ ID NOs: 7-18. In some embodiments, the HVR2 region comprises a residue corresponding to residue 265 of SEQ ID NO: 18. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of any one of SEQ ID NOs: 7-18.
  • the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 7-18. In some embodiments, the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 7-18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.
  • the second subunit comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of any one of SEQ ID NOs: 7-18.
  • the second subunit is a C-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of any one of SEQ ID NOs: 7-18.
  • the second subunit comprises a residue corresponding to residue 271 of any one of SEQ ID NOs: 8, 10, or 12-14. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of any one of SEQ ID NOs: 7, 10-12, 14, or 18. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of any one of SEQ ID NOs: 7-18. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of any one of SEQ ID NOs: 7-18.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 7-18. In some embodiments, the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 7-18.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-354 of any one of SEQ ID NOs: 7-18.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-354 of any one of SEQ ID NOs: 7-18.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of any one of SEQ ID NOs: 7-18.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of any one of SEQ ID NOs: 7-18.
  • the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 67-78. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 67-78.
  • the engineered meganuclease comprises a nuclear localization signal.
  • the nuclear localization signal is at the N-terminus of the engineered meganuclease.
  • the nuclear localization signal is at the C-terminus of the engineered meganuclease.
  • the nuclear localization signal comprises an amino acid sequence having at least 80% or at least 90% sequence identity to SEQ ID NO: 133 or 134.
  • the nuclear localization signal comprises SEQ ID NO: 133 or 134.
  • the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 145-148. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 145-148.
  • an engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 5 within a C90rf72 gene, wherein the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and wherein the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region.
  • HVR1 hypervariable
  • HVR2 hypervariable hypervariable
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 79-87. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 79-87.
  • the HVR1 region comprises 24-79 of any one of SEQ ID NOs: 79- 87. In some embodiments, the HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 79-87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.
  • the first subunit comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of any one of SEQ ID NOs: 79-87.
  • the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of any one of SEQ ID NOs: 79-87.
  • the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-153 of any one of SEQ ID NOs: 79-87.
  • the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of any one of SEQ ID NOs: 79-87.
  • the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-153 of any one of SEQ ID NOs: 79-87.
  • the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-153 of any one of SEQ ID NOs: 79-87.
  • the first subunit is an N- terminal subunit and the residue at position 1 of any one of SEQ ID NOs: 79-87 is modified from M to another amino acid.
  • the residue at position 1 is modified from M to A.
  • the first subunit comprises a residue corresponding to residue 19 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of any one of SEQ ID NOs: 81, 82, 85, or 87. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of any one of SEQ ID NOs: 79-87.
  • the first subunit comprises residues 7-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit comprises residues 7-153 of any one of SEQ ID NOs: 79-87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the first subunit is an N-terminal subunit that comprises residues 7- 153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 6-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 5-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 4- 153 of any one of SEQ ID NOs: 79-87.
  • the first subunit is an N-terminal subunit that comprises residues 3-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 2-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 1- 153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit comprises residues 1-153 of any one of SEQ ID NOs: 79-87.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 79-87.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 79-87. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 79-87.
  • the HVR2 region comprises a residue corresponding to residue 241 of any one of SEQ ID NOs: 79-87. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of any one of SEQ ID NOs: 79-87. In some embodiments, the HVR2 region comprises a residue corresponding to residue 264 of any one of SEQ ID NOs: 79-87. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of any one of SEQ ID NOs: 79-87.
  • the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 79-87. In some embodiments, the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 79-87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of any one of SEQ ID NOs: 79-87.
  • the second subunit is a C-terminal subunit that comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more sequence identity to residues 196-354 of any one of SEQ ID NOs: 79-87.
  • the second subunit comprises a residue corresponding to residue 271 of any one of SEQ ID NOs: 79, 80, 81, 83, 84, or 87. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 81 or 86. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of any one of SEQ ID NOs: 79-87. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of any one of SEQ ID NOs: 79-87.
  • the second subunit comprises residues 198-344 of any one of SEQ ID NOs: 79-87. In some embodiments, the second subunit comprises residues 198-344 of any one of SEQ ID NOs: 79-87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 196-354 of any one of SEQ ID NOs: 79-87.
  • the second subunit comprises residues 196-354 of any one of SEQ ID NOs: 79-87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the engineered meganuclease is a single chain meganuclease comprising a linker that covalently joins the first subunit and the second subunit.
  • the engineered meganuclease linker comprises an amino acid sequence according to SEQ ID NO: 196.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 79-87. In some embodiments, the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 79-87.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-354 of any one of SEQ ID NOs: 79-87. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-354 of any one of SEQ ID NOs: 79-87.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-354 of any one of SEQ ID NOs: 79-87. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of any one of SEQ ID NOs: 79-87.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-354 of any one of SEQ ID NOs: 79-87. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of any one of SEQ ID NOs: 79-87.
  • the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 124-132. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 124-132.
  • the engineered meganuclease comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 80.
  • the engineered meganuclease comprises a nuclear localization signal.
  • the nuclear localization signal is at the N-terminus of the engineered meganuclease.
  • the nuclear localization signal is at the C-terminus of the engineered meganuclease.
  • the nuclear localization signal comprises an amino acid sequence having at least 80% or at least 90% sequence identity to SEQ ID NO: 133 or 134.
  • the nuclear localization signal comprises SEQ ID NO: 133 or 134.
  • the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 149. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence according to SEQ ID NO: 149.
  • a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein.
  • the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the engineered meganuclease.
  • the promoter is a CNS cell-specific promoter.
  • the promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells.
  • the promoter is a CAG promoter or a human synapsin 1 (Syn-1) promoter.
  • a polynucleotide comprising a first nucleic acid sequence encoding a first engineered meganuclease described herein that binds and cleaves a recognition sequence comprising SEQ ID NO: 3, and a second nucleic acid sequence encoding a second engineered meganuclease described herein that binds and cleaves a recognition sequence comprising SEQ ID NO: 5.
  • the polynucleotide comprises, from 5’ to 3’, (i) the first nucleic acid sequence encoding the first engineered meganuclease, and (ii) the second nucleic acid sequence encoding the second engineered meganuclease.
  • the polynucleotide comprises, from 5’ to 3’, (i) the second nucleic acid sequence encoding the second engineered meganuclease and (ii) the first nucleic acid sequence encoding the first engineered meganuclease.
  • the polynucleotide comprises a promoter enhancer.
  • the promoter enhancer is a neuron-specific promoter enhancer.
  • the first nucleic acid sequence and/or the second nucleic acid sequence is codon modified to reduce the percent sequence identity between the first nucleic acid sequence and the second nucleic acid sequence, wherein the codon modification does not alter the amino acid sequence of the first engineered meganuclease or the second engineered meganuclease.
  • the first nucleic acid sequence has no more than about 40% to about 80% sequence identity to the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has no more than about 60% sequence identity to the second nucleic acid sequence.
  • the first engineered meganuclease comprises a nuclear localization sequence (NLS).
  • the NLS is attached to the N-terminus of the first engineered meganuclease.
  • the NLS is attached to the C-terminus of the second engineered meganuclease.
  • the NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134.
  • the NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 or 134.
  • the engineered meganuclease comprises a first NLS attached at the N-terminus and a second NLS attached at the C-terminus.
  • the first NLS and the second NLS are identical.
  • the first NLS and the second NLS comprise an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134.
  • the first NLS and the second NLS comprise an amino acid sequence set forth in SEQ ID NO: 133 or 134.
  • the first NLS and the second NLS are not identical.
  • the NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 134.
  • the first NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.
  • the first NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 134.
  • the first NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.
  • the second engineered meganuclease comprises a third NLS attached at the N-terminus and a fourth NLS attached at the C-terminus.
  • the third and fourth NLS are identical.
  • the third NLS and the fourth NLS comprise an amino acid sequence having at least 80% sequence identity to a second set forth in SEQ ID NO: 133 or 134.
  • the third and the fourth NLS comprise an amino acid sequence set forth in SEQ ID NO: 133 or 134.
  • the third NLS and the fourth NLS are not identical.
  • the third NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the fourth NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 134.
  • the third NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 and the fourth NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.
  • the third NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the fourth NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 134.
  • the third NLS comprises an amino acid sequence set forth IN SEQ ID NO: 133 and the fourth NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.
  • the first nucleic acid sequence and the second nucleic acid sequence are separated by an internal ribosome entry site (IRES) element or a nucleic acid sequence encoding a 2A peptide.
  • the first nucleic acid sequence and the second nucleic acid sequence are separated by a nucleic acid sequence encoding a furin cleavage motif and a nucleic acid sequence encoding a 2A peptide.
  • the first nucleic acid sequence and the second nucleic acid sequence are separated by a nucleic acid sequence encoding a furin cleavage motif, a nucleic acid sequence encoding a polypeptide linker, and a nucleic acid sequence encoding a 2A peptide.
  • the 2A sequence is a T2A, P2A, E2A, or F2A sequence.
  • the 2A peptide is a P2A peptide.
  • the first nucleic acid sequence and the second nucleic acid sequence are separated by a nucleic acid sequence encoding a P2A/furin peptide comprising an amino acid sequence set forth in SEQ ID NO: 135.
  • the first nucleic acid sequence and the second nucleic acid sequence are operably linked to a promoter.
  • the promoter is a CNS cell-specific promoter.
  • the promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells.
  • the promoter is a CAG promoter or a human Syn-1 promoter.
  • the first nucleic acid sequence is operably linked to a first promoter and the second nucleic acid sequence is operably linked to a second promoter.
  • the first and second promoters are identical.
  • the first and the second promoter is a CNS cell-specific promoter.
  • the first and the second promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells.
  • the first promoter and the second promoter are a CAG promoter or a human Syn-1 promoter.
  • the first promoter and the second promoter are a CNS cell-specific promoter. In some embodiments, the first promoter and the second promoter are active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells.
  • the first promoter is a CAG promoter
  • the second promoter is a human Syn-1 promoter. In some embodiments, the first promoter is a human Syn-1 promoter, and the second promoter is a CAG promoter.
  • the polynucleotide comprises a woodchuck hepatitis virus post- transcriptional regulatory element (WPRE). In some embodiments, the polynucleotide comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to according to SEQ ID NO: 194.
  • WPRE woodchuck hepatitis virus post- transcriptional regulatory element
  • the polynucleotide comprises an intron sequence.
  • the intron sequence is a chimeric or synthetic intron sequence.
  • the intron sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 136.
  • the polynucleotide comprises a termination sequence.
  • the termination sequence is a polyA sequence.
  • the polyA sequence comprises a nucleic acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 137.
  • the polyA sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 137.
  • the polynucleotide comprises, from 5' to 3': (a) a neuron-specific promoter enhancer; (b) a human Syn-1 promoter; (c) an intron sequence; (d) the first nucleic acid sequence encoding the first engineered meganuclease, wherein the first engineered meganuclease comprises an NLS; (e) a nucleic acid sequence encoding a P2A/furin peptide; (f) the second nucleic acid sequence encoding the second engineered meganuclease, wherein the second engineered meganuclease comprises an NLS; (g) a WPRE sequence; and (h) a polyA sequence.
  • the polynucleotide comprises, from 5' to 3': (a) a neuron-specific promoter enhancer; (b) a human Syn-1 promoter comprising a nucleic acid sequence set forth in SEQ ID NO: 138; (c) an intron sequence comprising a nucleic acid sequence set forth in SEQ ID NO: 136; (d) the first nucleic acid sequence encoding the first engineered meganuclease, wherein the first engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 7 or residues 2-354 of SEQ ID NO: 7, and wherein the first engineered meganuclease comprises an NLS; (e) a nucleic acid sequence encoding a P2A/furin peptide comprising an amino acid sequence set forth in SEQ ID NO: 135; (f) the second nucleic acid sequence encoding the second engineered meganuclease, wherein said second engineered meganuclease comprises an
  • the polynucleotide comprises, from 5' to 3': (a) a neuron-specific promoter enhancer; (b) a human Syn-1 promoter; (c) an intron sequence; (d) the second nucleic acid sequence encoding the second engineered meganuclease, wherein the second engineered meganuclease comprises an NLS; (e) a nucleic acid sequence encoding a P2A/furin peptide; (f) the first nucleic acid sequence encoding the first engineered meganuclease, wherein the first engineered meganuclease comprises an NLS; (g) a WPRE sequence; and (h) a polyA sequence.
  • the polynucleotide comprises, from 5' to 3': (a) a neuron-specific promoter enhancer; (b) a human Syn-1 promoter comprising a nucleic acid sequence set forth in SEQ ID NO: 138; (c) an intron sequence comprising a nucleic acid sequence set forth in SEQ ID NO: 136; (d) the second nucleic acid sequence encoding the second engineered meganuclease, wherein said second engineered meganuclease comprises an amino acid sequence set forth in SEQ ID NO: 80 or residues 2-354 of SEQ ID NO: 80, and wherein said second engineered meganuclease comprises an NLS; (e) a nucleic acid sequence encoding a P2A/furin peptide comprising an amino acid sequence set forth in SEQ ID NO: 135; (f) the first nucleic acid sequence encoding the first engineered meganuclease, wherein the first engineered meganucleas
  • the first engineered meganuclease comprises a first NLS attached at its C-terminus. In some such embodiments, the first NLS comprises a nucleic acid sequence set forth in SEQ ID NO: 134. In some embodiments, the second engineered meganuclease comprises a second NLS attached at its N-terminus. In some embodiments, the second NLS comprises a nucleic acid sequence set forth in SEQ ID NO: 133.
  • the polynucleotide is an mRNA.
  • the polynucleotide comprises a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease, wherein: (a) the first engineered meganuclease binds and cleaves a recognition sequence comprising SEQ ID NO: 3 and comprises an amino acid sequence of SEQ ID NO: 7 or residues 2-354 of SEQ ID NO: 7; and (b) the second engineered meganuclease binds and cleaves a recognition sequence comprising SEQ ID NO: 5 and comprises an amino acid sequence of SEQ ID NO: 80 or residues 2-354 of SEQ ID NO: 80.
  • a recombinant DNA construct comprising a polynucleotide described herein.
  • the recombinant DNA construct is a plasmid DNA.
  • the recombinant DNA construct encodes a recombinant virus comprising the polynucleotide.
  • the recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adeno-associated virus (AAV).
  • the recombinant virus is a recombinant AAV.
  • the recombinant AAV has an AAV9 capsid.
  • the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the engineered meganuclease.
  • the promoter is a CNS cell-specific promoter.
  • the promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells.
  • the promoter is a CAG promoter or a Syn-1 promoter.
  • the disclosure provides a recombinant virus comprising a polynucleotide described herein.
  • the polynucleotide is a polynucleotide described herein that comprises a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease.
  • the polynucleotide comprises a promoter operably linked to the first nucleic acid sequence and the second nucleic acid sequence.
  • the first nucleic acid sequence and the second nucleic acid sequence are separated by an IRES or 2A sequence.
  • the 2A sequence is a T2A, P2A, E2A, or F2A sequence.
  • the disclosure provides a lipid nanoparticle composition comprising lipid nanoparticles comprising a polynucleotide described herein.
  • the polynucleotide is an mRNA described herein. In some embodiments, the polynucleotide is a recombinant DNA construct described herein.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polynucleotide described herein.
  • the polynucleotide comprises an mRNA described herein. In some embodiments, the polynucleotide comprises a recombinant DNA construct described herein. In some embodiments, the pharmaceutical composition comprises a recombinant virus described herein. In some embodiments, the pharmaceutical composition comprises a lipid nanoparticle composition described herein.
  • composition comprising a pharmaceutically acceptable carrier and a recombinant DNA construct described herein.
  • composition comprising a pharmaceutically acceptable carrier and a recombinant virus described herein.
  • a host cell comprising a polynucleotide described herein.
  • a host cell comprising an engineered meganuclease described herein.
  • a method for producing a genetically-modified eukaryotic cell having a modified target sequence in a C90rf72 gene of the genetically-modified eukaryotic cell comprises introducing into a eukaryotic cell a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein, wherein the engineered meganuclease is expressed in the eukaryotic cell, and wherein the engineered meganuclease produces a cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3 or SEQ ID NO: 5.
  • the eukaryotic cell is a mammalian cell.
  • the mammalian cell is a CNS cell.
  • the CNS cell is a neuron or a motor neuron progenitor cell.
  • the mammalian cell is a human cell.
  • the polynucleotide is introduced into the eukaryotic cell by a recombinant virus, a lipid nanoparticle, or by an mRNA.
  • a method for producing a genetically-modified eukaryotic cell having a modified target sequence in a C90rf72 gene of the genetically-modified eukaryotic cell comprises introducing into a eukaryotic cell an engineered meganuclease described herein, wherein the engineered meganuclease produces a cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3 or SEQ ID NO: 5.
  • the eukaryotic cell is a mammalian cell.
  • the mammalian cell is a CNS cell.
  • the CNS cell is a neuron or a motor neuron progenitor cell.
  • the mammalian cell is a human cell.
  • a method for producing a genetically-modified eukaryotic cell comprising an exogenous sequence of interest inserted into a C90rf72 gene of the genetically-modified eukaryotic cell, the method comprising introducing into a eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding an engineered meganuclease described herein and a second nucleic acid sequence comprising the sequence of interest, wherein the engineered meganuclease produces a cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3 or SEQ ID NO: 5, wherein the sequence of interest is inserted into the C90rf72 gene at the cleavage site.
  • the second nucleic acid sequence further comprises nucleic acid sequences homologous to nucleic acid sequences flanking the cleavage site and the sequence of interest is inserted at the cleavage site by homologous recombination.
  • the eukaryotic cell is a mammalian cell.
  • the mammalian cell is a CNS cell.
  • the CNS cell is a neuron or a motor neuron progenitor cell.
  • the mammalian cell is a human cell.
  • the first and/or second polynucleotide is introduced into the eukaryotic cell by a recombinant virus, a lipid nanoparticle, or by an mRNA.
  • a method for producing a genetically-modified eukaryotic cell comprising an exogenous sequence of interest inserted into a C90rf72 gene of the genetically-modified eukaryotic cell, the method comprising introducing into a eukaryotic cell an engineered meganuclease described herein and a polynucleotide comprising the sequence of interest, wherein the engineered meganuclease produces a cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3 or SEQ ID NO: 5, wherein the sequence of interest is inserted into the C90rf72 gene at the cleavage site.
  • the polynucleotide further comprises nucleic acid sequences homologous to nucleic acid sequences flanking the cleavage site and the sequence of interest is inserted at the cleavage site by homologous recombination.
  • the eukaryotic cell is a mammalian cell.
  • the mammalian cell is a CNS cell.
  • the CNS cell is a neuron or a motor neuron progenitor cell.
  • the mammalian cell is a human cell.
  • the polynucleotide is introduced into the eukaryotic cell by a recombinant virus, a lipid nanoparticle, or by an mRNA.
  • a method for producing a genetically-modified eukaryotic cell comprising a modified C90rf72 gene comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease, wherein the first engineered meganuclease and the second engineered meganuclease are expressed in the eukaryotic cell, wherein the first engineered meganuclease produces a first cleavage site in an endogenous C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3, wherein the second engineered meganuclease produces a second cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 5, wherein an intervening genomic DNA between the first cleavage site and the second
  • the modified C90rf72 gene comprises a reduced number of GGGGCC hexanucleotide repeats relative to the endogenous C90rf72 gene.
  • the method comprises introducing into the eukaryotic cell a polynucleotide described herein comprising a first nucleic acid encoding the first engineered meganuclease and a second nucleic acid sequence encoding the second engineered meganuclease.
  • the polynucleotide is introduced into the eukaryotic cell by a recombinant virus described herein.
  • the recombinant virus is a recombinant AAV described herein.
  • the polynucleotide is a recombinant DNA construct.
  • the polynucleotide is an mRNA described herein.
  • the polynucleotide is introduced into the eukaryotic cell is a lipid nanoparticle.
  • the eukaryotic cell is a mammalian cell.
  • the eukaryotic cell is a human cell.
  • the eukaryotic cell is a CNS cell.
  • the CNS cell is a neuron or a motor neuron progenitor cell.
  • the method comprises introducing into the eukaryotic cell a first polynucleotide described herein comprising a first nucleic acid sequence encoding the first engineered meganuclease and a second polynucleotide described herein comprising the second nucleic acid sequence encoding the second engineered meganuclease.
  • the first polynucleotide comprises a first promoter operably linked to the first nucleic acid sequence encoding the first engineered meganuclease and/or the second polynucleotide comprises a second promoter operably linked to the second nucleic acid sequence encoding the second engineered meganuclease.
  • the first promoter and/or the second promoter is a CNS cell-specific promoter.
  • the first promoter and/or the second promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells.
  • the first promoter and/or the second promoter is a CAG promoter or a human Syn-1 promoter.
  • the first polynucleotide comprises a first promoter enhancer and/or the second polynucleotide comprises a second promoter enhancer.
  • the first and/or the second promoter enhancer is a neuron-specific promoter enhancer.
  • the first engineered meganuclease and/or the second engineered meganuclease comprises an NLS.
  • the NLS is attached to the N-terminus of the first engineered meganuclease and/or the second engineered meganuclease.
  • the NLS is attached to the C-terminus of the first engineered meganuclease and/or the second engineered meganuclease. In some embodiments, the NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134. In some embodiments, the NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 or 134.
  • the first engineered meganuclease and/or the second engineered meganuclease comprises a first NLS attached at the N-terminus and a second NLS attached at the C-terminus.
  • the first NLS and the second NLS are identical.
  • the first NLS and the second NLS comprise an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134.
  • the first NLS and the second NLS comprise an amino acid sequence set forth in SEQ ID NO: 133 or 134.
  • the first NLS and the second NLS are not identical.
  • the first NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO; 134.
  • the first NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.
  • the first polynucleotide and/or the second polynucleotide comprises a WPRE.
  • the first polynucleotide and/or the second polynucleotide comprises an intron sequence.
  • the intron sequence is a chimeric or synthetic intron sequence.
  • the intron sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 136.
  • the first polynucleotide and/or the second polynucleotide sequence comprises a termination sequence.
  • the termination sequence is a polyA sequence.
  • the polyA sequence comprises a nucleic acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 137.
  • the polyA sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 137.
  • the first polynucleotide is introduced into the eukaryotic cell by a first recombinant virus.
  • the second polynucleotide is introduced into the eukaryotic cell by a second recombinant virus.
  • the first recombinant virus is a first recombinant AAV and/or the second recombinant virus is a second recombinant AAV.
  • the first recombinant and/or the second recombinant AAV has an AAV9 capsid.
  • the first polynucleotide is a first mRNA.
  • the second polynucleotide is a second mRNA.
  • the first polynucleotide is a first recombinant DNA construct.
  • the second polynucleotide is a second recombinant DNA construct.
  • the first polynucleotide and the second polynucleotide are introduced into the eukaryotic cell by one or more lipid nanoparticles.
  • the first polynucleotide is introduced into the eukaryotic cell by a first lipid nanoparticle.
  • the second polynucleotide is introduced into the eukaryotic cell by a second lipid nanoparticle.
  • a method for modifying a C90rf72 gene in a target cell in a subject comprising delivering to the target cell one or more polynucleotides comprising a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease, wherein the first and the second engineered meganucleases are expressed in the target cell, wherein the first meganuclease produces a first cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3, wherein the second engineered meganuclease produces a second cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 5, wherein the intervening genomic DNA between the first cleavage site and the second cleavage site is excised from the C90rf72 gene, and wherein the C90rf72 gene is
  • the modified C90rf72 gene comprises a reduced number of GGGCC hexanucleotide repeats relative to the endogenous C90rf72 gene.
  • the method comprises introducing into the target cell a polynucleotide described herein comprising a first nucleic acid sequence encoding the first engineered meganuclease and a second nucleic acid sequence encoding the second engineered meganuclease.
  • the polynucleotide is introduced into the eukaryotic cell by a recombinant virus.
  • the recombinant virus is a recombinant AAV.
  • the polynucleotide is a recombinant DNA construct. In some embodiments, the polynucleotide is an mRNA. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the eukaryotic cell is a CNS cell. In some embodiments, the CNS cell is a neuron or a motor neuron progenitor cell.
  • the method comprises introducing into the target cell a first polynucleotide comprising a first nucleic acid sequence encoding the first engineered meganuclease and a second polynucleotide comprising the second nucleic acid sequence encoding the second engineered meganuclease.
  • the first polynucleotide comprises a first promoter operably linked to the first nucleic acid sequence encoding the first engineered meganuclease and/or the second polynucleotide comprises a second promoter operably linked to the second nucleic acid sequence encoding the second engineered meganuclease.
  • the first promoter and/or the second promoter is a CNS cell-specific promoter.
  • the first promoter and/or the second promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells.
  • the first promoter and/or the second promoter is a CAG promoter or a human Syn-1 promoter.
  • the first polynucleotide comprises a first promoter enhancer and/or the second polynucleotide comprises a second promoter enhancer.
  • the first and/or the second promoter enhancer is a neuron-specific promoter enhancer.
  • the first engineered meganuclease and/or the second engineered meganuclease comprises an NLS.
  • the NLS is attached to the N-terminus of the first engineered meganuclease and/or the second engineered meganuclease.
  • the NLS is attached to the C-terminus of the first engineered meganuclease and/or the second engineered meganuclease.
  • the NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134.
  • the NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 or 134.
  • the first engineered meganuclease and/or the second engineered meganuclease comprises a first NLS attached at the N-terminus and a second NLS attached at the C-terminus. In some embodiments, the first NLS and the second NLS are identical.
  • the first NLS and the second NLS comprise an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134. In some embodiments, the first NLS and the second NLS comprise an amino acid sequence set forth in SEQ ID NO: 133 or 134.
  • the first NLS and the second NLS are not identical.
  • the first NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO; 134.
  • the first NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.
  • the first polynucleotide and/or the second polynucleotide comprises a WPRE. In some embodiments, the first polynucleotide and/or the second polynucleotide comprises an intron sequence. In some embodiments, the intron sequence is a chimeric or synthetic intron sequence. In some embodiments, the intron sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 136. In some embodiments, the first polynucleotide and/or the second polynucleotide sequence comprises a termination sequence. In some embodiments, the termination sequence is a polyA sequence.
  • the polyA sequence comprises a nucleic acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 137. In some embodiments, the polyA sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 137.
  • the first polynucleotide is introduced into the eukaryotic cell by a first recombinant virus.
  • the second polynucleotide is introduced into the eukaryotic cell by a second recombinant virus.
  • the first recombinant virus is a first recombinant AAV and/or the second recombinant virus is a second recombinant AAV.
  • the first recombinant and/or the second recombinant AAV has an AAV9 capsid.
  • the first polynucleotide is a first mRNA.
  • the second polynucleotide is a second mRNA.
  • the first polynucleotide is a first recombinant DNA construct.
  • the second polynucleotide is a second recombinant DNA construct.
  • the first polynucleotide and the second polynucleotide are introduced into the eukaryotic cell by one or more lipid nanoparticles.
  • the first polynucleotide is introduced into the eukaryotic cell by a first lipid nanoparticle.
  • the second polynucleotide is introduced into the eukaryotic cell by a second lipid nanoparticle.
  • a method for treating a neurological disorder in a subject in need thereof wherein the neurological disorder is characterized by a mutation in a C90rf72 gene that increases the number of GGGGCC hexanucleotide repeats relative to a full- length wild-type C90rf72 gene, the method comprising: administering to the subject an effective amount of one or more polynucleotides comprising a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease, wherein the one or more polynucleotides are delivered to a target cell in the subject, wherein the first engineered meganuclease and the second engineered meganuclease are expressed in the target cell, wherein the first engineered meganuclease produces a first cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO:
  • the neurological disorder is amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).
  • the modified C9ORf72 gene comprises a reduced number of GGGGCC hexanucleotide repeats relative to the endogenous C90rf72 gene.
  • the method comprises delivering to the target cell a polynucleotide described herein comprising a first nucleic acid encoding the first engineered meganuclease and a second nucleic acid sequence encoding the second engineered meganuclease.
  • the polynucleotide is introduced into the eukaryotic cell by a recombinant virus.
  • the recombinant virus is an AAV.
  • the polynucleotide is a recombinant DNA construct.
  • the polynucleotide is an mRNA.
  • the polynucleotide is delivered to the target cell by a lipid nanoparticle.
  • the subject is a mammal.
  • the subject is a human.
  • the target cell is a CNS cell.
  • the CNS cell is a neuron or a motor neuron.
  • the method comprises introducing into the target cell a first polynucleotide comprising a first nucleic acid sequence encoding the first engineered meganuclease and a second polynucleotide comprising the second nucleic acid sequence encoding the second engineered meganuclease.
  • the first polynucleotide comprises a first promoter operably linked to the first nucleic acid sequence encoding the first engineered meganuclease and/or the second polynucleotide comprises a second promoter operably linked to the second nucleic acid sequence encoding the second engineered meganuclease.
  • the first promoter and/or the second promoter is a CNS cell-specific promoter. In some embodiments, the first promoter and/or the second promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells. In some embodiments, the first promoter and/or the second promoter is a CAG promoter or a human Syn-1 promoter. In some embodiments, the first polynucleotide comprises a first promoter enhancer and/or the second polynucleotide comprises a second promoter enhancer.
  • the first and/or the second promoter enhancer is a neuron-specific promoter enhancer.
  • the first engineered meganuclease and/or the second engineered meganuclease comprises an NLS.
  • the NLS is attached to the N-terminus of the first engineered meganuclease and/or the second engineered meganuclease.
  • the NLS is attached to the C-terminus of the first engineered meganuclease and/or the second engineered meganuclease.
  • the NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134.
  • the NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 or 134.
  • the first engineered meganuclease and/or the second engineered meganuclease comprises a first NLS attached at the N-terminus and a second NLS attached at the C-terminus.
  • the first NLS and the second NLS are identical.
  • the first NLS and the second NLS comprise an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134.
  • the first NLS and the second NLS comprise an amino acid sequence set forth in SEQ ID NO: 133 or 134.
  • the first NLS and the second NLS are not identical.
  • the first NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 134.
  • the first NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.
  • the first polynucleotide and/or the second polynucleotide comprises a WPRE. In some embodiments, the first polynucleotide and/or the second polynucleotide comprises an intron sequence. In some embodiments, the intron sequence is a chimeric or synthetic intron sequence. In some embodiments, the intron sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 136. In some embodiments, the first polynucleotide and/or the second polynucleotide sequence comprises a termination sequence. In some embodiments, the termination sequence is a polyA sequence.
  • the polyA sequence comprises a nucleic acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 137. In some embodiments, the polyA sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 137.
  • the first polynucleotide is introduced into the eukaryotic cell by a first recombinant virus.
  • the second polynucleotide is introduced into the eukaryotic cell by a second recombinant virus.
  • the first recombinant virus is a first recombinant AAV and/or the second recombinant virus is a second recombinant AAV.
  • the first recombinant and/or the second recombinant AAV has an AAV9 capsid.
  • the first polynucleotide is a first mRNA.
  • the second polynucleotide is a second mRNA.
  • the first polynucleotide is a first recombinant DNA construct.
  • the second polynucleotide is a second recombinant DNA construct.
  • the first polynucleotide and the second polynucleotide are introduced into the eukaryotic cell by one or more lipid nanoparticles.
  • the first polynucleotide is introduced into the eukaryotic cell by a first lipid nanoparticle.
  • the second polynucleotide is introduced into the eukaryotic cell by a second lipid nanoparticle.
  • the rate of motor function decline according to the ALS or FTD Functional Rating Scale is reduced compared to the decline prior to treatment or compared to an untreated subject having ALS or FTD.
  • the neurofilament light chain levels in the cerebral spinal fluid are reduced.
  • the method reduces the number of GGGGCC dipeptides in the CSF and/or neurons of the subject compared to an untreated subject having ALS or FTD. In some embodiments, the method reduces the number of GGGGCC RNA Foci in neurons of the subject compared to an untreated subject having ALS or FTD.
  • the disclosure provides engineered meganucleases described herein, or polynucleotides described herein encoding engineered meganucleases, or cells described herein expressing engineered meganucleases, for use as a medicament.
  • the medicament is useful for producing a modified C90rf72 gene in a subject. In some embodiments, the medicament is useful for the treatment of ALS or FTD.
  • the disclosure provides the use of engineered meganucleases described herein, or polynucleotides disclosed herein encoding engineered meganucleases, or cells described herein expressing engineered meganucleases, in the manufacture of a medicament for treating ALS or FTD, for increasing levels of a modified C90rf72 gene (i.e., lacking the GGGGCC hexanucleotide repeats relative to the endogenous C90rf72 gene), or reducing the symptoms associated with ALS or FTD.
  • a modified C90rf72 gene i.e., lacking the GGGGCC hexanucleotide repeats relative to the endogenous C90rf72 gene
  • FIG. 1 Schematic providing the approximate location of the CNR1-2 and CNR 21-22 meganuclease recognition sequences and illustrating the dual meganuclease approach for excising a region of DNA from the C90rf72 gene using either an upstream or downstream excision strategy.
  • a pair of engineered meganucleases that bind and cleave either upstream sites and the CNR 1-2 site (upstream excision approach) or that bind and cleave downstream CNR 21-22 sites and the CNR 1-2 site (downstream excision approach) are used.
  • the resultant C90rf72 transcript from the upstream excision approach is expressed from exon lb, thereby any mRNA lacks the hexanucleotide repeat region.
  • the resultant C90rf72 transcript from the downstream excision approach is expressed from exon la, but with an excised hexanucleotide repeat region.
  • FIG. 2 A schematic showing exemplary recognition sequences of the disclosure, which includes sense and anti-sense sequences for CNR 1-2 (SEQ ID NOs: 3 and 4) and CNR 21-22 (SEQ ID NOs: 5 and 6).
  • Each CNR recognition sequence targeted by engineered meganucleases described herein comprises two recognition half-sites.
  • Each recognition half-site comprises 9 base pairs, separated by a 4 base pair central sequence.
  • the CNR 1-2 recognition sequence has a 5’ CNR1 half-site and a 3’ CNR2 half-site with a four base pair center sequence ATAA.
  • the engineered meganucleases described herein comprise two subunits, with the first subunit having a hyper variable region (HVR), HVR1, that binds to a first recognition half-site and the second subunit having a HVR2 that binds to a second recognition half-site.
  • HVR hyper variable region
  • the first subunit comprising HVR1 can be positioned as either the N-terminal or C-terminal subunit connected via a linker to the second subunit comprising HVR2.
  • the second subunit comprising the HVR2 can be positioned as either the N-terminal or C-terminal subunit connected via a linker to the first subunit comprising HVR1.
  • Figures 4A-4B Figure 4 A provides an alignment of the sequences of the CNR 1-2 meganucleases described herein.
  • Figure 4B provides an alignment of the sequences of the CNR 21-22 meganucleases described herein. Asterisks indicate conserved residues amongst all aligned nucleases, and a space or colon indicates that at least one amino acid differed amongst the meganucleases.
  • Figure 5. Schematic of a reporter assay in CHO cells for evaluating engineered meganucleases targeting recognition sequences. For the engineered meganucleases described herein, a CHO cell line was produced in which a reporter cassette was integrated stably into the genome of the cell.
  • the reporter cassette comprised, in 5’ to 3’ order: an SV40 Early Promoter, the 5’ 2/3 of the green fluorescence protein (GFP) gene, the recognition sequence for an engineered meganuclease described herein (e.g., CNR 1-2 or CNR 21-22), the recognition sequence for the CHO-23/24 meganuclease (WO 2012/167192), and the 3’ 2/3 of the GFP gene.
  • GFP green fluorescence protein
  • the duplicated regions of the GFP gene recombined with one another to produce a functional GFP gene.
  • the percentage of GFP-expressing cells could then be determined by flow cytometry as an indirect measure of the frequency of genome cleavage by the meganucleases.
  • Figures 6A-6J Figure 6A, Figure 6B, Figure 6C, Figure 6D, Figure 6E, and Figure 6F provide the efficiency of engineered meganucleases for binding and cleaving the CNR 1-2 recognition sequence expressed in the CHO cell reporter assay.
  • Figure 6G, Figure 6H, Figure 61, and Figure 6J provide the efficiency of engineered meganucleases for binding and cleaving the CNR 21-22 recognition sequence expressed in the CHO cell reporter assay.
  • the relative activity index represents the %GFP positive cells for each cell line expressing the test meganuclease normalized to the cell line expressing the CHO-23/24 meganuclease, accounting for the toxicity of the meganuclease.
  • FIG. 7 A bar graph showing the percentage frequency of insertions and deletions (indels) of the tested meganucleases targeting the indicated recognition sequences in HEK293 cells. Each meganuclease was tested at two time points (days 2 and 6) following transfection of the meganuclease.
  • Figure 8 Provides a schematic of the oligocapture assay used to determine off-target effects of an engineered nuclease (e.g., an engineered meganuclease described herein).
  • an engineered nuclease e.g., an engineered meganuclease described herein.
  • the integration cassette or oligo anneals with a double-strand break (DSB) in the gnome that may be due to engineered nuclease cleavage.
  • the DNA is then sheared by sonication, adapters are ligated, and PCR amplified, followed by sequence analysis to determine location of the DSB.
  • DSB double-strand break
  • Figure 9 Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNRl-2xl, CNRl-2xl7, CNRl-2x25, CNRl-2x80, CNRl-2x87, CNRl-2x88, CNRl-2x91, and CNRl-2x93 meganucleases transfected in HEK293 cells.
  • the circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site.
  • the shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.
  • Figures 10A-10B Provide bar graphs showing the percentage of indels in a HEK293 cell line as assessed by digital PCR ( Figure 10A) or NGS targeted sequencing ( Figure 10B). The percentage of indels were assessed at day 2 and day 5 post transfection.
  • Figure 11 Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR1-2L.15, CNR1-2L.18, or CNR1-2L.56 meganucleases transfected in HEK293 cells.
  • the circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site.
  • the shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.
  • Figures 12A-12B Provides bar graphs showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR (ddPCR).
  • Figure 12A provides data using the downstream excision strategy (see Figure 1), with the combination of CNR1-2L.15 and CNR19- 20x.l l meganucleases, the CNR1-2L.98 and CNR19-20x.l l meganucleases, the CNR1-2L.100 and CNR19-20x.
  • Figure 12B provides data using the upstream excision strategy (see Figure 1), with the combination of CNR1-2L.15 and CNR17-18x.85 meganucleases, the CNR1- 2L.98 and CNR17-18x.85 meganucleases, the CNR1-2L.100 and CNR17-18x.85 meganucleases, the CNR1-2L.104 and CNR17-18x.85 meganucleases, the CNR1-2L.108 and CNR17-18x.85 meganucleases, the CNR1-2L.140 and CNR17-18x.85 meganucleases, and a mock or GFP control.
  • Figure 13 Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR1-2L100, CNR1-2L104, CNR1-2L108, CNR1-2L126, CNR1- 2L140, CNR1-2L141, or CNR1-2L198 meganucleases transfected in HEK293 cells.
  • the circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site.
  • the shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.
  • Figure 14A-14B Provide bar graphs showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR (ddPCR).
  • Figure 14A provides data using the upstream excision strategy (see Figure 1), with the combination of CNR1-2L.98 and CNR17- 18L.12 meganucleases, CNR1-2L.211 and CNR17-18L.12 meganucleases, CNR1-2L.213 and CNR17-18L.12 meganucleases, the CNR1-2L.217 and CNR17-18L.12 meganucleases, or mock or GFP controls.
  • Figure 14B provides data using the downstream excision strategy (see Figure 1), with the combination of CNR1-2L.98 and CNR19-20L.71 meganucleases, the CNR1-2L.211 and CNR19-20L.71 meganucleases, the CNR1-2L.213 and CNR19-20L.71 meganucleases, the CNR1- 2L.217 and CNR19-20L.71 meganucleases, or mock or GFP controls.
  • Figure 15 Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR1-2L.211, CNR1-2L.213, or CNR1-2L.217 meganucleases transfected in HEK293 cells.
  • the circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site.
  • the shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.
  • Figure 16 Provides a bar graph depicting multiplex targeted amplicon sequencing results of the intended site (chr9_27573614_27573672) and several top off-target sites as identified by oligocapture assays for CNR1-2L.211, CNR1-2L.213, CNR1-2L.217, or CNR1-2L.98.
  • Figure 17A-17B Provide bar graphs showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR (ddPCR).
  • Figure 17A provides data using the upstream excision strategy (see Figure 1), with the indicated CNR 1-2 meganucleases with GFP as a control or the combinations of indicated CNR1-2 and CNR17-18 engineered meganucleases and a mock or GFP controls.
  • Figure 17b provides data using the downstream excision strategy (see Figure 1), with the indicated CNR 1-2 meganucleases with GFP as a control or the combinations of indicated CNR1-2 and CNR19-20 engineered meganucleases and a mock or GFP controls.
  • the circled dots indicate the on-target site, and the non-circled dots indicate off- target sites, with the X axis representing the number of sequencing reads for each detected off- target site.
  • the shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.
  • Figure 19 Provides a bar graph depicting multiplex targeted amplicon sequencing results of the intended site (9_27573614_27573635) and several top off-target sites as identified by oligocapture assays for CNR1-2L.284 or CNR1-2L.297 meganucleases.
  • Figures 20A-20B Provide bar graphs showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR (ddPCR).
  • Figure 20A provides data using the upstream excision strategy (see Figure 1), with the combination of indicated CNR 1-2 and CNR 17- 18 meganucleases mock, or GFP controls.
  • Figure 20B provides data using the downstream excision strategy (see Figure 1), with the combination of indicated CNR1-2 and CNR21-22 meganucleases mock, or GFP controls.
  • Figure 22 Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR1-2L.371, CNR1-2L.403, or CNR1-2L.425 meganucleases transfected in HEK293 cells.
  • the circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site.
  • the shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.
  • Figure 23 Provides a bar graph depicting multiplex targeted amplicon sequencing results of the intended site (9_27573614_27573635) and several top off-target sites as identified by oligocapture assays for CNR1-2L.297, CNR1-2L.371, CNR1-2L.403, CNR1-2L.425 meganucleases or GFP control.
  • Figure 24A-24B Provide bar graphs showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR (ddPCR).
  • Figure 24A provides data using the upstream excision strategy (see Figure 1), with the combination of the indicated CNR1-2 and CNR 17-18 meganucleases, mock, or GFP controls.
  • Figure 24B provides data using the downstream excision strategy (see Figure 1), with the combination of CNR1-2 and CNR21-22 meganucleases mock, or GFP controls.
  • Figure 25 Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR1-2L.468 or CNR1-2L.531 meganucleases transfected in HEK293 cells.
  • the circled dots indicate the on-target site, and the non-circled dots indicate off- target sites, with the X axis representing the number of sequencing reads for each detected off- target site.
  • the shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.
  • Figure 26 Provides a bar graph depicting multiplex targeted amplicon sequencing results of the intended site (9_27573614_27573635) and several top off-target sites as identified by oligocapture assays for the CNR1-2L.425 or CNR1-2L.531 meganucleases or GFP control at the 5ng, 25ng, or 200ng dose.
  • Figure 27 Provides a bar graph showing the percentage of excision and ligation in the C90rf72 gene as assessed by ddPCR with the indicated combinations of CNR 1-2 and CNR 21-22 meganucleases or GFP control.
  • Figure 28A-28C Provide graphs depicting results from an oligo capture assay to identify off target cutting induced by the CNR1-2L.425 (Figure 28A), CNR1-2L.531 ( Figure 28B), or CNR1-2L.532 ( Figure 28C) meganucleases transfected in HEK293 cells.
  • the circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site.
  • the shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.
  • Figure 29 Provides a graph showing the percentage of indel formation in the C90rf72 gene as assessed by ddPCR for the CNR1-2L.425 (circle), CNR1-2L.531 (square), and CNR1-2L.532 (triangle) meganucleases at concentrations ranging from 0.39 ng to 400 ng.
  • Figure 30 Provides the percentage of indels at on-target (intended target) and off-target sites as assessed by MTA for CNR1-2L.425 and CNR1-2L.532 at concentrations ranging from 6.25 ng to 400 ng.
  • Figure 31 A-3 IB Provides bar graphs showing the percentage of excision and ligation in the C90rf72 gene in either healthy cells (Figure 31 A) or C9 patient-derived motor neuron progenitor cells (MNPCs) (Figure 3 IB) using the dual meganuclease approach as shown in Figure 1.
  • MNPCs motor neuron progenitor cells
  • Figure 32A-32B Provide bar graphs showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR (ddPCR).
  • Figure 32A provides data using the upstream excision strategy (see Figure 1), with the combination of CNR1-2L.211 and CNR17- 18L.120 meganucleases, the CNR1-2L.211 and CNR17-18L.132 meganucleases, the CNR9- 10L.257 and CNR17-18L.120 meganucleases, the CNR9-10L.257 and CNR17-18L.132 meganucleases, the CNR3-4L.212 and CNR29-30L.107 meganucleases, the CNR3-4L.212 and CNR25-26L.103 meganucleases, the CNR3-4L.212 and CNR25-26L.174 meganucleases, or the CNR3-4L.212 and CNR31-32L.103 meganucleases.
  • Figure 32B provides data using the downstream excision strategy (see Figure 1), with the combination of CNR1-2L.211 and CNR19- 20L.93 meganucleases, the CNR1-2L.211 and CNR21-22L.139 meganucleases, the CNR1-2L.211 and CNR21-22L.169 meganucleases, or the CNR3-4L.212 and CNR35-36L.176 meganucleases.
  • Figure 33 Provides a bar graph showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR for the combination of CNR1-2L.15 and CNR21- 22x.l3 meganucleases, the CNR1-2L.15 and CNR21-22x.45 meganucleases, the CNR1-2L.15 and CNR21-22x.84 meganucleases, mock, GFP, and single meganuclease controls.
  • Figure 34 Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR21-22xl3, CNR21-22x45, and CNR21-22x84 meganucleases transfected in HEK293 cells.
  • the circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site.
  • the shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.
  • Figure 35 Provides a bar graph showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR for the indicated combinations of CNR 21-22 and CNR 1-2 meganucleases, mock, or GFP controls.
  • Figure 36 Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR21-22L.13, CNR21-22L.27, CNR21-22L.34, CNR21-22L.37, CNR21-22L.38, and CNR21-22L.45 meganucleases transfected in HEK293 cells.
  • the circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site.
  • the shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.
  • Figure 37 Provides a bar graph showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR for the indicated combinations of CNR21-22 and CNR1-2 meganucleases, CNR 21-22 meganuclease with GFP, mock, or GFP alone controls.
  • Figure 38 Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR21-22L.109, CNR21-22L.117, CNR21-22L.121, CNR21- 22L.122, CNR21-22L.124, CNR21-22L.139, CNR21-22L.140, CNR21-22L.148, CNR21-22L.152, CNR21-22L.153, CNR21-22L.157, CNR21-22L.158, CNR21-22L.165, CNR21-22L.167, CNR21- 22L.169, CNR21-22L.176, and CNR21-22L.181 meganucleases transfected in HEK293 cells.
  • FIG. 39 Provides a bar graph showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR for the indicated combinations of CNR 21-22 and CNR 1-2 meganucleases, CNR 21-22 meganuclease with GFP, mock, or GFP controls.
  • Figure 40A-40B Provide graphs depicting results from an oligo capture assay to identify off target cutting induced by meganucleases described herein.
  • Figure 40A depicts results for the CNR21-22L.197, CNR21-22L.198, CNR21-22L.228, CNR21-22L.243, CNR21-22L.250, and CNR21-22L.274 meganucleases transfected in HEK293 cells.
  • Figure 40B depicts results for the CNR21-22L.212 and CNR21-22L.206 meganucleases transfected in HEK293 cells.
  • the circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site.
  • the shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.
  • Figure 41 Provides the percentage of indels at on-target (9_27571934_27571955; intended target) and off-target sites as assessed by MTA for CNR21-22L.197, CNR21-22L.212, and CNR21-22L.274 meganucleases.
  • Figure 42 Provides the percentage of indels at on-target (9_27571934_27571955; intended target) and off-target sites as assessed by MTA for CNR21-22L.197, CNR21-22L.212, and CNR21-22L.274 meganucleases at 5 ng, 25ng, and 200ng doses.
  • Figure 43 Provides an enlarged perspective of the percentage of indels at off-target sites of the meganucleases as shown in Figure 42.
  • Figure 44 Provides a bar graph showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR for the indicated CNR 21-22 meganucleases mock, or GFP controls in combination with meganucleases CNR1-2L.425 (black bar), CNR1- 2L.531 (light grey bar), or CNR1-2L.532 (dark grey bar).
  • Figure 45 Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR21-22L.277, CNR21-22L.279, CNR21-22L.281, CNR21- 22L.285, CNR21-22L.286, CNR21-22L.289, or CNR21-22L.290 meganucleases transfected in HEK293 cells.
  • the circled dots indicate the on-target site, and the non-circled dots indicate off- target sites, with the X axis representing the number of sequencing reads for each detected off- target site.
  • the shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.
  • Figure 46 Provides a graph showing the percentage of indels in the C90rf72 gene as assessed by ddPCR for the CNR21-22L.212 (diamond), CNR21-22L.286 (circle), and CNR21- 22L.290 (square) meganucleases at concentrations ranging from 0.39 ng to 400 ng.
  • Figure 47 Provides the percentage of indels at the on target (9:27571934-27571955) and one-off target site as assessed by MTA for CNR21-22L.286, CNR21-22L.290, and CNR21- 22L.212 at concentrations ranging from 6.25 ng to 400 ng.
  • Figures 48A-48D Provide bar graphs depicting the percentage of excision and ligation observed in an in vivo assay using a transgenic mouse line C9BAC. Mice were injected with one of six different constructs encapsulated by AAV9 or AAVrhlO.
  • Figure 48A depicts the percentage of excision and ligation in the cortex for Group 1 to Group 7 (G1 - G7).
  • Figure 48B depicts the percentage of excision and ligation in the brain stem for G1 to G7.
  • Figure 48C depicts the percentage of excision and ligation in the cervical spine for G1 to G7.
  • Figure 48D depicts the percentage of excision and ligation in the thoracic spine for G1 to G7.
  • the vector for G1 comprises a CAG promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease, and a WPRE 3’UTR encapsulated in an AAV9 particle;
  • the vector for G2 comprises a Synl promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease, and a WPRE 3’UTR encapsulated in an AAV9 particle;
  • the vector for G3 comprises a CAG promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease encapsulated in an AAV9 particle;
  • the vector for G4 comprises a Synl promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease encapsulated in an AAV9 particle;
  • the vector for G5 comprises a CAG promoter, a CNR1-2L.98 and a CNR21-22
  • Figures 49A-49B Provide bar graphs depicting the percentage of excision and ligation observed in an in vivo assay using a transgenic mouse line C9BAC. Mice were injected with one of six different constructs encapsulated by AAV9 or AAVrhlO.
  • Figure 49A depicts the percentage of excision and ligation in the liver for Group 1 to Group 7 (G1 - G7).
  • Figure 49B depicts the percentage of excision and ligation in the heart for G1 to G7.
  • the vector for G1 comprises a CAG promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease, and a WPRE 3’UTR encapsulated in an AAV9 particle;
  • the vector for G2 comprises a Synl promoter, a CNR1-2L.98 and a CNR21- 22L.38 meganuclease, and a WPRE 3’UTR encapsulated in an AAV9 particle;
  • the vector for G3 comprises a CAG promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease encapsulated in an AAV9 particle;
  • the vector for G4 comprises a Synl promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease encapsulated in an AAV9 particle;
  • the vector for G5 comprises a CAG promoter, a CNR1-2L.98 and a CNR21
  • Figure 50A-50D Provide bar graphs depicting the percentage of excision and ligation observed in an in vivo assay in non-human primates (cynomolgus macaques). NHP were injected with one of three different constructs encapsulated by AAV9 or AAVrhlO or a control of PBS.
  • Figure 50A depicts the percentage of excision and ligation in the target CNS.
  • Figure 50B depicts the percentage of excision and ligation in the other CNS.
  • Figure 50C depicts the percentage of excision and ligation in the peripheral nervous system.
  • Figure 50D depicts the percentage of excision and ligation in the peripheral tissues.
  • AAV9-CAG represents a viral vector comprising a CAG promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease, and a WPRE 3’UTR encapsulated in an AAV9 particle.
  • AAV9-Synl represents a viral vector comprising a Synl promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease, and a WPRE 3’ UTR encapsulated in an AAV9 particle.
  • AAV4hlO-Synl represents a viral vector comprising a Synl promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease, and a WPRE 3’UTR encapsulated in an AAVrhlO particle.
  • Figure 51A-51B Figure 51 A provides RNAscope staining of lower motor neurons from the spine with the CHAT marker in NHPs treated with either AAV vectors (upper left and right panel) or PBS control (lower left and lower right panel). CHAT expression is provided in the upper and lower left panels and meganuclease staining is provided in the upper right and lower right panels.
  • Figure 5 IB provides quantification of meganuclease positive motor neurons from the entire spine from RNAscope staining and analysis.
  • Figure 52A-52B Figure 52A provides RNA scope staining of upper motor neurons from the motor cortex of NHPs treated with either AAV vectors or PBS control. The left panel provides staining of upper motor neurons, and the right panel shows staining of upper motor neurons expressing the meganuclease.
  • Figure 52B provides quantification of the RNAscope staining of meganuclease positive upper motor neurons from RNAscope staining and analysis.
  • Figure 53A-53B Figure 53A provides RNA scope staining of motor neurons from the medulla in NHPs treated with AAV vectors that are positive for meganuclease expression.
  • Figure 53B provides quantification of the RNAscope staining of meganuclease motor neurons from RNAscope staining and analysis.
  • Figure 54 Provides a bar graph depicting the percentage of excision and ligation observed in an in vivo assay using a transgenic mouse line C9BAC across different tissue types. Mice were injected with one of four different constructs encapsulated by AAV9.
  • Group 1 was treated with a viral vector comprising a CNR1-2L.98 and CNR19-20L.71 meganuclease encapsulated in an AAV9 particle;
  • Group 2 was treated with a viral vector comprising a CNR1-2L.98 and CNR21- 22L.38 meganuclease encapsulated in an AAV9 particle;
  • Group 3 was treated with a viral vector comprising a CNR1-2L.98 and CNR23-24L.35 meganuclease encapsulated in an AAV9 particle;
  • Group 4 was treated with a viral vector comprising a CNR1-2L.98 and CNR17-18L.12 meganuclease encapsulated in an AAV9 particle.
  • Figure 55A-55E Provide survival curves depicting the percent survival of a transgenic mouse line C9-500 treated as follows. Mice were injected with one of four different constructs encapsulated by AAV9. Group 1 was a control group treated with PBS. Group 2 was treated with a viral vector comprising a CAG promoter, and a CNR1-2L.211 and a CNR21-22L.129 meganuclease encapsulated in an AAV9 particle. Group 3 was treated with a viral vector comprising a Synl promoter, and a CNR1-2L.211 and a CNR21-22L.139 meganuclease encapsulated in an AAV9 particle.
  • Group 4 was treated with a viral vector comprising a CAG promoter, and a CNR3-4L.212 and a CNR29-30L.107 meganuclease encapsulated in an AAV9 particle.
  • Group 5 was treated with a viral vector comprising a Synl promoter, and a CNR3-4L.212 and a CNR29-30L.107 meganuclease encapsulated in an AAV9 particle.
  • Figure 55A shows the survival curve for Group 1 (control);
  • Figure 55B shows the survival curve for Group 2;
  • Figure 55C shows the survival curve for Group 3;
  • Figure 55D shows the survival curve for Group 4; and
  • Figure 55E shows the survival curve for Group 5.
  • Figure 56 Provides bar graphs showing the excision and ligation of regions as detected by ddPCR in the spine and cortex of the surviving mice in group 1 (labelled as PBS), group 2 ( labelled as CAG CNR1 -2x21-22), group 3 (labelled as Synl CNR1 -2x21-22), and group 4 (labelled as CNR3-4x29-30) corresponding to those groups described in Figure 55A-55E.
  • Figure 57 Provides a bar graph showing expression levels of polyGP in the cortex of mice from Group 1 (control; circle), Group 2 (square), Group 3 (triangle) and Group 4 (inverted triangle) corresponding to those groups described in Figure 55A-55E.
  • Figures 58A-58B Provide bar graphs showing the number of RNA foci in the motor cortex (Figure 58A) or the lumbar spine ( Figure 58B) of mice from Group 1 (control; circle), Group 2 (square), Group 3 (triangle), and Group 4 (inverted triangle) corresponding to those groups described in Figure 55A-55E.
  • Figures 59A-59D Provide bar graphs showing the expression of C90rf72 mRNA f RNA foci in the cortex (Figure 59A) or the cervical spine ( Figure 59B).
  • Figure 59A depicts expression results for all the C90rf72 isoforms for mice from Group 1 (control; circle), Group 2 (square), Group 3 (triangle), and Group 4 (inverted triangle).
  • Figure 59B depicts expression results for all the C90rf72 isoforms for mice from group 1 (control; circle), Group 2 (square), Group 3 (triangle), and Group 4 (inverted triangle) .
  • Figure 59C depicts expression results for the C90rf72 long isoforms (isoform variant 2 and isoform variant 3 denoted as V23) for mice from group 1 (control; circle), Group 2 (square), Group 3 (triangle), and Group 4 (inverted triangle).
  • Figure 59D depicts expression results for the C90rf72 short isoform variant 1 (denoted as VI) for mice from group 1 (control; circle), Group 2 (square), Group 3 (triangle), and Group 4 (inverted triangle) corresponding to those groups described in Figure 55A-55E.
  • Figures 60A-60B Provide bar graphs showing the percentage of excision and ligation of the C90rf72 locus and RNA foci in motor neuron progenitor cells (MNPCs) derived from a C9-ALS patient or a healthy donor.
  • MNPCs motor neuron progenitor cells
  • Cells were treated with a viral vector comprising a neuron specific enhancer, a Synl promoter, a CNR21-22L.286 and a CNR1-2L.532 meganuclease encapsulated in an AAV9 particle.
  • Figure 60A depicts the percentage of percentage of excision and ligation of the C90rf72 in treated cells (square) vs untreated control (circle).
  • Figure 60B depicts the number of RNA Foci between healthy donor cells, untreated C9-ALS-derived cells, and treated C9-ALS- derived cells.
  • Figures 61 A-61B Provide bar graphs showing the percentage of excision and ligation of the C90rf72 locus and RNA foci in motor neuron progenitor cells (MNPCs) derived from a C9-ALS patient or a healthy donor.
  • MNPCs motor neuron progenitor cells
  • Cells were treated with a viral vector comprising a neuron specific enhancer, a Synl promoter, a CNR 1-2L.425 and a CNR 21-22L.212 meganuclease encapsulated in an AAV9 particle.
  • Figure 61 A depicts the percentage of excision and ligation of the C90rf72 in treated cells vs untreated control.
  • Figure 61B depicts the number of RNA Foci between healthy donor cells, untreated C9-ALS-derived cells, and treated C9-ALS-derived cells.
  • SEQ ID NO: 1 sets forth amino acid sequence of the wild-type I-Crel meganuclease from Chlamydomonas reinhardtii.
  • SEQ ID NO: 2 sets forth the amino acid sequence of the LAGLID ADG motif.
  • SEQ ID NO: 3 sets forth the nucleic acid sequence of the sense strand of the CNR 1-2 recognition sequence.
  • SEQ ID NO: 4 sets forth the nucleic acid sequence of the antisense strand of the CNR 1-2 recognition sequence.
  • SEQ ID NO: 5 sets forth the nucleic acid sequence of the sense strand of the CNR 21-22 recognition sequence.
  • SEQ ID NO: 6 sets forth the nucleic acid sequence of the antisense strand of the CNR 21-22 recognition sequence.
  • SEQ ID NO: 7 sets forth the amino acid sequence of the CNR 1-2L.532 engineered meganuclease.
  • SEQ ID NO: 8 sets forth the amino acid sequence of the CNR 1-2L.468 engineered meganuclease.
  • SEQ ID NO: sets forth the amino acid sequence of the CNR 1-2L.531 engineered meganuclease.
  • SEQ ID NO: 10 sets forth the amino acid sequence of the CNR 1-2L.371 engineered meganuclease.
  • SEQ ID NO: 11 sets forth the amino acid sequence of the CNR 1-2L.425 engineered meganuclease.
  • SEQ ID NO: 12 sets forth the amino acid sequence of the CNR 1-2L.284 engineered meganuclease.
  • SEQ ID NO: 13 sets forth the amino acid sequence of the CNR 1-2L.297 engineered meganuclease.
  • SEQ ID NO: 14 sets forth the amino acid sequence of the CNR 1-2L.211 engineered meganuclease.
  • SEQ ID NO: 15 sets forth the amino acid sequence of the CNR 1-2L.98 engineered meganuclease.
  • SEQ ID NO: 16 sets forth the amino acid sequence of the CNR 1-2L.108 engineered meganuclease.
  • SEQ ID NO: 17 sets forth the amino acid sequence of the CNR 1-2L15 engineered meganuclease.
  • SEQ ID NO: 18 sets forth the amino acid sequence of the CNR l-2x.88 engineered meganuclease.
  • SEQ ID NO: 19 sets forth the amino acid sequence of the CNR 1-2L.532 engineered meganuclease CNR1 binding subunit.
  • SEQ ID NO: 20 sets forth the amino acid sequence of the CNR 1-2L.468 engineered meganuclease CNR1 binding subunit.
  • SEQ ID NO: 21 sets forth the amino acid sequence of the CNR 1-2L.531 engineered meganuclease CNR1 binding subunit.
  • SEQ ID NO: 22 sets forth the amino acid sequence of the CNR 1-2L.371 engineered meganuclease CNR1 binding subunit.
  • SEQ ID NO: 23 sets forth the amino acid sequence of the CNR 1-2L.425 engineered meganuclease CNR1 binding subunit.
  • SEQ ID NO: 24 sets forth the amino acid sequence of the CNR 1-2L.284 engineered meganuclease CNR1 binding subunit.
  • SEQ ID NO: 25 sets forth the amino acid sequence of the CNR 1-2L.297 engineered meganuclease CNR1 binding subunit.
  • SEQ ID NO: 26 sets forth the amino acid sequence of the CNR 1-2L.211 engineered meganuclease CNR1 binding subunit.
  • SEQ ID NO: 27 sets forth the amino acid sequence of the CNR 1-2L.98 engineered meganuclease CNR1 binding subunit.
  • SEQ ID NO: 28 sets forth the amino acid sequence of the CNR 1-2L.108 engineered meganuclease CNR1 binding subunit.
  • SEQ ID NO: 29 sets forth the amino acid sequence of the CNR 1-2L15 engineered meganuclease CNR1 binding subunit.
  • SEQ ID NO: 30 sets forth the amino acid sequence of the CNR l-2x.88 engineered meganuclease CNR1 binding subunit.
  • SEQ ID NO: 31 sets forth the amino acid sequence of the CNR 1-2L.532 engineered meganuclease CNR2 binding subunit.
  • SEQ ID NO: 32 sets forth the amino acid sequence of the CNR 1-2L.468 engineered meganuclease CNR2 binding subunit.
  • SEQ ID NO: 33 sets forth the amino acid sequence of the CNR 1-2L.531 engineered meganuclease CNR2 binding subunit.
  • SEQ ID NO: 34 sets forth the amino acid sequence of the CNR 1-2L.371 engineered meganuclease CNR2 binding subunit.
  • SEQ ID NO: 35 sets forth the amino acid sequence of the CNR 1-2L.425 engineered meganuclease CNR2 binding subunit.
  • SEQ ID NO: 36 sets forth the amino acid sequence of the CNR 1-2L.284 engineered meganuclease CNR2 binding subunit.
  • SEQ ID NO: 37 sets forth the amino acid sequence of the CNR 1-2L.297 engineered meganuclease CNR2 binding subunit.
  • SEQ ID NO: 38 sets forth the amino acid sequence of the CNR 1-2L.211 engineered meganuclease CNR2 binding subunit.
  • SEQ ID NO: 39 sets forth the amino acid sequence of the CNR 1-2L.98 engineered meganuclease CNR2 binding subunit.
  • SEQ ID NO: 40 sets forth the amino acid sequence of the CNR 1-2L.108 engineered meganuclease CNR2 binding subunit.
  • SEQ ID NO: 41 sets forth the amino acid sequence of the CNR 1-2L15 engineered meganuclease CNR2 binding subunit.
  • SEQ ID NO: 42 sets forth the amino acid sequence of the CNR l-2Lx.88 engineered meganuclease CNR2 binding subunit.
  • SEQ ID NO: 43 sets forth the amino acid sequence of the CNR 1-2L.532 engineered meganuclease CNR1 binding subunit HVR1 region.
  • SEQ ID NO: 44 sets forth the amino acid sequence of the CNR 1-2L.468 engineered meganuclease CNR1 binding subunit HVR1 region.
  • SEQ ID NO: 45 sets forth the amino acid sequence of the CNR 1-2L.531 engineered meganuclease CNR1 binding subunit HVR1 region.
  • SEQ ID NO: 46 sets forth the amino acid sequence of the CNR 1-2L.371 engineered meganuclease CNR1 binding subunit HVR1 region.
  • SEQ ID NO: 47 sets forth the amino acid sequence of the CNR 1-2L.425 engineered meganuclease CNR1 binding subunit HVR1 region.
  • SEQ ID NO: 48 sets forth the amino acid sequence of the CNR 1-2L.284 engineered meganuclease CNR1 binding subunit HVR1 region.
  • SEQ ID NO: 49 sets forth the amino acid sequence of the CNR 1-2L.297 engineered meganuclease CNR1 binding subunit HVR1 region.
  • SEQ ID NO: 50 sets forth the amino acid sequence of the CNR 1-2L.211 engineered meganuclease CNR1 binding subunit HVR1 region.
  • SEQ ID NO: 51 sets forth the amino acid sequence of the CNR 1-2L.98 engineered meganuclease CNR1 binding subunit HVR1 region.
  • SEQ ID NO: 52 sets forth the amino acid sequence of the CNR 1-2L.108 engineered meganuclease CNR1 binding subunit HVR1 region.
  • SEQ ID NO: 53 sets forth the amino acid sequence of the CNR 1-2L15 engineered meganuclease CNR1 binding subunit HVR1 region.
  • SEQ ID NO: 54 sets forth the amino acid sequence of the CNR l-2x.88 engineered meganuclease CNR1 binding subunit HVR1 region.
  • SEQ ID NO: 55 sets forth the amino acid sequence of the CNR 1-2L.532 engineered meganuclease CNR2 binding subunit HVR2 region.
  • SEQ ID NO: 56 sets forth the amino acid sequence of the CNR 1-2L.468 engineered meganuclease CNR2 binding subunit HVR2 region.
  • SEQ ID NO: 57 sets forth the amino acid sequence of the CNR 1-2L.531 engineered meganuclease CNR2 binding subunit HVR2 region.
  • SEQ ID NO: 58 sets forth the amino acid sequence of the CNR 1-2L.371 engineered meganuclease CNR2 binding subunit HVR2 region.
  • SEQ ID NO: 59 sets forth the amino acid sequence of the CNR 1-2L.425 engineered meganuclease CNR2 binding subunit HVR2 region.
  • SEQ ID NO: 60 sets forth the amino acid sequence of the CNR 1-2L.284 engineered meganuclease CNR2 binding subunit HVR2 region.
  • SEQ ID NO: 61 sets forth the amino acid sequence of the CNR 1-2L.297 engineered meganuclease CNR2 binding subunit HVR2 region.
  • SEQ ID NO: 62 sets forth the amino acid sequence of the CNR 1-2L.211 engineered meganuclease CNR2 binding subunit HVR2 region.
  • SEQ ID NO: 64 sets forth the amino acid sequence of the CNR 1-2L.98 engineered meganuclease CNR2 binding subunit HVR2 region.
  • SEQ ID NO: 64 sets forth the amino acid sequence of the CNR 1-2L.108 engineered meganuclease CNR2 binding subunit HVR2 region.
  • SEQ ID NO: 65 sets forth the amino acid sequence of the CNR 1-2L15 engineered meganuclease CNR2 binding subunit HVR2 region.
  • SEQ ID NO: 66 sets forth the amino acid sequence of the CNR l-2x.88 engineered meganuclease CNR2 binding subunit HVR2 region.
  • SEQ ID NO: 67 sets forth a nucleic acid sequence encoding the CNR 1-2L.532 engineered meganuclease.
  • SEQ ID NO: 68 sets forth a nucleic acid sequence encoding the CNR 1-2L.468 engineered meganuclease.
  • SEQ ID NO: 69 sets forth a nucleic acid sequence encoding the CNR 1-2L.531 engineered meganuclease.
  • SEQ ID NO: 70 sets forth a nucleic acid sequence encoding the CNR 1-2L.371 engineered meganuclease.
  • SEQ ID NO: 71 sets forth a nucleic acid sequence encoding the CNR 1-2L.425 engineered meganuclease.
  • SEQ ID NO: 72 sets forth a nucleic acid sequence encoding the CNR 1-2L.284 engineered meganuclease.
  • SEQ ID NO: 73 sets forth a nucleic acid sequence encoding the CNR 1-2L.297 engineered meganuclease.
  • SEQ ID NO: 74 sets forth a nucleic acid sequence encoding the CNR 1-2L.211 engineered meganuclease.
  • SEQ ID NO: 75 sets forth a nucleic acid sequence encoding the CNR 1-2L.98 engineered meganuclease.
  • SEQ ID NO: 76 sets forth a nucleic acid sequence encoding the CNR 1-2L.108 engineered meganuclease.
  • SEQ ID NO: 77 sets forth a nucleic acid sequence encoding the CNR 1-2L15 engineered meganuclease.
  • SEQ ID NO: 78 sets forth a nucleic acid sequence encoding the CNR l-2x.88 engineered meganuclease.
  • SEQ ID NO: 79 sets forth the amino acid sequence of the CNR 21-22L.290 engineered meganuclease.
  • SEQ ID NO: 80 sets forth the amino acid sequence of the CNR 21-22L.286 engineered meganuclease.
  • SEQ ID NO: 81 sets forth the amino acid sequence of the CNR 21-22L.274 engineered meganuclease.
  • SEQ ID NO: 82 sets forth the amino acid sequence of the CNR 21-22L.212 engineered meganuclease.
  • SEQ ID NO: 83 sets forth the amino acid sequence of the CNR 21-22L.197 engineered meganuclease.
  • SEQ ID NO: 84 sets forth the amino acid sequence of the CNR 21-22L.169 engineered meganuclease.
  • SEQ ID NO: 85 sets forth the amino acid sequence of the CNR 21-22L.139 engineered meganuclease.
  • SEQ ID NO: 86 sets forth the amino acid sequence of the CNR 21-22L.38 engineered meganuclease.
  • SEQ ID NO: 87 sets forth the amino acid sequence of the CNR 21-22x.13 engineered meganuclease.
  • SEQ ID NO: 88 sets forth the amino acid sequence of the CNR 21-22L.290 engineered meganuclease CNR21 binding subunit.
  • SEQ ID NO: 89 sets forth the amino acid sequence of the CNR 21-22L.286 engineered meganuclease CNR21 binding subunit.
  • SEQ ID NO: 90 sets forth the amino acid sequence of the CNR 21-22L.274 engineered meganuclease CNR21 binding subunit.
  • SEQ ID NO: 91 sets forth the amino acid sequence of the CNR 21-22L.212 engineered meganuclease CNR21 binding subunit.
  • SEQ ID NO: 92 sets forth the amino acid sequence of the CNR 21-22L.197 engineered meganuclease CNR21 binding subunit.
  • SEQ ID NO: 93 sets forth the amino acid sequence of the CNR 21-22L.169 engineered meganuclease CNR21 binding subunit.
  • SEQ ID NO: 94 sets forth the amino acid sequence of the CNR 21-22L.139 engineered meganuclease CNR21 binding subunit.
  • SEQ ID NO: 95 sets forth the amino acid sequence of the CNR 21-22L.38 engineered meganuclease CNR21 binding subunit.
  • SEQ ID NO: 96 sets forth the amino acid sequence of the CNR 21-22x.13 engineered meganuclease CNR21 binding subunit.
  • SEQ ID NO: 97 sets forth the amino acid sequence of the CNR 21-22L.290 engineered meganuclease CNR22 binding subunit.
  • SEQ ID NO: 98 sets forth the amino acid sequence of the CNR 21-22L.286 engineered meganuclease CNR22 binding subunit.
  • SEQ ID NO: 99 sets forth the amino acid sequence of the CNR 21-22L.274 engineered meganuclease CNR22 binding subunit.
  • SEQ ID NO: 100 sets forth the amino acid sequence of the CNR 21-22L.212 engineered meganuclease CNR22 binding subunit.
  • SEQ ID NO: 101 sets forth the amino acid sequence of the CNR 21-22L.197 engineered meganuclease CNR22 binding subunit.
  • SEQ ID NO: 102 sets forth the amino acid sequence of the CNR 21-22L.169 engineered meganuclease CNR22 binding subunit.
  • SEQ ID NO: 103 sets forth the amino acid sequence of the CNR 21-22L.139 engineered meganuclease CNR22 binding subunit.
  • SEQ ID NO: 104 sets forth the amino acid sequence of the CNR 21-22L.38 engineered meganuclease CNR22 binding subunit.
  • SEQ ID NO: 105 sets forth the amino acid sequence of the CNR 21-22Lx. l3 engineered meganuclease CNR22 binding subunit.
  • SEQ ID NO: 106 sets forth the amino acid sequence of the CNR 21-22L.290 engineered meganuclease CNR21 binding subunit HVR1 region.
  • SEQ ID NO: 107 sets forth the amino acid sequence of the CNR 21-22L.286 engineered meganuclease CNR21 binding subunit HVR1 region.
  • SEQ ID NO: 108 sets forth the amino acid sequence of the CNR 21-22L.274 engineered meganuclease CNR21 binding subunit HVR1 region.
  • SEQ ID NO: 109 sets forth the amino acid sequence of the CNR 21-22L.212 engineered meganuclease CNR21 binding subunit HVR1 region.
  • SEQ ID NO: 110 sets forth the amino acid sequence of the CNR 21-22L.197 engineered meganuclease CNR21 binding subunit HVR1 region.
  • SEQ ID NO: 111 sets forth the amino acid sequence of the CNR 21-22L.169 engineered meganuclease CNR21 binding subunit HVR1 region.
  • SEQ ID NO: 112 sets forth the amino acid sequence of the CNR 21-22L.139 engineered meganuclease CNR21 binding subunit HVR1 region.
  • SEQ ID NO: 113 sets forth the amino acid sequence of the CNR 21-22L.38 engineered meganuclease CNR21 binding subunit HVR1 region.
  • SEQ ID NO: 114 sets forth the amino acid sequence of the CNR 21-22x.l3 engineered meganuclease CNR21 binding subunit HVR1 region.
  • SEQ ID NO: 115 sets forth the amino acid sequence of the CNR 21-22L.290 engineered meganuclease CNR22 binding subunit HVR2 region.
  • SEQ ID NO: 116 sets forth the amino acid sequence of the CNR 21-22L.286 engineered meganuclease CNR22 binding subunit HVR2 region.
  • SEQ ID NO: 117 sets forth the amino acid sequence of the CNR 21-22L.274 engineered meganuclease CNR22 binding subunit HVR2 region.
  • SEQ ID NO: 118 sets forth the amino acid sequence of the CNR 21-22L.212 engineered meganuclease CNR22 binding subunit HVR2 region.
  • SEQ ID NO: 119 sets forth the amino acid sequence of the CNR 21-22L.197 engineered meganuclease CNR22 binding subunit HVR2 region.
  • SEQ ID NO: 120 sets forth the amino acid sequence of the CNR 21-22L.169 engineered meganuclease CNR22 binding subunit HVR2 region.
  • SEQ ID NO: 121 sets forth the amino acid sequence of the CNR 21-22L.139 engineered meganuclease CNR22 binding subunit HVR2 region.
  • SEQ ID NO: 122 sets forth the amino acid sequence of the CNR 21-22L.38 engineered meganuclease CNR22 binding subunit HVR2 region.
  • SEQ ID NO: 123 sets forth the amino acid sequence of the CNR 21-22x.l3 engineered meganuclease CNR22 binding subunit HVR2 region.
  • SEQ ID NO: 124 sets forth a nucleic acid sequence encoding the CNR 21-22L.290 engineered meganuclease.
  • SEQ ID NO: 125 sets forth a nucleic acid sequence encoding the CNR 21-22L.286 engineered meganuclease.
  • SEQ ID NO: 126 sets forth a nucleic acid sequence encoding the CNR 21-22L.274 engineered meganuclease.
  • SEQ ID NO: 127 sets forth a nucleic acid sequence encoding the CNR 21-22L.212 engineered meganuclease.
  • SEQ ID NO: 128 sets forth a nucleic acid sequence encoding the CNR 21-22L.197 engineered meganuclease.
  • SEQ ID NO: 129 sets forth a nucleic acid sequence encoding the CNR 21-22L.169 engineered meganuclease.
  • SEQ ID NO: 130 sets forth a nucleic acid sequence encoding the CNR 21-22L.139 engineered meganuclease.
  • SEQ ID NO: 131 sets forth a nucleic acid sequence encoding the CNR 21-22L.38 engineered meganuclease.
  • SEQ ID NO: 132 sets forth a nucleic acid sequence encoding the CNR 21-22x.l3 engineered meganuclease.
  • SEQ ID NO: 133 sets forth the amino acid sequence of a SV40 nuclear localization signal.
  • SEQ ID NO: 134 sets forth the amino acid sequence of a CMYC nuclear localization signal.
  • SEQ ID NO: 135 sets forth the amino acid sequence of a P2A/furin peptide.
  • SEQ ID NO: 136 sets forth the nucleic acid sequence of a chimeric intron.
  • SEQ ID NO: 137 sets forth the nucleic acid sequence of a poly A tail.
  • SEQ ID NO: 138 sets forth the nucleic acid sequence of a hSynl promoter.
  • SEQ ID NO: 139 sets forth the nucleic acid sequence of a CAG promoter.
  • SEQ ID NO: 140 sets forth the nucleic acid sequence of a SV40 nuclear localization signal.
  • SEQ ID NO: 141 sets forth the nucleic acid sequence of a CMV enhancer.
  • SEQ ID NO: 142 sets forth the nucleic acid sequence of a CMYC nuclear localization signal.
  • SEQ ID NO: 143 sets forth the nucleic acid sequence of a SV40 nuclear localization signal that has been codon modified.
  • SEQ ID NO: 144 sets forth the nucleic acid sequence of a CMYC nuclear localization signal that has been codon modified.
  • SEQ ID NO: 145 sets forth the nucleic acid sequence of a CNR 1-2L.425 engineered meganuclease that has been codon modified.
  • SEQ ID NO: 146 sets forth the nucleic acid sequence of a CNR 1-2L.211 engineered meganuclease that has been codon modified.
  • SEQ ID NO: 147 sets forth the nucleic acid sequence of a CNR 1-2L.98 engineered meganuclease that has been codon modified.
  • SEQ ID NO: 148 sets forth the nucleic acid sequence of a CNR 1-2L15 engineered meganuclease that has been codon modified.
  • SEQ ID NO: 149 sets forth the nucleic acid sequence of a CNR 21-22L.286 engineered meganuclease that has been codon modified.
  • SEQ ID NO: 150 sets forth the nucleic acid sequence of a CNR1-2 Fl primer
  • SEQ ID NO: 151 sets forth the nucleic acid sequence of a CNR1-2 F2 primer
  • SEQ ID NO: 152 sets forth the nucleic acid sequence of a CNR1-2 F3 primer
  • SEQ ID NO: 153 sets forth the nucleic acid sequence of a CNR1-2 F4 primer
  • SEQ ID NO: 154 sets forth the nucleic acid sequence of a CNR1-2 R1 primer
  • SEQ ID NO: 155 sets forth the nucleic acid sequence of a CNR1-2 R2 primer
  • SEQ ID NO: 156 sets forth the nucleic acid sequence of a CNR1-2 R3 primer
  • SEQ ID NO: 157 sets forth the nucleic acid sequence of a CNR1-2 R4 primer
  • SEQ ID NO: 158 sets forth the nucleic acid sequence of a CNR1-2 R5 primer
  • SEQ ID NO: 159 sets forth the nucleic acid sequence of a CNR1-2 R
  • SEQ ID NO: 168 sets forth the nucleic acid sequence of a CNR17-18 Fl primer
  • SEQ ID NO: 169 sets forth the nucleic acid sequence of a P2 probe
  • SEQ ID NO: 170 sets forth the nucleic acid sequence of a F2 primer
  • SEQ ID NO: 171 sets forth the nucleic acid sequence of a R2 primer.
  • SEQ ID NO: 172 sets forth the nucleic acid sequence of a CNR21-22 R1 primer
  • SEQ ID NO: 173 sets forth the nucleic acid sequence of a CNR23-24 R1 primer
  • SEQ ID NO: 174 sets forth the nucleic acid sequence of a CNR35-36 R1 primer
  • SEQ ID NO: 175 sets forth the nucleic acid sequence of a CNR29-30 Fl primer
  • SEQ ID NO: 176 sets forth the nucleic acid sequence of a CNR25-26 Fl primer
  • SEQ ID NO: 177 sets forth the nucleic acid sequence of a CNR31-32 Fl primer
  • SEQ ID NO: 178 sets forth the nucleic acid sequence of a CNR21-22 Fl primer
  • SEQ ID NO: 179 sets forth the nucleic acid sequence of a CNR21-22 R2 primer.
  • SEQ ID NO: 180 sets forth the nucleic acid sequence of a CNR21-22 Pl primer.
  • SEQ ID NO: 181 sets forth the nucleic acid sequence of a CNR21-22 R3 primer.
  • SEQ ID NO: 182 sets forth the nucleic acid sequence of an APC Fl primer.
  • SEQ ID NO: 183 sets forth the nucleic acid sequence of an APC F2 primer.
  • SEQ ID NO: 184 sets forth the nucleic acid sequence of an APC Pl primer.
  • SEQ ID NO: 185 sets forth the nucleic acid sequence of a region of the C90rf72 gene that has been re-ligated following excision with a CNR1-2 and CNR21-22 engineered meganuclease described herein.
  • SEQ ID NO: 186 sets forth the nucleic acid sequence of a region of the C90rf72 gene that has been re-ligated following excision with a CNR1-2 and CNR21-22 engineered meganuclease described herein.
  • SEQ ID NO: 187 sets forth the nucleic acid sequence of a region of the C90rf72 gene that has been re-ligated following excision with a CNR1-2 and CNR21-22 engineered meganuclease described herein.
  • SEQ ID NO: 188 sets forth the nucleic acid sequence of a region of the C90rf72 gene that has been re-ligated following excision with a CNR1-2 and CNR21-22 engineered meganuclease described herein.
  • SEQ ID NO: 189 sets forth the nucleic acid sequence of a region of the C90rf72 gene that has been re-ligated following excision with a CNR1-2 and CNR21-22 engineered meganuclease described herein.
  • SEQ ID NO: 190 sets forth the nucleic acid sequence of a minimal promoter region of the C90rf72 gene that is immediately 5' upstream from exon la of the C90rf72 gene.
  • SEQ ID NO: 191 sets forth the nucleic acid sequence of exon la of the C90rf72 gene corresponding to mRNA variant 3.
  • SEQ ID NO: 192 sets forth the nucleic acid sequence of an exon la of the C90rf72 gene corresponding to mRNA variant 1.
  • SEQ ID NO: 193 sets forth the nucleic acid sequence of an exon lb of the C90rf72 gene corresponding to mRNA variant 2.
  • SEQ ID NO: 194 sets forth the nucleic acid sequence of a WPRE sequence.
  • SEQ ID NO: 195 sets forth an alternative amino acid sequence of the CNR 1-2L.15 engineered meganuclease when the methionine start codon of the engineered meganuclease is replaced with an alanine residue.
  • the methionine start codon is a part of an N-terminal domain such as an NLS.
  • SEQ ID NO: 196 sets forth the amino acid sequence of a peptide linker that is useful for linking a first subunit and a second subunit of the engineered meganucleases described herein.
  • a can mean one or more than one.
  • a cell can mean a single cell or a multiplicity of cells.
  • nuclease and “endonuclease” are used interchangeably to refer to naturally-occurring or engineered enzymes, which cleave a phosphodiester bond within a polynucleotide chain.
  • Engineered nucleases can include, without limitation, engineered meganucleases, zinc finger nucleases, TALENs, compact TALENs, CRISPR system nucleases, and megaTALs.
  • any engineered nuclease is envisioned that is capable of generating overhangs at its cleavage site.
  • cleavage refers to the hydrolysis of phosphodiester bonds within the backbone of a recognition sequence within a target sequence that results in a double-stranded break within the target sequence, referred to herein as a “cleavage site”.
  • the term “meganuclease” refers to an endonuclease that binds doublestranded DNA at a recognition sequence that is greater than 12 base pairs. In some embodiments, the recognition sequence for a meganuclease of the present disclosure is 22 base pairs.
  • a meganuclease can be an endonuclease that is derived from I-Crel (SEQ ID NO: 1), and can refer to an engineered variant of I-Crel that has been modified relative to natural I-Crel with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties.
  • a meganuclease as used herein binds to double-stranded DNA as a heterodimer.
  • a meganuclease may also be a “single-chain meganuclease” in which a pair of DNA-binding domains is joined into a single polypeptide using a peptide linker.
  • homing endonuclease is synonymous with the term “meganuclease.”
  • Meganucleases of the present disclosure are substantially non-toxic when expressed in the targeted cells as described herein such that cells can be transfected and maintained at 37°C without observing deleterious effects on cell viability or significant reductions in meganuclease cleavage activity when measured using the methods described herein.
  • single-chain meganuclease refers to a polypeptide comprising a pair of nuclease subunits joined by a linker.
  • a single-chain meganuclease has the organization: N- terminal subunit - Linker - C-terminal subunit.
  • the two meganuclease subunits will generally be non-identical in amino acid sequence and will bind non-identical DNA sequences.
  • singlechain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences.
  • a single-chain meganuclease may be referred to as a “single-chain heterodimer” or “single-chain heterodimeric meganuclease” although it is not, in fact, dimeric.
  • the term “meganuclease” can refer to a dimeric or single-chain meganuclease.
  • linker refers to an exogenous peptide sequence used to join two nuclease subunits into a single polypeptide.
  • a linker may have a sequence that is found in natural proteins or may be an artificial sequence that is not found in any natural protein.
  • a linker may be flexible and lacking in secondary structure or may have a propensity to form a specific three- dimensional structure under physiological conditions.
  • a linker can include, without limitation, those encompassed by U.S. Patent Nos. 8,445,251, 9,340,777, 9,434,931, and 10,041,053, each of which is incorporated by reference in its entirety.
  • a linker may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 196, which sets forth residues 154-195 of any one of SEQ ID NOs: 7-18 or 79-87.
  • the terms “recombinant” or “engineered,” with respect to a protein means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids that encode the protein and cells or organisms that express the protein.
  • the term “recombinant” or “engineered” means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation, and other gene transfer technologies; homologous recombination; site- directed mutagenesis; and gene fusion.
  • a protein having an amino acid sequence identical to a naturally-occurring protein but produced by cloning and expression in a heterologous host is not considered recombinant or engineered.
  • wild-type refers to the most common naturally occurring allele (z.e., polynucleotide sequence) in the allele population of the same type of gene, wherein a polypeptide encoded by the wild-type allele has its original functions.
  • wild-type also refers to a polypeptide encoded by a wild-type allele. Wild-type alleles (z.e., polynucleotides) and polypeptides are distinguishable from mutant or variant alleles and polypeptides, which comprise one or more mutations and/or substitutions relative to the wild-type sequence(s).
  • Wild-type nucleases are distinguishable from recombinant or non-naturally-occurring nucleases.
  • wild-type can also refer to a cell, an organism, and/or a subject which possesses a wild-type allele of a particular gene, or a cell, an organism, and/or a subject used for comparative purposes.
  • the term “genetically modified” refers to a cell or organism in which, or in an ancestor of which, a genomic DNA sequence has been deliberately modified by recombinant technology. As used herein, the term “genetically modified” encompasses the term “transgenic.”
  • modification means any insertion, deletion, or substitution of an amino acid residue in the recombinant sequence relative to a reference sequence (e.g., a wild-type or a native sequence).
  • a recognition sequence or “recognition site” refers to a DNA sequence that is bound and cleaved by a nuclease.
  • a recognition sequence comprises a pair of inverted, 9 basepair “half-sites,” which are separated by four basepairs.
  • the N-terminal domain of the protein contacts a first half-site and the C-terminal domain of the protein contacts a second half-site. Cleavage by a meganuclease produces four basepair 3' overhangs.
  • “Overhangs,” or “sticky ends” are short, single-stranded DNA segments that can be produced by endonuclease cleavage of a double-stranded DNA sequence.
  • the overhang comprises bases 10-13 of the 22 basepair recognition sequence.
  • target site or “target sequence” refers to a region of the chromosomal DNA of a cell comprising a recognition sequence for a nuclease.
  • DNA-binding affinity or “binding affinity” means the tendency of a nuclease to non-covalently associate with a reference DNA molecule (e.g., a recognition sequence or an arbitrary sequence). Binding affinity is measured by a dissociation constant, Kd. As used herein, a nuclease has “altered” binding affinity if the Kd of the nuclease for a reference recognition sequence is increased or decreased by a statistically significant percent change relative to a reference nuclease.
  • the term “specificity” means the ability of a nuclease to bind and cleave double-stranded DNA molecules only at a particular sequence of base pairs referred to as the recognition sequence, or only at a particular set of recognition sequences.
  • the set of recognition sequences will share certain conserved positions or sequence motifs but may be degenerate at one or more positions.
  • a highly-specific nuclease is capable of cleaving only one or a very few recognition sequences. Specificity can be determined by any method known in the art.
  • C90rf72 gene refers to the gene associated with National Center for Biotechnology Information (NCBI) gene ID 203228, as well as naturally occurring variants thereof.
  • C90rf72 polypeptide refers to a polypeptide encoded by the C90rf72 gene.
  • the C90rf72 gene is edited with a pair of engineered meganucleases, resulting in the excision of the hexanucleotide repeat region and exon lb and subsequent perfect ligation of the C90rf72 gene.
  • re-ligation refers to the ligation (z.e., annealing) of a portion or all of the bases of a 3' overhang of a first cleavage site with bases of a 3' overhang of a second cleavage site in a C90rf72 gene following cleavage by a pair of engineered meganucleases described herein.
  • the four base pair center sequence of the pair of recognition sequences selected are 100%, 75%, 50%, or 25% complementary to each other.
  • a first recognition sequence comprises a four base pair center sequence that is 75% complementary to a four base pair center sequence of a second recognition sequence.
  • a first recognition sequence comprises the four base pair center sequence ATAA and a second recognition sequence comprises the four base pair center sequence ATAT.
  • the recognition sequences targeted by the disclosed engineered meganucleases have identical (i.e., 100% complementarity), such that the first and second cleavage sites will have complementary four basepair 3' overhangs. Accordingly, each basepair of the first 3' overhang pairs with its complement basepair on the second 3' overhang, and ligation occurs through a DNA ligase enzyme.
  • C90rf72 associated disease means any disease associated with any C90rf72 nucleic acid or expression product thereof. Such diseases may include a neurodegenerative disease. Such neurodegen erative diseases may include ALS and FTD.
  • C90rf72 hexanucleotide repeat expansion associated disease means any disease associated with a C90rf72 nucleic acid containing a hexanucleotide repeat expansion.
  • the hexanucleotide repeat expansion may comprise GGGGCC, GGGGGG, GGGGGC, or GGGGCG repeated at least 30 times.
  • diseases may include a neurodegenerative disease.
  • Such neurodegenerative diseases may include ALS and FTD.
  • C90rf72 nucleic acid means any nucleic acid encoding C90rf72.
  • a C90rf72 nucleic acid includes a DNA sequence encoding C90rf72, an RNA sequence transcribed from DNA encoding C90rf72 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding C90rf72.
  • C90rf72 mRNA means an mRNA encoding a C90rf72 protein.
  • “Hexanucleotide repeat expansion” (also GGGGCCfn] RNA repeat) means a series of six bases (for example, GGGGCC, GGGGGG, GGGGCG, or GGGGGC) repeated at least twice.
  • the hexanucleotide repeat expansion may be located in intron 1 of a C90rf72 nucleic acid.
  • a pathogenic hexanucleotide repeat expansion includes at least 30 repeats of GGGGCC, GGGGGG, GGGGCG, or GGGGGC in a C90rf72 nucleic acid and is associated with disease. In certain embodiments, the repeats are consecutive.
  • the repeats are interrupted by 1 or more nucleobases.
  • a wildtype hexanucleotide repeat expansion includes 23 or fewer repeats of GGGGCC, GGGGGG, GGGGCG, or GGGGGC in a C90rf72 nucleic acid.
  • the repeats are consecutive.
  • the repeats are interrupted by 1 or more nucleobases.
  • exon la when in reference to the C90rf72 gene refers to a noncoding exon that is transcribed as a part of mRNA variants 1 and 3 of the C90rf72 gene.
  • exon la comprises a sequence according to mRNA variant 3 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 191.
  • exon la comprises a sequence according to SEQ ID NO: 191.
  • exon la comprises a sequence according to mRNA variant 1 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity SEQ ID NO: 192. In some embodiments, exon la comprises a sequence according to SEQ ID NO: 192.
  • exon lb when in reference to the C90rf72 gene refers to a noncoding exon that is transcribed as a part of mRNA variants 2 of the C90rf72 gene.
  • exon lb comprises a sequence according to mRNA variant 2 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 193.
  • exon lb comprises a sequence according to SEQ ID NO: 191.
  • homologous recombination refers to the natural, cellular process in which a double-stranded DNA-break is repaired using a homologous DNA sequence as the repair template (see, e.g., Cahill et al. (2006) Front. Biosci. 11 : 1958-76).
  • the homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid that was delivered to the cell.
  • non-homologous end-joining refers to the natural, cellular process in which a double-stranded DNA-break is repaired by the direct joining of two non- homologous DNA segments (see, e.g., Cahill et al. (2006)). DNA repair by non-homologous endjoining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair. In some instances, cleavage at a target recognition sequence results in NHEJ at a target recognition site.
  • NHEJ non-homologous end joining
  • homology arms or “sequences homologous to sequences flanking a nuclease cleavage site” refer to sequences flanking the 5' and 3' ends of a nucleic acid molecule, which promote insertion of the nucleic acid molecule into a cleavage site generated by a nuclease.
  • homology arms can have a length of at least 50 base pairs, preferably at least 100 base pairs, and up to 2000 base pairs or more, and can have at least 90%, preferably at least 95%, or more, sequence homology to their corresponding sequences in the genome. In some embodiments, the homology arms are about 500 base pairs.
  • the term with respect to both amino acid sequences and nucleic acid sequences refers to a measure of the degree of similarity of two sequences based upon an alignment of the sequences that maximizes similarity between aligned amino acid residues or nucleotides, and which is a function of the number of identical or similar residues or nucleotides, the number of total residues or nucleotides, and the presence and length of gaps in the sequence alignment.
  • a variety of algorithms and computer programs are available for determining sequence similarity using standard parameters.
  • sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/), and are described in, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-10; Gish & States (1993) Nature Genet. 3:266-72; Madden et al. (1996) Meth. Enzymol. 266: 131-41; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402; and Zhang et al. (2000) J. Comput. Biol. 7:203-14.
  • the term “corresponding to” with respect to modifications of two proteins or amino acid sequences is used to indicate that a specified modification in the first protein is a substitution of the same amino acid residue as in the modification in the second protein, and that the amino acid position of the modification in the first protein corresponds to or aligns with the amino acid position of the modification in the second protein when the two proteins are subjected to standard sequence alignments (e.g., using the BLASTp program).
  • the modification of residue “X” to amino acid “A” in the first protein will correspond to the modification of residue “Y” to amino acid “A” in the second protein if residues X and Y correspond to each other in a sequence alignment and despite the fact that X and Y may be different numbers.
  • the term “recognition half-site,” “recognition sequence half-site,” or simply “half-site” means a nucleic acid sequence in a double-stranded DNA molecule that is recognized and bound by a monomer of a homodimeric or heterodimeric meganuclease or by one subunit of a single-chain meganuclease or by one subunit of a single-chain meganuclease.
  • the term post-transcriptional regulatory element refers to a nucleotide sequence, which functions to increase the stability of mRNA and cytoplasmic transport of the mRNA. Stability is of an intron-less gene is typically increased by promoting mRNA exportation from the nucleus to the cytoplasm, enhancing 3' end processing and stability.
  • Suitable PTRE(s) include PTREs derived from viruses including but not limited to the Hepatitis B virus (HPRE) and Woodchuck Hepatitis virus (WPRE). In some embodiments described herein, the PTRE is a WPRE.
  • the WPRE comprises a nucleotide sequence according to a sequence set forth in SEQ ID NO: 194
  • the term “hypervariable region” refers to a localized sequence within a meganuclease monomer or subunit that comprises amino acids with relatively high variability.
  • a hypervariable region can comprise about 50-60 contiguous residues, about 53-57 contiguous residues, or preferably about 56 residues.
  • the residues of a hypervariable region may correspond to positions 24-79 or positions 215-270 of any one of SEQ ID NOs: 7-18 or 79-87.
  • a hypervariable region can comprise one or more residues that contact DNA bases in a recognition sequence and can be modified to alter base preference of the monomer or subunit.
  • a hypervariable region can also comprise one or more residues that bind to the DNA backbone when the meganuclease associates with a double-stranded DNA recognition sequence. Such residues can be modified to alter the binding affinity of the meganuclease for the DNA backbone and the target recognition sequence.
  • a hypervariable region may comprise between 1-20 residues that exhibit variability and can be modified to influence base preference and/or DNA-binding affinity.
  • a hypervariable region comprises between about 15-20 residues that exhibit variability and can be modified to influence base preference and/or DNA-binding affinity.
  • variable residues within a hypervariable region correspond to one or more of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7-18 or 79-87.
  • variable residues within a hypervariable region can further correspond to residues 48, 50, and 71-73 of any one of SEQ ID NOs: 7-18 or 79-87.
  • variable residues within a hypervariable region correspond to one or more of positions 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 239, 241, 259, 261, 262, 263, 264, 266, and 268 of any one of SEQ ID NOs: 7- 18 or 79-87.
  • variable residues within a hypervariable region can further correspond to residues 239, 241, and 263-265 of any one of SEQ ID NOs: 7-18 or 79-87.
  • C90rf72 gene or mRNA levels refers to any increase in the levels of C90rf72 gene or mRNA expression relative to a reference level including an increase of C90rf72 gene or mRNA expression of at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more, when compared to a reference level or control.
  • an increase in C90rf72 gene or mRNA levels refers to an increase in a shortened C90rf72 gene or mRNA transcript, for example, missing a portion of the gene encoded by at least one intron (e.g., a portion comprising the hexanucleotide repeat region) or missing a portion of mRNA corresponding to the hexanucleotide repeat region and exon lb compared to the wild-type C90rf72 polypeptide or gene.
  • the term “reference level” in the context of C90rf72 gene or mRNA levels refers to a level of C90rf72 gene or mRNA as measured in, for example, a control cell, control cell population or a control subject, at a previous time point in the control cell, the control cell population or the subject undergoing treatment (e.g., a pre-dose baseline level obtained from the control cell, control cell population or subject), or a pre-defined threshold level of C90rf72 gene or mRNA (e.g., a threshold level identified through previous experimentation).
  • control refers to a cell that provides a reference point for measuring changes in genotype or phenotype of a genetically modified cell.
  • a control cell may comprise, for example: (a) a wild-type cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the genetically modified cell; (b) a cell of the same genotype as the genetically modified cell but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest); or, (c) a cell genetically identical to the genetically modified cell but which is not exposed to conditions or stimuli or further genetic modifications that would induce expression of altered genotype or phenotype.
  • a control subject may comprise, for example: a wild-type subject, i.e., of the same genotype as the starting subject for the genetic alteration which resulted in the genetically modified subject (e.g., a subject having the same mutation in a C90rf72 gene), which is not exposed to conditions or stimuli or further genetic modifications that would induce expression of altered genotype or phenotype in the subject.
  • a wild-type subject i.e., of the same genotype as the starting subject for the genetic alteration which resulted in the genetically modified subject
  • the genetically modified subject e.g., a subject having the same mutation in a C90rf72 gene
  • recombinant DNA construct As used herein, the term “recombinant DNA construct,” “recombinant construct,” “expression cassette,” “expression construct,” “chimeric construct,” “construct,” and “recombinant DNA fragment” are used interchangeably herein and are single or double-stranded polynucleotides.
  • a recombinant construct comprises an artificial combination of nucleic acid fragments, including, without limitation, regulatory and coding sequences that are not found together in nature.
  • a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source and arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector.
  • vector or “recombinant DNA vector” may be a construct that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art.
  • Vectors can include, without limitation, plasmid vectors and recombinant AAV vectors, or any other vector known in the art suitable for delivering a gene to a target cell. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleotides or nucleic acid sequences of the disclosure.
  • a “vector” also refers to a viral vector.
  • Viral vectors can include, without limitation, retroviral vectors, lentiviral vectors, adenoviral vectors, and AAV.
  • operably linked is intended to mean a functional linkage between two or more elements.
  • an operable linkage between a nucleic acid sequence encoding a nuclease as disclosed herein and a regulatory sequence is a functional link that allows for expression of the nucleic acid sequence encoding the nuclease.
  • Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame.
  • treatment refers to the administration of an engineered meganuclease described herein, or a polynucleotide encoding an engineered meganuclease described herein, or a pair of such engineered meganucleases or polynucleotides, to a subject having ALS or FTD, or having increased susceptibility to ALS or FTD, for the purpose of increasing levels of a modified C90rf72 gene or mRNA in the subject.
  • expression of a shortened version e.g., missing amino acids encoded by multiple exons
  • expression of a version of the C90rf72 gene or mRNA, lacking the non-coding sequence comprising the hexanucleotide repeat region is increased.
  • Such treatment prevents development of a neurological condition or neurodegenerative disorder, such as ALS and/or FTD.
  • gc/kg or “gene copies/kilogram” refers to the number of copies of a nucleic acid sequence encoding an engineered meganuclease described herein per weight in kilograms of a subject that is administered a polynucleotide comprising the nucleic acid sequence.
  • the term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
  • the therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated.
  • an effective amount of an engineered meganuclease or pair of engineered meganucleases described herein, or polynucleotide or pair of polynucleotides encoding the same, or pharmaceutical compositions disclosed herein increases the level of expression of a modified, shortened C90rf72 gene or mRNA (e.g., a shortened C90rf72 gene or RNA lacking the non-coding sequence comprising the hexanucleotide repeat region) and ameliorates at least one symptom associated with development of FTD or ALS.
  • lipid nanoparticle refers to a lipid composition having a typically spherical structure with an average diameter between 10 and 1000 nm.
  • lipid nanoparticles can comprise at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid.
  • Lipid nanoparticles known in the art that are suitable for encapsulating nucleic acids, such as mRNA, are contemplated for use in the embodiments described herein.
  • a polynucleotide or nucleic acid, such as an mRNA may be encapsulated in the lipid portion of the lipid nanoparticle or aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle.
  • Lipid nanoparticles may further be conjugated with a targeting moiety, such as an antibody or a ligand, to direct said lipid nanoparticle to a target cell or target tissue.
  • a targeting moiety such as an antibody or a ligand
  • variable As used herein, the recitation of a numerical range for a variable is intended to convey that the present disclosure may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable that is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range.
  • the present disclosure is based, in part, on the hypothesis that certain mutations located in the non-coding portion of the C90rf72 gene associated with neurodegenerative disorders such as frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) can be removed by utilizing pairs of endonucleases to strategically delete exons and/or introns, or portions of both, within the C90rf72 gene in order to remove and/or prevent transcription of hexanucleotide repeat mutation(s) associated with neurological disorders including FTD and ALS.
  • FTD frontotemporal dementia
  • ALS amyotrophic lateral sclerosis
  • the hexanucleotide repeat is located between non-coding C90rf72 exons la and lb, and expansion of the hexanucleotide repeat has been regarded as the most frequent cause of sporadic ALS and sporadic FTD identified (Majounie, Elisa et al. “Frequency of the C90rf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study.” The Lancet. Neurology vol. 11,4 (2012): 323-30. doi: 10.1016/S 1474-4422(12)70043-1; DeJesus-Hemandez, Mariely et al.
  • C90rf72 repeat expansions have also been identified as a rare cause of other neurodegenerative diseases, including Parkinson’s disease, progressive supranuclear palsy, ataxia, corticobasal syndrome, Huntington’s disease-like syndrome, Creutzfeldt- Jakob disease and Alzheimer’s disease.
  • the intron, or portions thereof, containing the hexanucleotide repeat of the C90rf72 gene will be removed in order to prevent disease- associated outcomes associated with expansion of this non-coding region within C90rf72 RNA.
  • the intron comprising the hexanucleotide repeat region as well as exon lb are excised from the C90rf72 gene by utilizing a recognition sequence that is 5’ upstream from the hexanucleotide repeat region and a recognition sequence that is 3’ downstream of exon lb.
  • This excision strategy referred to herein as “downstream” excision allows for the C90rf72 coding sequence to be under control of genetic regulatory elements that are present 5’ of the hexanucleotide repeat region in wild-type C90rf72 gene (e.g., exon la). Following excision of the hexanucleotide repeat region, C90rf72 transcription will be controlled by exon la, and the RNA produced will lack the hexanucleotide repeat region as well as non-coding exon lb ( Figure 1).
  • recognition sequences located 5’ upstream from a minimal promoter and exon la of the C90rf72 gene are utilized in combination with recognition sequences 3’ downstream of exon la.
  • the resulting RNA lacks the minimal promoter and exon la, which prevents transcription of the hexanucleotide repeat region. Transcription then proceeds normally from exon lb. This excision strategy as described herein is referred to as “upstream” excision.
  • the hexanucleotide repeat region is associated with disease mechanisms, its removal from C90rf72 RNA may function to prevent development of neurogenerative disease, due to any toxicity, or loss of function of the C90rf72 protein, associated with hexanucleotide repeats forming RNA foci or di-peptide repeat proteins (DPRs) being present in C90rf72 RNA.
  • DPRs di-peptide repeat proteins
  • introns and/or exons are achieved by the expression of a pair of engineered meganucleases in neural cells or neural precursor cells that generate a pair of cleavage sites in the non-coding sequence of the COrf72 gene, 5’ upstream of the hexanucleotide repeat (e.g., CNR 1-2 recognition sequence; SEQ ID NO: 3) and downstream of exon lb (e.g., CNR 21-22 recognition sequence; SEQ ID NO: 5), allowing for excision of the intervening genomic region (i.e., hexanucleotide repeat and exon lb).
  • hexanucleotide repeat e.g., CNR 1-2 recognition sequence; SEQ ID NO: 3
  • exon lb e.g., CNR 21-22 recognition sequence; SEQ ID NO: 5
  • a genetically modified cell e.g., a neural cell in a treated subject
  • a genetically modified cell will comprise a modified COrf72 gene lacking the hexanucleotide repeat region that is susceptible to extension mutation that is correlated with neurological disease, such as FTD and ALS.
  • This removal of the hexanucleotide repeat mutation may be sufficient to rescue disease permanently by removing the sequence susceptible to mutation.
  • the minimal promoter sequence comprises SEQ ID NO: 190.
  • exon la comprises SEQ ID NO: 191.
  • the minimal promoter sequence having a sequence comprising SEQ ID NO: 190 and exon la having a sequence comprising SEQ ID NO: 191 of the C90rf72 gene has been excised.
  • the minimal promoter region and exon la is excised from the C90rf72 gene utilizing a first engineered nuclease that targets a recognition sequence 5’ upstream from the minimal promoter region and second engineered nuclease that targets a recognition sequence 3’ downstream from exon lb of the C90rf72 gene.
  • a genetically modified cell having a modified C90rf72 gene wherein a G4C2 hexanucleotide repeat region of the C90rf72 gene upstream of exon lb of the C90rf72 gene has been excised.
  • a G4C2 hexanucleotide repeat region and exon lb having a sequence comprising SEQ ID NO: 192 of the C90rf72 gene has been excised.
  • the hexanucleotide repeat region and exon lb is excised from the C90rf72 gene utilizing a first engineered nuclease that targets a recognition sequence 5’ upstream from the G4C2 hexanucleotide repeat region and second engineered nuclease that targets a recognition sequence 3’ downstream from exon lb of the C90rf72 gene.
  • the first engineered nuclease targets a CNR 1-2 recognition sequence comprising SEQ ID NO: 3
  • the second engineered nuclease targets a CNR 21-22 recognition sequence comprising SEQ ID NO: 5.
  • a single treatment will permanently delete the hexanucleotide repeat mutation from a percentage of cells in a subject.
  • these cells will be neurons (z.e., neuronal cells) or neuroglia, or neural precursor cells or central nervous system (CNS) cells that are capable of replicating and giving rise to the C90rf72 polypeptide.
  • neurons z.e., neuronal cells
  • neuroglia or neuroglia
  • neural precursor cells or central nervous system (CNS) cells that are capable of replicating and giving rise to the C90rf72 polypeptide.
  • CNS central nervous system
  • NHEJ can produce mutagenesis at the cleavage site, resulting in inactivation of the allele.
  • NHEJ-associated mutagenesis may inactivate an allele via generation of early stop codons, frameshift mutations producing aberrant non-functional proteins, or could trigger mechanisms such as nonsense-mediated mRNA decay.
  • nucleases to induce mutagenesis via NHEJ can be used to target a specific mutation or a sequence present in a wild-type allele. Further, the use of nucleases to induce a double-strand break in a target locus is known to stimulate homologous recombination, particularly of transgenic DNA sequences flanked by sequences that are homologous to the genomic target. In this manner, exogenous polynucleotides can be inserted into a target locus. Such exogenous polynucleotides can encode any sequence or polypeptide of interest.
  • engineered meganucleases of the disclosure have been designed to bind and cleave a CNR 1-2 recognition sequence (SEQ ID NO: 3), or a CNR 21-22 recognition sequence (SEQ ID NO: 5).
  • engineered meganucleases have been designed to bind and cleave a recognition sequence upstream of a minimal promoter/exon la.
  • Exemplary meganucleases that bind and cleave the CNR 1-2 recognition sequence are provided in SEQ ID NOs: 7-18.
  • Exemplary meganucleases that bind and cleave the CNR 21-22 recognition sequence are provided in SEQ ID NOs: 79-87.
  • the first engineered meganuclease is an engineered meganuclease described herein that binds and cleaves a recognition sequence comprising SEQ ID NO: 3
  • the second engineered meganuclease is an engineered meganuclease described herein that binds and cleaves a recognition sequence comprising SEQ ID NO: 5.
  • the first engineered meganuclease and the second engineered meganuclease are selected from the combinations of meganucleases (and variants thereof described herein) provided in Table 1.
  • a pair of engineered meganucleases described herein are utilized together in the same cell to modify the C90rf72 gene.
  • Such pairs of engineered meganucleases were designed to generate a first cleavage site in an intron upstream of exon lb (i.e., at a site between exon la and exon lb) and a second cleavage site downstream of exon lb (i.e., at a site between exon lb and C90rf72 coding sequence), allowing for removal of the intervening genomic sequence (i.e., a portion of the nucleotide sequence between exon la and the C90rf2 coding sequence).
  • the disclosed engineered meganucleases can be used to generate genetically modified eukaryotic cells having a modified C90rf72 locus according to formula I below. Accordingly, in some embodiments described herein, is a genetically modified eukaryotic cell comprising a contiguous nucleic acid sequence within its genome according to Formula I:
  • Xi-Xs is absent. In some embodiments Xi is selected from A, T, G, or C and X2-X8 is absent. In some embodiments X1-X2 is selected from A, T, G, or C and X3-X8 is absent. In some embodiments X1-X3 is selected from A, T, G, or C and X4-X8 is absent. In some embodiments X1-X4 is selected from A, T, G, or C and X5-X8 is absent. In some embodiments Xi- X5 is selected from A, T, G, or C and Xe-Xs is absent. In some embodiments X1-X7 is selected from A, T, G, or C and Xs is absent.
  • Xi is a T
  • X2 is a G
  • X3 is an A
  • X4 is a T
  • X5 is an A
  • Xe is a T
  • X7 is a G
  • Xs is absent.
  • Xi is a T
  • X2 is a G
  • X3 is an A
  • X4 is a T
  • X5 is an A
  • Xe is an A
  • X7 is a T
  • Xs is a G.
  • Xi is a T
  • X2 is a G
  • X3-X8 is absent.
  • Xi is a T
  • X2 is a G
  • X3 is an A
  • X4 is a T
  • X5 is a G
  • Xe-Xs is absent.
  • Xi is a T
  • X2 is a G
  • X3 is an A
  • X4 is a T
  • Xs-Xs is absent.
  • the genetically modified eukaryotic cell comprises a contiguous nucleic acid sequence comprising SEQ ID NO: 185. In some embodiments, the genetically modified eukaryotic cell comprises a contiguous nucleic acid sequence comprising SEQ ID NO: 186. In some embodiments, the genetically modified eukaryotic cell comprises a contiguous nucleic acid sequence comprising SEQ ID NO: 187. In some embodiments, the genetically modified eukaryotic cell comprises a contiguous nucleic acid sequence comprising SEQ ID NO: 188. In some embodiments, the genetically modified eukaryotic cell comprises a contiguous nucleic acid sequence comprising SEQ ID NO: 189. Exemplary Engineered Meganucleases
  • Engineered meganucleases of the disclosure comprise a first subunit, comprising a HVR1 region, and a second subunit, comprising a HVR2 region. Further, the first subunit binds to a first recognition half-site in the recognition sequence (e.g., the CNR1 half-site), and the second subunit binds to a second recognition half-site in the recognition sequence (e.g., the CNR2 half-site).
  • a first recognition half-site in the recognition sequence e.g., the CNR1 half-site
  • the second subunit binds to a second recognition half-site in the recognition sequence (e.g., the CNR2 half-site).
  • the meganucleases used to practice the disclosure are singlechain meganucleases.
  • a single-chain meganuclease comprises an N-terminal subunit and a C- terminal subunit (z.e., the first and second subunits discussed above) joined by a linker peptide.
  • Each of the two subunits recognizes and binds to a half-site of the recognition sequence and the site of DNA cleavage is at the middle of the recognition sequence near the interface of the two subunits.
  • DNA strand breaks are offset by four base pairs such that DNA cleavage by a meganuclease generates a pair of four basepair 3' single-strand overhangs.
  • the first and second subunits can be oriented such that the first subunit, which comprises the HVR1 region and binds the first half-site, is positioned as the N-terminal subunit, and the second subunit, which comprises the HVR2 region and binds the second half-site, is positioned as the C-terminal subunit.
  • the first and second subunits can be oriented such that the first subunit, which comprises the HVR1 region and binds the first half-site, is positioned as the C- terminal subunit, and the second subunit, which comprises the HVR2 region and binds the second half-site, is positioned as the N-terminal subunit.
  • Table 4 Exemplary engineered meganucleases that bind and cleave the CNR 1-2 recognition sequence (SEQ ID NO: 3).
  • CNR1 Subunit % and “CNR2 Subunit %” represent the amino acid sequence identity between the CNR1 -binding and CNR2 -binding subunit regions of each meganuclease and the CNR1- binding and CNR2-binding subunit regions, respectively, of the CNR 1-2L.532 meganuclease.
  • CNR21 Subunit % and “CNR22 Subunit %” represent the amino acid sequence identity between the CNR21 -binding and CNR22-binding subunit regions of each meganuclease and the CNR21- binding and CNR22 -binding subunit regions, respectively, of the CNR 21-22L.286 meganuclease.
  • the engineered meganuclease binds and cleaves a recognition sequence comprising SEQ ID NO: 3 (z.e., the CNR 1-2 recognition sequence) within a C90rf72gene, wherein the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a HVR1 region, and wherein the second subunit binds to a second recognition halfsite of the recognition sequence and comprises a HVR2 region.
  • Exemplary CNR 1-2 meganucleases are described below.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 7.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 7.
  • the HVR1 region comprises a residue corresponding to residue 29 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises a residue corresponding to residue 41 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 7.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 7.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 7. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 7. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 7. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 7.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 7. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 7. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 7.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 7.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 7.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 7.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 7.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 7.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 7.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 7. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 7. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 7.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 7. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 7. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 7. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 7.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 7.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 67. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 67.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 8.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 8.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises a residue corresponding to residue 29 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises a residue corresponding to residue 41 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 8.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 8.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 8. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 8. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 8. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 8.
  • the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 8. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 8. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 8. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 8.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 8.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 8.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 8.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 8.
  • the HVR2 region comprises a residue corresponding to residue 258 of SEQ ID NO: 8.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 8.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 8.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 8.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 8. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 8. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 8.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 8. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 8. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 8. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 8.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 8.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 68. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 68.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 9.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 9.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 9.
  • the HVR1 region comprises a residue corresponding to residue 29 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises a residue corresponding to residue 41 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 9.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 9.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 9. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 9. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 9. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 9.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 9. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 9. In some embodiments, the first subunit comprises a residue corresponding to residue 142 of SEQ ID NO: 9. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 9. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 9.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 9.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 9.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 9.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 9.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 9.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 9.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 9.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 9. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 9. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 9.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 9.
  • the second subunit comprises residues 198- 344 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the second subunit comprises residues 198-344 of SEQ ID NO: 9.
  • the second subunit comprises residues 196-354 of SEQ ID NO: 9.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 9.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 9.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 69. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 69.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 10.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 10.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 10.
  • the HVR1 region comprises a residue corresponding to residue 29 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises a residue corresponding to residue 41 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 10.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 10.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 10. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 10. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 10. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 10.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 10. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 10. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 10. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 10.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 10.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 10.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 10.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 10.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 10.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 10.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 10.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 10. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 10.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 10. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 10. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 10.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 10.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NOs: 70. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 70.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 11.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 11.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 11.
  • the HVR1 region comprises a residue corresponding to residue 29 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 41 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 11.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 11 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 11.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 11. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 11. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 11. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 11.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 11. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 11. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 11 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 11. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 11.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 11.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 11.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 11.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 11.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 11.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 11.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 11 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 11. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 11.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 11.
  • the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 11.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 11.
  • the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 11.
  • the second subunit comprises residues 198-344 of SEQ ID NO: 11 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the second subunit comprises residues 198-344 of SEQ ID NO: 11.
  • the second subunit comprises residues 196-354 of SEQ ID NO: 11.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 11. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 11.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 71. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 71.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 12.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 12.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 12.
  • the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 12.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 12 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 12.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 12. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 12. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 12. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 12.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 12. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 12. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 12 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 12. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 12.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 12.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 12.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 12.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 12.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 12.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 12.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 12 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 12.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 12. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 12. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 12.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 12. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 12. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 12. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 12 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 12. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 12.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 12. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 12.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 72. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 72. CNR 1-2L.297 (SEQ ID NO: 13)
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 13.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 13.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 13.
  • the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 13.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 13 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 13.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 13. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 13. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 13. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 13.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 13. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 13 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 13. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 13.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 13.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 13.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 13.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 13.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 13.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 13.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 13 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 13.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 13. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 13. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 13.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 13. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 13. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 13 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 13. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 13.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 13. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 13. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 13.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 73. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 73.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 14.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 14.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 14.
  • the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 14.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 14 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 14.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 14. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 14. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 14. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 14.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 14. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 14. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 43 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 14. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 14.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 14.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 14.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 14.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 14.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 14.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 14.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 14 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 14.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 14. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 14. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 14.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 14. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 14. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 14. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 14 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 14. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 14.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 14. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 14. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 14.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 74. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 74.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 15.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 15.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 15.
  • the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 15.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 15 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 15.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 15. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 15. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 15. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 15.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 15. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 15 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 15. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 15.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 15.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 15.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 15.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 15.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 15.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 15.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 15 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 15.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 15. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 15. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 15.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 15.
  • the second subunit comprises residues 198- 344 of SEQ ID NO: 15 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the second subunit comprises residues 198-344 of SEQ ID NO: 15.
  • the second subunit comprises residues 196-354 of SEQ ID NO: 15.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 15.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 15. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 15. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 15.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 75. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 75.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 16.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 16.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 16.
  • the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 16.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 16 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 16.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 16. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 16. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 16. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 16.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 16. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 16 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 16. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 16.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 16.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 16.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 16.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 16.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 16.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 16.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 16 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 16.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 16. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 16. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 16.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 16.
  • the second subunit comprises residues 198- 344 of SEQ ID NO: 16 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the second subunit comprises residues 198-344 of SEQ ID NO: 16.
  • the second subunit comprises residues 196-354 of SEQ ID NO: 16.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 16.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 16. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 16.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 76. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 76.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 17.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 17.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 17.
  • the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 17.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 17 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 17.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 17. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 17. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 17. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 17.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 17. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 17 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 17. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 17.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 17.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 17.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 17.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 17.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 17.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 17.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 17 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 17. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 17.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 17.
  • the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 17.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 17.
  • the second subunit comprises residues 198- 344 of SEQ ID NO: 17 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the second subunit comprises residues 198-344 of SEQ ID NO: 17.
  • the second subunit comprises residues 196-354 of SEQ ID NO: 17.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 17.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 17. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 17. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 17.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 77. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 77.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 18.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 18.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 18.
  • the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 42.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 18.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 18. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 18. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 18. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 18.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 18. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 18. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 18. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 18.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 18.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 18.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 18.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 18.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 18.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 18.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 18.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 18. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 18. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 18.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 18. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 18. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
  • the second subunit comprises residues 198-344 of SEQ ID NO: 18. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 18.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 18.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 18. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 18. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 18.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 78. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 78.
  • the engineered meganuclease can comprise a nuclear localization signal.
  • the nuclear localization signal is at the N-terminus of the engineered meganuclease.
  • the nuclear localization signal comprises an amino acid sequence having at least 80% or at least 90% sequence identity to SEQ ID NO: 133 or 134.
  • the nuclear localization signal comprises SEQ ID NO: 133 or 134.
  • the engineered meganuclease binds and cleaves a recognition sequence comprising SEQ ID NO: 5 (z.e., the CNR 21-22 recognition sequence) within a C90rf72 gene, wherein the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and wherein the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region.
  • HVR1 hypervariable
  • HVR2 hypervariable hypervariable
  • Exemplary CNR 21-22 meganucleases are described below.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 79.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 79.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 79.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 79.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 79 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 79.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 79. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 79. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 79.
  • the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 79. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 79. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 79 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 79. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 79.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 79.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 79.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 79.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 79.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 79.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 79.
  • the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 79.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 79 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215- 270 of SEQ ID NO: 79.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 79. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 79. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 79.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 79. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 79. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 79 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 79. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 79.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 79.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 79. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 79. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 79.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 124. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 124.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 80.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 80.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 80.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 80.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 80 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 80.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 80. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 80. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 80.
  • the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 80. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 80. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 80 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 80. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 80.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 80.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 80.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 80.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 80.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 80.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 80.
  • the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 80.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 80 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215- 270 of SEQ ID NO: 80.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 80. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 80. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 80.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 80. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 80. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 80 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 80. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 80.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 80.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 80.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ IDNO: 125. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 125. In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 80.
  • the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 80. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ IDNO: 125. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 125.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 81.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 81.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 81.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 81.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 81 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 81.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 81. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 81. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 81.
  • the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 81. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 81. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 81. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 81 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 81. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 81.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 81.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 81.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 81.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 81.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 81.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 81.
  • the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 81.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 81 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215- 270 of SEQ ID NO: 81.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 81. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 81. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 81.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 81. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 81. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 81. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 81 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 81. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 81.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 81.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 81. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 81. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 81.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ IDNO: 126. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 126.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 82.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 82.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 82.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 82 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 82.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 82.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 82.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 82.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 82.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 82.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 82.
  • the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 82.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 82.
  • the second subunit comprises residues 198- 344 of SEQ ID NO: 82 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the second subunit comprises residues 198-344 of SEQ ID NO: 82.
  • the second subunit comprises residues 196-354 of SEQ ID NO: 82.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 82.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 82. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 82. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 82.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 127. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 127.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 83.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 83.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 83.
  • the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 83 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 83.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 83. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 83. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 83.
  • the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 83. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 83. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 83 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 83. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 83.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 83.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 83.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 83.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 83.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 83.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 83.
  • the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 83.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 83. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 83. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 83.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 128. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 128.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 84 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 84.
  • the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 84. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 84. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 84 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 84. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 84.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 84.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 84.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 84.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 84.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 84.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 84.
  • the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 84.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 84 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215- 270 of SEQ ID NO: 84.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 84. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 84. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 84.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 84. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 84. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 84 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 84. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 84.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 84. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 84. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 SEQ ID NO: 84.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 129. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 129.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 85.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 85.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 85.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 85. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 85. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 85. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 85. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 85. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 85.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 85 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 85.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 85. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 85. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 85.
  • the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 85. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 85. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 85. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 85 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 85. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 85.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 85.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 85.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 85.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 85.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 85.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 85.
  • the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 85.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 85 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215- 270 of SEQ ID NO: 85.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 85. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 85. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 85.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 85.
  • the second subunit comprises residues 198- 344 of SEQ ID NO: 85 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the second subunit comprises residues 198-344 of SEQ ID NO: 85.
  • the second subunit comprises residues 196-354 of SEQ ID NO: 85.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 85.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 85. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 85. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 85.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 130. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 130.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 86.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 86.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 86.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 86. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 86. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 86. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 86. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 86. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 86.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 86 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 86.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 86. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 86. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 86.
  • the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 86. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 86. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 86 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 86. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 86.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 86.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 86.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 86.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 86.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 86.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 86.
  • the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 86.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 86 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215- 270 of SEQ ID NO: 86.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 86. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 86. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 86.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 86. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 86. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 86 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 86. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 86.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 86.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 86. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 86. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 86.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 131. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 131.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 87.
  • the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 87.
  • the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 87.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 87. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 87. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 87. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 87. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 87.
  • the HVR1 region comprises residues 24-79 of SEQ ID NO: 87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 87.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 87. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 87. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 87.
  • the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 87. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 87. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 87. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 87. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 87.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 87.
  • the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 87.
  • the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 87.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 87.
  • the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 87.
  • the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 87.
  • the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 87.
  • the HVR2 region comprises residues 215-270 of SEQ ID NO: 87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215- 270 of SEQ ID NO: 87.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 87. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 87. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 87.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 87. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 87. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 87. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 87.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 87.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 87. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 87. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 87.
  • the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 132. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 132.
  • the engineered meganuclease can comprise a nuclear localization signal (NLS).
  • the nuclear localization signal is at the N- terminus of the engineered meganuclease.
  • the nuclear localization signal is at the C-terminus of the engineered meganuclease.
  • the engineered meganuclease can comprise a first NLS at the N-terminus of the engineered meganuclease and a second NLS at the C-terminus of the engineered meganuclease.
  • the nuclear localization signal comprises an amino acid sequence having at least 80% or at least 90% sequence identity to SEQ ID NO: 133 or 134.
  • the nuclear localization signal comprises SEQ ID NO: 133 or 134.
  • the first engineered meganuclease is an engineered meganuclease described herein that binds and cleaves a recognition sequence comprising SEQ ID NO: 3
  • the second engineered meganuclease is an engineered meganuclease described herein that binds and cleaves a recognition sequence comprising SEQ ID NO: 5.
  • the first engineered meganuclease is CNR 1-2L.532 (SEQ ID NO: 7), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21- 22L.290 (SEQ ID NO: 79), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.532 (SEQ ID NO: 7), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.286 (SEQ ID NO: 80), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1- 2L.532 (SEQ ID NO: 7), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.274 (SEQ ID NO: 81), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.532 (SEQ ID NO: 7), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.212 (SEQ ID NO: 82), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.532 (SEQ ID NO: 7), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.197 (SEQ ID NO: 83), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.532 (SEQ ID NO: 7), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.169 (SEQ ID NO: 84), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.532 (SEQ ID NO: 7), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.139 (SEQ ID NO: 85), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1- 2L.532 (SEQ ID NO: 7), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.39 (SEQ ID NO: 86), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.532 (SEQ ID NO: 7), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22x.l3 (SEQ ID NO: 86), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.532 (SEQ ID NO: 7), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21- 22L.286 (SEQ ID NO: 80), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.468 (SEQ ID NO: 8), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.286 (SEQ ID NO: 80), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1- 2L.531 (SEQ ID NO: 9), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.286 (SEQ ID NO: 80), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.371 (SEQ ID NO: 10), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.286 (SEQ ID NO: 80), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.425 (SEQ ID NO: 11), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.286 (SEQ ID NO: 80), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.284 (SEQ ID NO: 12), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.286 (SEQ ID NO: 80), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.297 (SEQ ID NO: 13), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.286 (SEQ ID NO: 80), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1- 2L.211 (SEQ ID NO: 14), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.286 (SEQ ID NO: 80), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.98 (SEQ ID NO: 15), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.286 (SEQ ID NO: 80), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L.108 (SEQ ID NO: 16), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.286 (SEQ ID NO: 80), or a variant thereof described herein.
  • the first engineered meganuclease is CNR 1-2L15 (SEQ ID NO: 17), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.286 (SEQ ID NO: 80), or a variant thereof described herein.
  • the first engineered meganuclease is CNR l-2x.88 (SEQ ID NO: 18), or a variant thereof described herein
  • the second engineered meganuclease is CNR 21-22L.286 (SEQ ID NO: 80), or a variant thereof described herein.
  • the disclosed engineered meganucleases comprise (i) an inactivating amino acid in the N-terminal subunit that reduces or abolishes cleavage activity; (ii) an inactivating amino acid in the C-terminal subunit that reduces or abolishes cleavage activity; or (iii) an inactivating amino acid in the N-terminal subunit that reduces or abolishes cleavage activity and an inactivating amino acid in the C-terminal subunit that reduces or abolishes cleavage activity.
  • an inactivating amino acid that “reduces” cleavage activity of an engineered meganuclease inactivates only the subunit comprising that amino acid, while not affecting the ability of the other subunit to cleave its DNA strand.
  • the other subunit remains active and the engineered meganuclease becomes a nickase that remains capable of cleaving one strand of the double-stranded DNA.
  • both subunits comprise an inactivating amino acid that reduces cleavage activity
  • neither subunit is active
  • the engineered meganuclease does not comprise any cleavage activity, and it cannot generate a single-strand or double-strand break in the DNA.
  • an inactivating amino acid that “abolishes” cleavage activity of an engineered meganuclease can be present in only one subunit but will inactivate both subunits of the engineered meganuclease, such that it does not comprise any cleavage activity and cannot generate a single-strand or double-strand break in the DNA.
  • the inactivating amino acid is an A at a position corresponding to position 20 or position 211 of any one of SEQ ID NOs: 7-18 or 79-87. In some embodiments, the inactivating amino acid is an E at a position corresponding to position 47 or position 238 of any one of SEQ ID NOs: 7-18 or 79-87.
  • the N-terminal subunit comprises an E at a position corresponding to position 47 of any one of SEQ ID NOs: 7-18 or 79-87
  • the C-terminal subunit comprises an E at a position corresponding to position 238 of any one of SEQ ID NOs: 7-18 or 79-87, wherein the engineered meganuclease does not comprise cleavage activity (i.e., activity is abolished).
  • the N-terminal subunit comprises an A at a position corresponding to position 20 of any one of SEQ ID NOs: 7-18 or 79-87
  • the C-terminal subunit comprises an A at a position corresponding to position 211 of any one of SEQ ID NOs: 7-18 or 79-87, wherein the engineered meganuclease does not comprise cleavage activity (i.e., activity is abolished).
  • the N-terminal subunit comprises an E at a position corresponding to position 47 of any one of SEQ ID NOs: 7-18 or 79-87 and the C-terminal subunit does not comprise an inactivating amino acid, wherein the engineered meganuclease is a nickase that is only capable of cleaving the antisense strand of a dsDNA target site.
  • the C-terminal subunit comprises an E at a position corresponding to position 238 of any one of SEQ ID NOs: 7-18 or 79-87 and the N-terminal subunit does not comprise an inactivating amino acid, wherein the engineered meganuclease is a nickase that is only capable of cleaving the sense strand of a dsDNA target site.
  • engineered meganuclease does not comprise cleavage activity (i.e., activity is abolished) due to one or more inactivating amino acid modifications
  • engineered meganucleases are capable of binding to a double-stranded DNA comprising the recognition sequence of SEQ ID NO: 3 or SEQ ID NO: 5 without cleaving the double-stranded DNA.
  • engineered meganuclease comprises an inactivating amino acid modification such that only one subunit has cleavage activity
  • the engineered meganuclease is a nickase
  • engineered meganucleases are capable of binding to a doublestranded DNA comprising the recognition sequence of SEQ ID NO: 3 or SEQ ID NO: 5 and cleaving either the sense or antisense strand of the DNA.
  • the disclosure provides engineered meganucleases described herein that are useful for binding and cleaving recognition sequences within a C90rf72 gene of a cell (e.g., the human C90rf72 gene).
  • the disclosure provides various methods for modifying a C90rf72 gene in cells using engineered meganucleases described herein, methods for making genetically modified cells comprising a modified C90rf72 gene, and methods of modifying a C90rf72 gene in a target cell in a subject.
  • the disclosure provides methods for treating FTD and/or ALS in a subject by administering the engineered meganucleases described herein, or polynucleotides encoding the same, to a subject, in some cases as part of a pharmaceutical composition.
  • the engineered meganucleases, or polynucleotides encoding the same are introduced into cells, such as neural cells of the central nervous system (CNS), including neurons, motor neurons (e.g., upper and lower motor neurons) or neuroglia cell, or neural precursor cells capable of expressing a C90rf72 protein.
  • CNS central nervous system
  • Any cell that expresses a C90rf72 gene is envisaged within the scope of the present disclosure for introducing the engineered meganucleases, or polynucleotides encoding the same, disclosed herein.
  • Engineered meganucleases described herein can be delivered into a cell in the form of protein or, preferably, as a polynucleotide encoding the engineered meganuclease.
  • Such polynucleotides can be, for example, DNA (e.g., circular or linearized plasmid DNA, PCR products, or a viral genome) or RNA (e.g., mRNA).
  • modified C90rf72 z.e., a gene or RNA lacking the hexanucleotide repeat region and exon lb non-coding sequences
  • levels of such modified C90rf72 may be assessed based on the level of any variable associated with C90rf72 gene expression, e.g., C90rf72 mRNA levels or C90rf72 protein levels. Increased levels or expression of such modified C90rf72 may be assessed by an increase in an absolute or relative level of one or more of these variables compared with a reference level.
  • modified C90rf72 levels may be measured in a biological sample isolated from a subject, such as a tissue biopsy or a bodily fluid including blood, serum, plasma, cerebrospinal fluid, or urine.
  • a biological sample isolated from a subject such as a tissue biopsy or a bodily fluid including blood, serum, plasma, cerebrospinal fluid, or urine.
  • modified C90rf72 levels are normalized to a standard polynucleotide or protein or substance in the sample. Further, such modified C90rf72 levels can be assessed any time before, during, or after treatment in accordance with the methods herein.
  • the methods described herein can increase polynucleotide levels of a modified C90rf72 gene (z.e., lacking non-coding nucleotide sequence comprising the hexanucleotide repeat region and exon lb) in a genetically modified cell, target cell, or subject (e.g., as measured in a cell, a tissue, an organ, or a biological sample obtained from the subject), to at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more, of a reference level (z.e., polynucleotide level of C90rf72 in a wild-type cell or subject).
  • a reference level z.e., polynucleotide level of C90rf72 in a wild-type cell or subject.
  • the methods herein are effective to increase the level of such modified C90rf72 polynucleotide gene sequence to about 10% to about 100% (e.g., 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-100%, or more) of a reference level of C90rf72 polynucleotide sequence (z.e., polynucleotide level of C90rf72 in a wild-type cell or subject).
  • the reference gene can be a wild-type C90rf72 gene comprising a hexanucleotide repeat region or a mutant, or mRNA levels associated with gene expression.
  • the reference can be a C90rf72 mRNA comprising a hexanucleotide repeat mutation, such as an extension mutation.
  • Methods of detecting the modified C90rf72 can comprise detection of the re-ligated sequence of Table 3.
  • the modified C90rf72 gene lacking the hexanucleotide sequence and exon lb would be identifiable by the absence of these nucleotide sequences compared at their location in a wild-type C90rf72 gene sequence.
  • Methods of determining the presence of specific polynucleotide sequence regions are readily available in the art.
  • a person having skill in the art would understand how to identify the modified C90rf72 gene sequence disclosed herein.
  • Engineered meganuclease proteins disclosed herein, or polynucleotides encoding the same, can be delivered into cells to cleave genomic DNA by a variety of different mechanisms known in the art, including those further detailed herein below.
  • Engineered meganucleases disclosed herein can be delivered into a cell in the form of protein or, preferably, as a polynucleotide comprising a nucleic acid sequence encoding the engineered meganuclease.
  • Such polynucleotides can be, for example, DNA (e.g., circular or linearized plasmid DNA, PCR products, or viral genomes) or RNA (e.g., mRNA).
  • said polynucleotide comprising a nucleic acid sequence encoding the engineered meganuclease is DNA.
  • said polynucleotide is RNA.
  • said polynucleotide is mRNA.
  • the engineered meganuclease coding sequence is delivered in DNA form, it should be operably linked to a promoter to facilitate transcription of the meganuclease gene.
  • the engineered meganuclease coding sequence can be delivered to a mammalian cell.
  • the engineered meganuclease coding sequence when it is delivered in DNA form, it can be operably linked to a promoter that facilities transcription in the mammalian cell.
  • Mammalian promoters suitable for the disclosure include constitutive promoters such as the cytomegalovirus early (CMV) promoter (Thomsen et al. (1984) Proc Natl Acad Sci USA.
  • CMV cytomegalovirus early
  • the promoter can be a CMV hybrid promoter, such as a CAG promoter, which is a hybrid of the CMV enhancer element and chicken beta(P)- actin promoter (Niwa, H et al. “Efficient selection for high-expression transfectants with a novel eukaryotic vector.” Gene vol. 108,2 (1991): 193-9).
  • an engineered meganuclease of the disclosure can also be operably linked to a synthetic promoter.
  • Synthetic promoters can include, without limitation, the JeT promoter (WO 2002/012514).
  • the mammalian cell is a neural tissue-specific (i.e., nervous tissue) cell.
  • the neural tissue cell can be a cell in the central nervous system (CNS), such as the brain or spinal cord, or the peripheral nervous system (PNS) which lies outside of the brain and spinal cord.
  • exemplary, non-limiting neural tissue cells include neurons (i.e., neuronal cells, nerve cells or neurons), motor neurons (i.e., upper and lower motor neurons) or neuroglia (i.e., glial cells, or glia), or precursors thereof.
  • Neurons have traditionally been classified according to structure and function, while some neurons are classified by location.
  • Exemplary classifications according to structure include unipolar neurons, bipolar neurons, multipolar neurons, anaxonic neurons and pseudounipolar neurons.
  • Exemplary classifications according to location include Basket cells, Betz cells, Lugaro cells, medium spiny neurons, Purkinje cells, Pyramidal cells, Rosehip cells, unipolar brush cells, granule cells, anterior horn cells, and spindle cells.
  • Exemplary neuron cells classified according to function include afferent neurons (i.e., sensory neurons), efferent neurons (i.e., motor neurons), and interneurons.
  • the motor neurons disclosed herein can be lower motor neurons or upper motor neurons.
  • the classifications disclosed herein are not intended to be limiting.
  • mammalian cells can include any cell known to within neural tissue containing a C90rf72 gene coding sequence and/or capable of producing a polypeptide encoded by a C90rf72 coding sequence is envisaged within the scope of the present disclosure for delivering meganucleases, or polynucleotides encoding the same.
  • a nucleic acid sequence encoding an engineered nuclease of the disclosure is operably linked to a tissue-specific promoter, such as a nerve tissue-specific promoter.
  • the promoter is a cell-specific promoter, such as a CNS-specific promoter, neuron cell-specific promoter or a glia-cell specific promoter.
  • the promoter is capable of expressing an engineered meganuclease described herein in a neural tissue cell, such as a neuronal cell, a neuroglia cell, or a neural tissue precursor cell.
  • promoters can demonstrate cell-specific functionality and can be selected in a cell-specific manner for expression of the meganucleases, or polynucleotides encoding the same, disclosed herein based on cell-specific functionality in a specific cell type.
  • CNS-cell specific promoters are known in the art (Nonnenmacher M, Wang W, Child MA, Ren XQ, Huang C, Ren AZ, Tocci J, Chen Q, Bittner K, Tyson K, Pande N, Chung CH, Paul SM, Hou J. Rapid evolution of blood-brain-barrier-penetrating AAV capsids by RNA-driven biopanning. Mol Ther Methods Clin Dev. 2020 Dec 23;20:366-378).
  • GFAP glial fibrillary acidic protein
  • the neural-specific promoter comprises an enolase (NSE) promoter, or a variant or derivative thereof.
  • NSE enolase
  • the neural-specific promoter comprises a sequence 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an enolase (NSE) promoter sequence.
  • the neural-specific promoter comprises a calcium/calmodulin-dependent kinase subunit alpha (a) (CaMKII) promoter, or a variant or derivative thereof.
  • the neural-specific promoter comprises a sequence 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a calcium/calmodulin-dependent kinase subunit alpha (a) (CaMKII) promoter sequence.
  • the neural-specific promoter comprises a synapsin I (Synl) promoter, or a variant or derivative thereof.
  • the neural-specific promoter comprises a sequence 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to synapsin I promoter sequence.
  • the synapsin 1 promoter comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 138.
  • the synapsin 1 promoter comprises a sequence according to SEQ ID NO: 138.
  • the neural-specific promoter comprises a glial fibrillary acidic protein (GFAP) promoter, or a variant or derivative thereof.
  • the neural- specific promoter comprises a sequence 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a glial fibrillary acidic protein (GFAP) promoter sequence.
  • the promoter is a CNS cell-specific promoter. In some embodiments, the promoter is active in any of the nerve system cells disclosed herein. In some embodiments, the promoter is active in neurons and/or neuroglial cells, or precursor cells thereof. The promoter can be active in neurons, such as motor neurons (e.g., upper motor neurons or lower motor neurons) or neuron progenitor cells, such as motor neuron progenitor cells. In some embodiments, the promoter is active in excitatory and/or inhibitory neurons. In some embodiments, the promoter can be active in cortex cells, spinal cells, astrocytes, and/or oligodendrocytes.
  • the polynucleotides disclosed herein can comprise a promoter enhancer.
  • a person having skill in the art would understand an enhancer to be a regulatory element that is distinct from a promoter. While promoters include specific DNA motifs where transcription factors (TFs) and their complexes can access (Hudson, Matthew E, and Peter H Quail. “Identification of promoter motifs involved in the network of phytochrome A-regulated gene expression by combined analysis of genomic sequence and microarray data.” Plant physiology vol. 133,4 (2003): 1605-16), enhancers, on the other hand, are defined as DNA regions that amplify transcription initiation by directly interplaying with their target promoters (Blackwood, E M, and J T Kadonaga.
  • the polynucleotide disclosed herein comprises a promoter enhancer.
  • the enhancer sequence is a CMV enhancer.
  • the promoter enhancer comprises a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 138.
  • the promoter enhancer comprises a nucleic acid sequence according to SEQ ID NO: 138.
  • Engineered meganucleases disclosed herein can be delivered into a cell in the form of protein or, preferably, as a polynucleotide comprising a nucleic acid sequence encoding a first and a second engineered meganuclease.
  • the first engineered meganuclease can be any engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 within a C90rf72 gene (i.e., CNR 1-2 meganuclease) disclosed herein.
  • the second engineered meganuclease can be any engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 5 within a C90rf72 gene (i.e., CNR 21-22 meganuclease) disclosed herein.
  • Such polynucleotides can be, for example, DNA (e.g., circular or linearized plasmid DNA, PCR products, or viral genomes) or RNA (e.g., mRNA).
  • said polynucleotide comprising a nucleic acid sequence encoding the engineered meganuclease is DNA.
  • said polynucleotide is RNA.
  • said polynucleotide is mRNA.
  • a single polynucleotide comprises two separate nucleic acid sequences each encoding an engineered meganuclease described herein
  • the meganuclease genes are operably linked to two separate promoters.
  • said first nucleic acid sequence is operably linked to a first promoter
  • said second nucleic acid sequence is operably linked to a second promoter.
  • the first and second promoter can be identical. Alternatively, in some embodiments, the first and second promoter are not identical.
  • the first and/or second promoter can be any promoter disclosed herein, such as a neural-specific promoter, or neuron-specific promoter.
  • the promoter can be any promoter that is active in a neural or CNS cell as described herein. In some instances, the promoter can be a constitute and/or synthetic promoter disclosed herein. In some embodiments, wherein said first and second promoter are identical, said first promoter is a central nervous system (CNS) cell-specific promoter and said second promoter is a central nervous system (CNS) cell-specific promoter.
  • said first promoter can be a CAG promoter and said second promoter can be a human Syn-1 promoter. In some instances, said first promoter and second promoter can be a human Syn-1 promoter or a CAG promoter. In some instances, said first promoter and second promoter is a human Syn-1 promoter.
  • said first promoter and second promoter is a CAG promoter.
  • said first promoter is a central nervous system (CNS) cell-specific promoter and said second promoter is a central nervous system (CNS) cell-specific promoter.
  • said first promoter can be a CAG promoter and said second promoter can be a human Syn-1 promoter.
  • said first promoter can be a human Syn-1 promoter and said second promoter can be a CAG promoter.
  • the two meganuclease genes are operably linked to a single promoter (e.g., a neural-specific or neuron-specific promoter), and in some examples can be separated by an internal-ribosome entry site (IRES) or a 2A peptide sequence (Szymczak & Vignali (2005) Expert Opin Biol Ther. 5:627-38).
  • IRS internal-ribosome entry site
  • 2A peptide sequences can include, for example, a T2A, P2A, E2A, or F2A sequence.
  • the two meganucleases genes are separated by a nucleic acid sequence encoding a furin cleavage motif and a nucleic acid sequence encoding a 2A peptide.
  • the two meganuclease genes can be separated by polypeptide linker, or a nucleic acid encoding a polypeptide linker.
  • said first nucleic acid sequence and said second nucleic acid sequence are separated by a nucleic acid sequence encoding a furin cleavage motif, a nucleic acid sequence encoding a polypeptide linker, and a nucleic acid sequence encoding a 2A peptide.
  • said 2A peptide can comprise a P2A peptide.
  • said 2A peptide is a P2A peptide.
  • the two genes can be separated by a P2A peptide and furin peptide.
  • said first nucleic acid sequence and said second nucleic acid sequence are separated by a nucleic acid sequence encoding a P2A/furin peptide comprising an amino acid sequence set forth in SEQ ID NO: 135.
  • the polynucleotide further comprises a post transcriptional regulatory element as described herein.
  • the post transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) described herein.
  • WPRE woodchuck hepatitis virus post-transcriptional regulatory element
  • the WPRE comprises a nucleic acid sequence that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 194.
  • the WPRE comprises a nucleic acid sequence according to a sequence set forth in SEQ ID NO: 194.
  • the polynucleotide further comprises a termination sequence.
  • said termination sequence is a poly A termination sequence.
  • the poly A termination sequence is an SV40 poly A.
  • the poly A termination sequence comprises a nucleic acid sequence that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 137.
  • the poly A termination sequence comprises a nucleic acid sequence according to a sequence set forth in SEQ ID NO: 137.
  • the polynucleotide comprises, from 5' to 3', (i) said first nucleic acid sequence encoding said first engineered meganuclease; and (ii) said second nucleic acid sequence encoding said second engineered meganuclease. In some embodiments, the polynucleotide comprises, from 5' to 3', (i) said second nucleic acid sequence encoding said second engineered meganuclease; and (ii) said first nucleic acid sequence encoding said first engineered meganuclease.
  • the single polynucleotide can encode a single-chain meganuclease.
  • the first and second meganucleases are not joined by a linker (i.e., the polynucleotide does not encode a single-chain meganuclease).
  • a single polynucleotide comprises two separate nucleic acid sequences each encoding an engineered meganuclease described herein
  • said first nucleic acid sequence and/or said second nucleic acid sequence is codon modified to reduce the percent sequence identity between said first nucleic acid sequence and said second nucleic acid sequence, wherein said codon modification does not alter the amino acid sequence of said first engineered meganuclease or said second engineered meganuclease.
  • said first nucleic acid sequence has no more than about 40% to about 80% sequence identity to said second nucleic acid sequence.
  • said first nucleic acid sequence has no more than about 60% sequence identity to said second nucleic acid sequence.
  • the polynucleotide can comprise a first and/or second nucleic acid encoding an engineered meganuclease(s) which comprises a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • Any NLS that is capable of is envisaged within the scope of the present disclosure.
  • Exemplary NLS sequence are known in the art and can include, but are not limited to, the NLS sequence from the c-myc protein (i.e., c-myc NLS) or simian virus 40 (sv40).
  • the nuclear localization signal is at the N-terminus of the engineered meganuclease. In some embodiments, the nuclear localization signal is at the C- terminus of the engineered meganuclease. In some embodiments, the nuclear localization signal is at the N-terminus of the first engineered meganuclease.
  • the nuclear localization signal is at the C-terminus of the first engineered meganuclease. In some embodiments, the nuclear localization signal is at the N-terminus of the second engineered meganuclease. In some embodiments, the nuclear localization signal is at the C-terminus of the second engineered meganuclease. In some embodiments, the nuclear localization signal is at the N-terminus of the first and second engineered meganuclease. In some embodiments, the nuclear localization signal is at the C-terminus of the first and second engineered meganuclease.
  • the nuclear localization signal is at the N-terminus of the first engineered meganuclease and the C-terminus of the second engineered meganuclease. In some embodiments, the nuclear localization signal is at the C-terminus of the first engineered meganuclease and the N-terminus of the second engineered meganuclease.
  • the nuclear localization signal at the N-terminus and/or C-terminus of the first and/or second engineered meganuclease can comprise an amino acid sequence of an sv40 or c-myc nuclear localization sequence (NLS), or a variant thereof.
  • the NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 133.
  • the NLS comprises an amino acid sequence set forth in SEQ ID NO: 133.
  • the NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 134.
  • the NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.
  • the NLS can be located on the N-terminus and/or C-terminus of the first engineered meganuclease.
  • the first engineered meganuclease comprises a first NLS attached at the N-terminus and a second NLS attached at the C-terminus.
  • said first and second NLS can be identical.
  • said first and second NLS are not identical.
  • the first engineered meganuclease comprises a first NLS attached at the N-terminus and a second NLS attached at the C-terminus, and said first and second NLS are identical
  • said first NLS and said second NLS comprise an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133.
  • said first NLS and said second NLS comprise an amino acid sequence set forth in SEQ ID NO: 133.
  • said first NLS and said second NLS comprise an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 134.
  • said first NLS and said second NLS comprise an amino acid sequence set forth in SEQ ID NO: 134.
  • the first engineered meganuclease comprises a first NLS attached at the N-terminus and a second NLS attached at the C-terminus
  • said first NLS and said second NLS are not identical.
  • said first NLS can comprise an amino acid sequence of an sv40 NLS and said second NLS can comprise an amino acid sequence of a c-myc NLS sequence.
  • said first NLS can comprise an amino acid sequence of a c- myc NLS and said second NLS can comprise an amino acid sequence of an sv40 NLS sequence.
  • said first NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133
  • said second NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 134.
  • said first NLS comprises an amino acid sequence set forth in SEQ ID NO: 133
  • said second NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.
  • said first NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 134
  • said second NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 133.
  • said first NLS comprises an amino acid sequence set forth in SEQ ID NO: 134
  • said second NLS comprises an amino acid sequence set forth in SEQ ID NO: 133.
  • said second engineered meganuclease can comprise a third NLS attached at the N-terminus and a fourth NLS attached at the C-terminus.
  • the third and fourth NLS attached at the N-terminus and C-terminus of said second engineered meganuclease can be identical.
  • the third and fourth NLS attached at the N-terminus and C-terminus of said second engineered meganuclease are not identical.
  • said third and fourth NLS can comprise an amino acid sequence of an sv40 or a c-myc NLS.
  • said third NLS and said fourth NLS comprise an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134. In some embodiments, said third NLS and said fourth NLS comprise an amino acid sequence set forth in SEQ ID NO: 133 or 134. In some embodiments, wherein the third and fourth NLS attached at the N-terminus and C- terminus of said second engineered meganuclease are not identical, said third NLS can comprise an amino acid sequence of an sv40 NLS and said fourth NLS can comprise an amino acid sequence of a c-myc NLS.
  • said third NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133
  • said fourth NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 134.
  • said third NLS comprises an amino acid sequence set forth in SEQ ID NO: 133
  • said fourth NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.
  • said third NLS can comprise an amino acid sequence of a c-myc NLS and said fourth NLS can comprise an amino acid sequence of an sv40 NLS.
  • said third NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 134
  • said fourth NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 133.
  • said third NLS comprises an amino acid sequence set forth in SEQ ID NO: 134
  • said fourth NLS comprises an amino acid sequence set forth in SEQ ID NO: 133.
  • a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein is delivered on a recombinant DNA construct or expression cassette.
  • the recombinant DNA construct can comprise an expression cassette (z.e., “cassette”) comprising a promoter and a nucleic acid sequence encoding an engineered meganuclease described herein.
  • the polynucleotide delivered on a recombinant DNA construct or expression cassette can be any polynucleotide encoding an engineered meganuclease described herein.
  • the polynucleotide encodes any engineered meganuclease described herein.
  • the polynucleotide comprises a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease.
  • the first engineered meganuclease can be any engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 within a C90rf72 gene (i.e., CNR 1-2 meganuclease) disclosed herein
  • the second engineered meganuclease can be any engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 5 within a C90rf72 gene (i.e., CNR 21-22 meganuclease) disclosed herein.
  • Such polynucleotides can be, for example, DNA (e.g., circular or linearized plasmid DNA, PCR products, or viral genomes) or RNA (e.g., mRNA). Accordingly, in some embodiments, said polynucleotide comprising a nucleic acid sequence encoding the engineered meganuclease is DNA. In some embodiments, said polynucleotide is RNA. In some embodiments, the recombinant DNA construct is a plasmid DNA. In some embodiments, the recombinant DNA construct encodes a recombinant virus comprising the polynucleotide, described herein.
  • the recombinant virus can be a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adeno-associated virus (AAV) disclosed herein.
  • AAV adeno-associated virus
  • a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein is introduced into the cell using a singlestranded DNA template.
  • the single-stranded DNA can further comprise a 5' and/or a 3' AAV inverted terminal repeat (ITR) upstream and/or downstream of the sequence encoding the engineered nuclease.
  • the single-stranded DNA can further comprise a 5' and/or a 3' homology arm upstream and/or downstream of the sequence encoding the engineered meganuclease.
  • a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein can be introduced into a cell using a linearized DNA template.
  • linearized DNA templates can be produced by methods known in the art.
  • a plasmid DNA encoding a nuclease can be digested by one or more restriction enzymes such that the circular plasmid DNA is linearized prior to being introduced into a cell.
  • mRNA encoding an engineered meganuclease described herein is delivered to a cell because this reduces the likelihood that the gene encoding the engineered meganuclease will integrate into the genome of the cell.
  • mRNA can be produced using methods known in the art such as in vitro transcription.
  • the mRNA is 5' capped using 7-methyl-guanosine, anti-reverse cap analogs (ARCA) (US 7,074,596), CleanCap® analogs such as Cap 1 analogs (Trilink, San Diego, CA), or enzymatically capped using vaccinia capping enzyme or similar.
  • the mRNA may be polyadenylated.
  • the mRNA may contain various 5’ and 3’ untranslated sequence elements to enhance expression of the encoded engineered meganuclease and/or stability of the mRNA itself.
  • Such elements can include, for example, posttranslational regulatory elements such as a woodchuck hepatitis virus posttranslational regulatory element.
  • the mRNA may contain nucleoside analogs or naturally- occurring nucleosides, such as pseudouridine, 5-methylcytidine, N6-methyladenosine, 5- methyluridine, or 2-thiouridine. Additional nucleoside analogs include, for example, those described in US Pat. No. 8,278,036.
  • the meganuclease proteins, or DNA/mRNA encoding the meganuclease are coupled to a cell penetrating peptide or targeting ligand to facilitate cellular uptake.
  • cell penetrating peptides known in the art include poly-arginine (Jearawiriyapaisarn et al. (2008) Mol Ther. 16: 1624-29), TAT peptide from the HIV virus (Hudecz et al. (2005) VW. Res. Rev. 25:679-736), MPG (Simeoni et al. (2003) Nucleic Acids Res. 31 :2717- 24), Pep-1 (Deshayes et al.
  • engineered nucleases are coupled covalently or non-covalently to an antibody that recognizes a specific cell-surface receptor expressed on target cells such that the nuclease protein/DNA/mRNA binds to and is internalized by the target cells.
  • engineered nuclease protein/DNA/mRNA can be coupled covalently or non-covalently to the natural ligand (or a portion of the natural ligand) for such a cell-surface receptor.
  • meganuclease proteins are encapsulated within biodegradable hydrogels for injection or implantation within the desired region of the liver (e.g., in proximity to hepatic sinusoidal endothelial cells or hematopoietic endothelial cells, or progenitor cells which differentiate into the same).
  • Hydrogels can provide sustained and tunable release of the therapeutic payload to the desired region of the target tissue without the need for frequent injections, and stimuli-responsive materials (e.g., temperature- and pH-responsive hydrogels) can be designed to release the payload in response to environmental or externally applied cues (Derwent et al. (2008) Trans Am. Ophthalmol. Soc. 106:206-14).
  • meganuclease proteins, or DNA/mRNA encoding meganucleases are coupled covalently or, preferably, non-covalently to a nanoparticle or encapsulated within such a nanoparticle using methods known in the art (Sharma et al. (2014) Biomed. Res. Int. 2014: 156010).
  • a nanoparticle is a nanoscale delivery system whose length scale is ⁇ 1 pm, preferably ⁇ 100 nm.
  • Such nanoparticles may be designed using a core composed of metal, lipid, polymer, or biological macromolecule, and multiple copies of the meganuclease proteins, mRNA, or DNA can be attached to or encapsulated with the nanoparticle core.
  • Nanoparticles may be further modified with polymers or lipids (e.g., chitosan, cationic polymers, or cationic lipids) to form a core-shell nanoparticle whose surface confers additional functionalities to enhance cellular delivery and uptake of the payload (Jian et al. (2012) Biomaterials. 33:7621-30).
  • Nanoparticles may additionally be advantageously coupled to targeting molecules to direct the nanoparticle to the appropriate cell type and/or increase the likelihood of cellular uptake. Examples of such targeting molecules include antibodies specific for cell-surface receptors and the natural ligands (or portions of the natural ligands) for cell surface receptors.
  • the meganuclease proteins, or DNA/mRNA encoding meganucleases are encapsulated within liposomes or complexed using cationic lipids (see, e.g., LIPOFECT AMINETM, Life Technologies Corp., Carlsbad, CA; Zuris et al. (2015) Nat. Biotechnol. 33:73-80; Mishra et al. (2011) J. DrugDeliv. 2011 :863734).
  • the liposome and lipoplex formulations can protect the payload from degradation, enhance accumulation and retention at the target site, and facilitate cellular uptake and delivery efficiency through fusion with and/or disruption of the cellular membranes of the target cells.
  • meganuclease proteins are encapsulated within polymeric scaffolds (e.g., PLGA) or complexed using cationic polymers (e.g., PEI, PLL) (Tamboli et al. (2011) Ther Deliv. 2:523-36).
  • Polymeric carriers can be designed to provide tunable drug release rates through control of polymer erosion and drug diffusion, and high drug encapsulation efficiencies can offer protection of the therapeutic payload until intracellular delivery to the desired target cell population.
  • meganuclease proteins are combined with amphiphilic molecules that self-assemble into micelles (Tong et al. (2007) J. Gene Med. 9:956-66).
  • Polymeric micelles may include a micellar shell formed with a hydrophilic polymer (e.g., polyethyleneglycol) that can prevent aggregation, mask charge interactions, and reduce nonspecific interactions.
  • a hydrophilic polymer e.g., polyethyleneglycol
  • meganuclease proteins or DNA/mRNA encoding meganucleases, are formulated into an emulsion or a nanoemulsion (z.e., having an average particle diameter of ⁇ Inm) for administration and/or delivery to the target cell.
  • emulsion refers to, without limitation, any oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil dispersions or droplets, including lipid structures that can form as a result of hydrophobic forces that drive apolar residues (e.g., long hydrocarbon chains) away from water and polar head groups toward water, when a water immiscible phase is mixed with an aqueous phase.
  • lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases.
  • Emulsions are composed of an aqueous phase and a lipophilic phase (typically containing an oil and an organic solvent). Emulsions also frequently contain one or more surfactants. Nanoemulsion formulations are well known, for example, as described in US Pat. Nos. 6,015,832, 6,506,803, 6,635,676, 6,559,189, and 7,767,216, each of which is incorporated herein by reference in its entirety.
  • meganuclease proteins are covalently attached to, or non-covalently associated with, multifunctional polymer conjugates, DNA dendrimers, and polymeric dendrimers (Mastorakos et al. (2015) Nanoscale . 7:3845-56; Cheng et al. (2008) J. Pharm Sci. 97: 123-43).
  • the dendrimer generation can control the payload capacity and size and can provide a high payload capacity.
  • display of multiple surface groups can be leveraged to improve stability, reduce nonspecific interactions, and enhance cellspecific targeting and drug release.
  • polynucleotides comprising a nucleic acid sequence encoding an engineered meganuclease described herein are introduced into a cell using a recombinant virus (z.e., a recombinant viral vector).
  • a recombinant virus z.e., a recombinant viral vector.
  • the recombinant virus comprises any polynucleotide encoding an engineered meganuclease described herein.
  • the recombinant virus can be encoded by a recombinant DNA disclosed herein.
  • recombinant viruses are known in the art and include recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, and recombinant AAVs (reviewed in Vannucci et al. (2013) New Microbiol. 36: 1-22).
  • Recombinant AAVs useful in the disclosure can have any serotype that allows for transduction of the virus into a target cell type and expression of the meganuclease gene in the target cell.
  • cells i.e., host cells
  • any of the recombinant AAVs comprising polynucleotides encoding an engineered meganuclease disclosed herein.
  • recombinant AAVs have a serotype (z.e., a capsid) of AAV1, AAV2, AAV5 AAV6, AAV7, AAV8, AAV9, AAV12, or AAVrh.74. It is known in the art that different AAVs tend to localize to different tissues (Wang et al. (2014) Expert Opin DrugDeliv 11 :345-34.). AAV variants capable of penetrating the blood brain barrier (BBB) and transducing cells of the central and peripheral nervous system are known in the art (Chan, Ken Y et al. “Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.” Nature neuroscience vol.
  • BBB blood brain barrier
  • AAV9 has been shown to transduce astrocytes when delivered intravenously to adult mice and non-human primates, but it also transduces neurons in several regions (Samaranch, Lluis et al.
  • the AAV serotype is AAV9. Additionally, it is known that modifications made to the AAV capsid can improve the ability to penetrate the BBB and transduce cells of the CNS (Nonnenmacher M, Wang W, Child MA, Ren XQ, Huang C, Ren AZ, Tocci J, Chen Q, Bittner K, Tyson K, Pande N, Chung CH, Paul SM, Hou J.
  • the AAV is derived from an AAV9 serotype.
  • Other AAV capsids such as AAVrh8 and AAVrhlO, also share this property (Yang, Bin et al. “Global CNS transduction of adult mice by intravenously delivered rAAVrh.8 and rAAVrh.10 and nonhuman primates by rAAVrh.10 ” Molecular therapy: the journal of the American Society of Gene Therapy vol. 22,7 (2014): 1299-1309; and Zhang, Hongwei et al.
  • the AAV serotype is AAV1. In some embodiments, the AAV serotype is AAV2. In some embodiments, the AAV serotype is AAV5. In some embodiments, the AAV serotype is AAV6. In some embodiments, the AAV serotype is AAV7. In some embodiments, the AAV serotype is AAV8. In some embodiments, the AAV serotype is AAV9. In some embodiments, the AAV serotype is AAV12.
  • the AAV serotype is AAVrh.74.
  • AAVs can also be self-complementary such that they do not require second-strand DNA synthesis in the host cell (McCarty et al. (2001) Gene Ther. 8: 1248-54).
  • Polynucleotides delivered by recombinant AAVs can include left (5') and right (3') inverted terminal repeats as part of the viral genome.
  • the recombinant viruses are injected directly into target tissues. In alternative embodiments, the recombinant viruses are delivered systemically via the circulatory system.
  • the AAV has the ability to penetrate the blood brain barrier (BBB) and transduce cells in the central nervous system (CNS).
  • BBB blood brain barrier
  • CNS central nervous system
  • the AAV is capable of BBB penetration and CNS delivery following intravascular delivery.
  • the AAV should be able to transduce cells within the brain or spinal cord.
  • the AAV can effectively transduce cells in any region of the brain, including, but not limited to, the cortex, thalamus, medulla, pons, hippocampus, olfactory bulb, putamen, striatum, cerebellum, or brainstem.
  • the AAV is able to transduce neuronal cells, such as neurons, and/or glia cells.
  • the AAV comprises a capsid with high tropism for neurons or glia cells.
  • the AAV capsid can comprise a peptide sequence that enhances tropism for CNS tissue.
  • the presence of the peptide sequence can enhance BBB penetration and/or neural cell tropism.
  • a recombinant virus used for meganuclease gene delivery is a selflimiting recombinant virus.
  • a self-limiting virus can have limited persistence time in a cell or organism due to the presence of a recognition sequence for an engineered meganuclease within the viral genome.
  • a self-limiting recombinant virus can be engineered to provide a coding sequence for a promoter, an engineered meganuclease described herein, and a meganuclease recognition site within the ITRs.
  • the self-limiting recombinant virus delivers the meganuclease gene to a cell, tissue, or organism, such that the meganuclease is expressed and able to cut the genome of the cell at an endogenous recognition sequence within the genome.
  • the delivered meganuclease will also find its target site within the self-limiting recombinant viral genome and cut the recombinant viral genome at this target site. Once cut, the 5' and 3' ends of the viral genome will be exposed and degraded by exonucleases, thus killing the virus and ceasing production of the meganuclease.
  • a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein is delivered to a cell by a recombinant virus (e.g. an AAV)
  • the nucleic acid sequence encoding the engineered meganuclease can be operably linked to a promoter.
  • this can be a viral promoter such as endogenous promoters from the recombinant virus (e.g. the LTR of a lentivirus) or the well-known cytomegalovirus- or SV40 virus-early promoters.
  • nucleic acid sequences encoding the engineered meganucleases are operably linked to a promoter that drives gene expression preferentially in the target cells (e.g., neural -tissue cell, such as aneuronal cells, glia cell, or precursor cells thereof).
  • a promoter that drives gene expression preferentially in the target cells
  • the target cells e.g., neural -tissue cell, such as aneuronal cells, glia cell, or precursor cells thereof.
  • neuronal-specific tissue promoters include but are not limited to those neuronalspecific promoters previously described, including enolase (NSE) promoter, calcium/calmodulin- dependent kinase subunit alpha (a) (CaMKII) promoter, or synapsin I (SYN I) promoter.
  • NSE enolase
  • CaMKII calcium/calmodulin- dependent kinase subunit alpha
  • SYN I synapsin I
  • the neural-specific promoter comprises an enolase (NSE) promoter, or a variant or derivative thereof. In some embodiments, the neural-specific promoter comprises a sequence 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an enolase (NSE) promoter sequence. In some embodiments, the neural-specific promoter comprises a calcium/calmodulin-dependent kinase subunit alpha (a) (CaMKII) promoter, or a variant or derivative thereof.
  • a calcium/calmodulin-dependent kinase subunit alpha
  • the neural-specific promoter comprises a sequence 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a calcium/calmodulin-dependent kinase subunit alpha (a) (CaMKII) promoter sequence.
  • the neural-specific promoter comprises a synapsin I promoter, or a variant or derivative thereof.
  • the neural-specific promoter comprises a sequence 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to synapsin I promoter sequence.
  • the neural-specific promoter comprises a glial fibrillary acidic protein (GFAP) promoter, or a variant or derivative thereof. In some embodiments, the neural-specific promoter comprises a sequence 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a glial fibrillary acidic protein (GFAP) promoter sequence.
  • GFAP glial fibrillary acidic protein
  • a single polynucleotide comprises two separate nucleic acid sequences each encoding an engineered meganuclease described herein
  • the meganuclease genes are operably linked to two separate promoters.
  • the two meganuclease genes are operably linked to a single promoter, and in some examples can be separated by an internal-ribosome entry site (IRES) or a 2A peptide sequence (Szymczak & Vignali (2005) Expert Opin Biol Ther. 5:627-38).
  • IRS internal-ribosome entry site
  • 2A peptide sequences can include, for example, a T2A, P2A, E2A, or F2A sequence.
  • the methods include delivering an engineered meganuclease described herein, or a polynucleotide encoding the same, to a cell in combination with a second polynucleotide comprising an exogenous nucleic acid sequence encoding a sequence of interest, wherein the engineered meganuclease is expressed in the cells, recognizes and cleaves a recognition sequence described herein (e.g., SEQ ID NO: 3 or SEQ ID NO: 5) within a C90rf72 gene of the cell, and generates a cleavage site, wherein the exogenous nucleic acid and sequence of interest are inserted into the genome at the cleavage site (e.g., by homologous recombination).
  • a recognition sequence described herein e.g., SEQ ID NO: 3 or SEQ ID NO: 5
  • the polynucleotide can comprise sequences homologous to nucleic acid sequences flanking the meganuclease cleavage site in order to promote homologous recombination of the exogenous nucleic acid and sequence of interest into the genome.
  • Such polynucleotides comprising exogenous nucleic acids can be introduced into a cell and/or delivered to a target cell in a subject by any of the means previously discussed.
  • such polynucleotides comprising exogenous nucleic acid molecules are introduced by way of a recombinant virus (z.e., a viral vector), such as a recombinant lentivirus, recombinant retrovirus, recombinant adenovirus, or a recombinant AAV.
  • Recombinant AAVs useful for introducing a polynucleotide comprising an exogenous nucleic acid molecule can have any serotype (z.e., capsid) that allows for transduction of the virus into the cell and insertion of the exogenous nucleic acid molecule sequence into the cell genome.
  • recombinant AAVs have a serotype of AAV1, AAV2, AAV5 AAV6, AAV7, AAV8, AAV9, AAV12, or AAVrh.74.
  • the AAV serotype is AAV1.
  • the AAV serotype is AAV2.
  • the AAV serotype is AAV5.
  • the AAV serotype is AAV6. In some embodiments, the AAV serotype is AAV7. In some embodiments, the AAV serotype is AAV8. In some embodiments, the AAV serotype is AAV9. In some embodiments, the AAV serotype is AAV12. In some embodiments, the AAV serotype is AAVrh.74. In particular embodiments, the AAV has the ability to penetrate the blood brain barrier (BBB) and transduce cells in the central nervous system (CNS). In particular, the AAV should be able to transduce cells within the brain or spinal cord.
  • BBB blood brain barrier
  • CNS central nervous system
  • the AAV can effectively transduce cells in any region of the brain, including, but not limited to, the cortex, thalamus, medulla, pons, hippocampus, olfactory bulb, putamen, striatum, cerebellum, or brainstem.
  • the AAV is capable of BBB penetration and CNS delivery following intravascular delivery.
  • the AAV is able to transduce neuronal cells, such as neurons, and/or glia cells.
  • the AAV comprises a capsid with high tropism for neurons or glia cells.
  • the AAV capsid can comprise a peptide sequence that enhances tropism for CNS tissue.
  • the presence of the peptide sequence can enhance BBB penetration and/or neural cell tropism.
  • the recombinant AAV can also be self-complementary such that it does not require second-strand DNA synthesis in the host cell.
  • Exogenous nucleic acid molecules introduced using a recombinant AAV can be flanked by a 5' (left) and 3' (right) inverted terminal repeat in the viral genome.
  • an exogenous nucleic acid molecule can be introduced into a cell using a single-stranded DNA template.
  • the single-stranded DNA can comprise the exogenous nucleic acid molecule and, in particular embodiments, can comprise 5' and 3' homology arms to promote insertion of the nucleic acid sequence into the nuclease cleavage site by homologous recombination.
  • the single-stranded DNA can further comprise a 5' AAV ITR sequence 5' upstream of the 5' homology arm, and a 3' AAV ITR sequence 3' downstream of the 3' homology arm.
  • genes encoding a nuclease of the disclosure and/or an exogenous nucleic acid molecule of the disclosure can be introduced into a cell by transfection with a linearized DNA template.
  • a plasmid DNA encoding an engineered nuclease and/or an exogenous nucleic acid molecule can, for example, be digested by one or more restriction enzymes such that the circular plasmid DNA is linearized prior to transfection into the cell.
  • an exogenous nucleic acid of the disclosure When delivered to a cell, an exogenous nucleic acid of the disclosure can be operably linked to any promoter suitable for expression of the encoded polypeptide in the cell, including those mammalian promoters and inducible promoters previously discussed.
  • An exogenous nucleic acid of the disclosure can also be operably linked to a synthetic promoter.
  • Synthetic promoters can include, without limitation, the JeT promoter (WO 2002/012514).
  • a nucleic acid sequence encoding an engineered meganuclease as disclosed herein can be operably linked to a neural-specific promoter discussed herein.
  • the target tissue(s) or target cell(s) include, without limitation, neural tissue and neural cells, such as neurons, neuron progenitor cells, cortex cells, upper motor neurons, spinal cells, lower motor neurons, motor neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, or oligodendrocytes.
  • the target cell is a neural progenitor cell that gives rise to any glial or neuronal cell type that populates the central nervous system (CNS).
  • CNS central nervous system
  • Such neural progenitor cells have been described in the art and can either be present in a subject or derived from another stem cell population such as an induced pluripotent stem cell or an embryonic stem cell (Zhao, Xinyu, and Darcie L Moore. “Neural stem cells: developmental mechanisms and disease modeling.” Cell and tissue research vol. 371,1 (2018): 1-6.).
  • engineered meganucleases described herein, or polynucleotides encoding the same are delivered to a cell in vitro. In some embodiments, engineered meganucleases described herein, or polynucleotides encoding the same, are delivered to a cell in a subject in vivo. As discussed herein, meganucleases of the disclosure can be delivered as purified protein or as a polynucleotide (e.g., RNA or DNA) comprising a nucleic acid sequence encoding the meganuclease.
  • meganuclease proteins, or polynucleotides encoding meganucleases are supplied to target cells (e.g., a neuron cell, glial cell, or neuronal progenitor cell) via injection directly to the target tissue.
  • target cells e.g., a neuron cell, glial cell, or neuronal progenitor cell
  • meganuclease proteins, or polynucleotides encoding meganucleases can be delivered systemically via the circulatory system.
  • compositions described herein such as the engineered meganucleases described herein, polynucleotides encoding the same, recombinant viruses comprising such polynucleotides, or lipid nanoparticles comprising such polynucleotides, can be administered via any suitable route of administration known in the art.
  • routes of administration can include, for example, intravenous, intramuscular, intraperitoneal, subcutaneous, intrahepatic, transmucosal, transdermal, intraarterial, and sublingual.
  • the engineered meganuclease proteins, polynucleotides encoding the same, recombinant viruses comprising such polynucleotides, or lipid nanoparticles comprising such polynucleotides are supplied to target cells (e.g., neuronal cells or neuronal precursor cells) via injection directly to the target tissue (e.g., neuronal tissue).
  • target cells e.g., neuronal cells or neuronal precursor cells
  • target tissue e.g., neuronal tissue.
  • Other suitable routes of administration can be readily determined by the treating physician as necessary.
  • a therapeutically effective amount of an engineered nuclease described herein, or a polynucleotide encoding the same is administered to a subject in need thereof for the treatment of a disease.
  • the dosage or dosing frequency of the engineered meganuclease, or the polynucleotide encoding the same may be adjusted over the course of the treatment, based on the judgment of the administering physician.
  • Appropriate doses will depend, among other factors, on the specifics of any AAV chosen (e.g., serotype, etc.), any lipid nanoparticle chosen, on the route of administration, on the subject being treated (i.e., age, weight, sex, and general condition of the subject), and the mode of administration.
  • the appropriate dosage may vary from patient to patient.
  • An appropriate effective amount can be readily determined by one of skill in the art or treating physician.
  • Dosage treatment may be a single dose schedule or, if multiple doses are required, a multiple dose schedule.
  • the subject may be administered as many doses as appropriate.
  • One of skill in the art can readily determine an appropriate number of doses.
  • the dosage may need to be adjusted to take into consideration an alternative route of administration or balance the therapeutic benefit against any side effects.
  • the methods further include administration of a polynucleotide comprising a nucleic acid sequence encoding a secretion-impaired hepatotoxin, or encoding tPA, which stimulates hepatocyte regeneration without acting as a hepatotoxin.
  • a subject is administered a pharmaceutical composition
  • a pharmaceutical composition comprising a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein, wherein the encoding nucleic acid sequence is administered at a dose of about IxlO 10 gc/kg to about IxlO 14 gc/kg (e.g., about IxlO 10 gc/kg, about IxlO 11 gc/kg, about IxlO 12 gc/kg, about IxlO 13 gc/kg, or about IxlO 14 gc/kg).
  • a subject is administered a pharmaceutical composition
  • a pharmaceutical composition comprising a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein, wherein the encoding nucleic acid sequence is administered at a dose of about IxlO 10 gc/kg, about IxlO 11 gc/kg, about IxlO 12 gc/kg, about IxlO 13 gc/kg, or about IxlO 14 gc/kg.
  • a subject is administered a pharmaceutical composition
  • a pharmaceutical composition comprising a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein, wherein the encoding nucleic acid sequence is administered at a dose of about IxlO 10 gc/kg to about IxlO 11 gc/kg, about IxlO 11 gc/kg to about IxlO 12 gc/kg, about IxlO 12 gc/kg to about IxlO 13 gc/kg, or about IxlO 13 gc/kg to about IxlO 14 gc/kg.
  • these doses can relate to the administration of a single polynucleotide comprising a single nucleic acid sequence encoding a single engineered meganuclease described herein or, alternatively, can relate to a single polynucleotide comprising a first nucleic acid sequence encoding a first engineered meganuclease described herein and a second nucleic acid sequence encoding a second engineered meganuclease described herein, wherein each of the two encoding nucleic acid sequences is administered at the indicated dose.
  • a subject is administered a lipid nanoparticle formulation comprising an mRNA comprising a nucleic acid sequence encoding an engineered meganuclease described herein, wherein the dose of the mRNA is about 0.1 mg/kg to about 3 mg/kg.
  • a subject is administered a lipid nanoparticle formulation comprising an mRNA comprising a nucleic acid sequence encoding an engineered meganuclease described herein, wherein the dose of the mRNA is about 0.1 mg/kg, about 0.25 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, or about 3.0 mg/kg.
  • a subject is administered a lipid nanoparticle formulation comprising an mRNA comprising a nucleic acid sequence encoding an engineered meganuclease described herein, wherein the dose of the mRNA is about 0.1 mg/kg to about 0.25 mg/kg, about 0.25 mg/kg to about 0.5 mg/kg, about 0.5 mg/kg to about 0.75 mg/kg, about 0.75 mg/kg to about 1.0 mg/kg, about 1.0 mg/kg to about 1.5 mg/kg, about 1.5 mg/kg to about 2.0 mg/kg, about 2.0 mg/kg to about 2.5 mg/kg, or about 2.5 mg/kg to about 3.0 mg/kg.
  • BBB blood-brain barrier
  • CNS central nervous system
  • BBB blood-brain barrier
  • Several options for delivery across the BBB have been identified as efficient approaches to bypass the BBB, including the use of drugs encapsulated in lipid nanoparticles, as they allow the passage of drugs through this barrier, improving brain bioavailability (Correia, A C et al. “Lipid nanoparticles strategies to modify pharmacokinetics of central nervous system targeting drugs: Crossing or circumventing the bloodbrain barrier (BBB) to manage neurological disorders.” Advanced drug delivery reviews vol. 189 (2022): 114485).
  • any lipid nanoparticle that can effectively deliver the meganucleases, or a polynucleotides encoding the same, disclosed herein would be particularly useful for delivery of the meganucleases, or polynucleotides encoding the same, to the CNS tissue (e.g., brain and/or spinal cord).
  • CNS tissue e.g., brain and/or spinal cord.
  • Multiple approaches have been undertaken to optimize LNPs for improved brain delivery and accumulation in brain tissue (Khare, Purva et al. “Lipid nanoparticle-mediated drug delivery to the brain.” Advanced drug delivery reviews vol. 197 (2023): 114861.).
  • lipid nanoparticles that particularly effective at delivery to the CNS tissue are known in the art, and are described, for example, in WO 2015/061461, the entirety of which is incorporated herein by reference as it pertains to delivery to the CNS tissue. Any method of CNS tissue delivery that delivers the meganucleases, or polynucleotides encoding the same, disclosed herein across the blood brain barrier and/or to the CNS tissue, such as the brain or spinal cord and cells located within the CNS tissue is envisaged within the scope of the present disclosure.
  • the lipid nanoparticle used can penetrate the blood brain barrier (BBB) and entering the central nervous system (CNS) tissue, such as the brain and/or spinal cord.
  • BBB blood brain barrier
  • CNS central nervous system
  • the lipid nanoparticle delivers engineered meganucleases described herein, or polynucleotides encoding the same, are delivered to a cell in the central nervous system. In some embodiments, the engineered meganucleases, or polynucleotides encoding the same, are delivered to a neuron cell or a neuroglia cell in the CNS. In some embodiments, the cell is in the brain. In some embodiments, the cell is in the spinal cord.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an engineered meganuclease described herein, or a pharmaceutically acceptable carrier and a polynucleotide described herein that comprises a nucleic acid sequence encoding an engineered meganuclease described herein.
  • Such polynucleotides can be, for example, mRNA or DNA as described herein.
  • the polynucleotide in the pharmaceutical composition can be comprised by a lipid nanoparticle or can be comprised by a recombinant virus (e.g., a recombinant AAV).
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a genetically modified cell of the disclosure, which can be delivered to a target tissue where the cell expresses the engineered meganuclease as disclosed herein.
  • Such pharmaceutical compositions are formulated, for example, for systemic administration, or administration to target tissues.
  • the pharmaceutical compositions can be useful for preventing or treating frontotemporal dementia (FTD) and/or amyotrophic lateral sclerosis (ALS) and/or reducing the symptoms associated with FTD and/or ALS in a subject.
  • FTD frontotemporal dementia
  • ALS amyotrophic lateral sclerosis
  • Such pharmaceutical compositions can be prepared in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (21st ed., Philadelphia, Lippincott, Williams & Wilkins, 2005).
  • engineered meganucleases described herein polynucleotides encoding the same, or cells expressing the same, are typically admixed with a pharmaceutically acceptable carrier and the resulting composition is administered to a subject.
  • the carrier must be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject.
  • the carrier can be a solid or a liquid, or both, and can be formulated with the compound as a unit-dose formulation.
  • compositions of the disclosure can further comprise one or more additional agents or biological molecules useful in the treatment of a disease in the subject.
  • additional agent(s) and/or biological molecule(s) can be co-administered as a separate composition.
  • compositions described herein can include a therapeutically effective amount of any engineered meganuclease disclosed herein, or any polynucleotide described herein encoding any engineered meganuclease described herein.
  • the pharmaceutical composition can include polynucleotides described herein at any of the doses (e.g., gc/kg of an encoding nucleic acid sequence or mg/kg of mRNA) described herein.
  • the pharmaceutical composition can comprise one or more recombinant viruses (e.g., recombinant AAVs) described herein that comprise one or more polynucleotides described herein (i.e., packaged within the viral genome).
  • the pharmaceutical composition comprises two or more recombinant viruses (e.g., recombinant AAVs) described herein, each comprising a polynucleotide comprising a nucleic acid sequence encoding a different engineered meganuclease described herein.
  • a first recombinant virus may comprise a first polynucleotide comprising a first nucleic acid sequence encoding a first engineered meganuclease described herein having specificity for the CNR 1-2 recognition sequence
  • a second recombinant virus e.g., recombinant AAV
  • a second polynucleotide comprising a second polynucleotide comprising a second nucleic acid sequence encoding a second engineered meganuclease described herein having specificity for the CNR 21-22 recognition sequence.
  • a pair of engineered meganucleases in the same cell e.g., a neural-tissue cell or CNS cell, such as a neuron or neuroglia cell, or precursor thereof
  • a pair of engineered meganucleases in the same cell would allow for the excision of the hexanucleotide repeat region and exon lb from the C90rf72 gene according to the disclosure.
  • the pharmaceutical composition can comprise a recombinant virus (e.g., recombinant AAV) described herein that comprises a polynucleotide (i.e., packaged within the viral genome) that comprises two nucleic acid sequences encoding two separate engineered meganucleases described herein.
  • a recombinant virus e.g., recombinant AAV
  • polynucleotide i.e., packaged within the viral genome
  • the recombinant virus (e.g., recombinant AAV) can comprise a polynucleotide comprising a first nucleic acid sequence encoding a first engineered meganuclease described herein having specificity for the CNR 1-2 recognition sequence, and a second nucleic acid sequence encoding a second engineered meganuclease described herein having specificity for the CNR 21-22 recognition sequence.
  • the expression of such a pair of engineered meganucleases would allow for the excision of the hexanucleotide repeat region and exon Ibfrom the C90rf72 gene according to the disclosure.
  • the pharmaceutical composition can comprise one or more polynucleotides (e.g., mRNAs) described herein encapsulated within lipid nanoparticles.
  • lipid nanoparticles can comprise two or more polynucleotides (e.g., mRNAs) described herein, each comprising a nucleic acid sequence encoding a different engineered meganuclease described herein.
  • a first polynucleotide (e.g., mRNA) in the lipid nanoparticle may encode a first engineered meganuclease described herein having specificity for the CNR 1-2 recognition sequence
  • a second polynucleotide (e.g., mRNA) in the lipid nanoparticle may encode a second engineered meganuclease described herein having specificity for the CNR 21-22 recognition sequence.
  • a pair of engineered meganucleases in the same cell e.g., a neural -tissue cell or CNS cell, such as a neuron or neuroglia cell, or precursor thereof
  • a pair of engineered meganucleases in the same cell would allow for the excision of the hexanucleotide repeat region and exon lb from the C90rf72 gene according to the disclosure.
  • the pharmaceutical composition can comprise two separate populations of lipid nanoparticles, each comprising a different polynucleotide (e.g., mRNA) described herein, wherein a first population of lipid nanoparticles comprise a first polynucleotide (e.g., mRNA) described herein encoding a first engineered meganuclease having specificity for the CNR 1-2 recognition sequence, and the second population of lipid nanoparticles comprise a second polynucleotide (e.g., mRNA) described herein encoding a second engineered meganuclease having specificity for the CNR 21-22 recognition sequence.
  • a first population of lipid nanoparticles comprise a first polynucleotide (e.g., mRNA) described herein encoding a first engineered meganuclease having specificity for the CNR 1-2 recognition sequence
  • the second population of lipid nanoparticles comprise a second polynucleot
  • lipid nanoparticles can comprise one polynucleotide (e.g., mRNA) described herein that comprises two nucleic acid sequences encoding two separate engineered meganucleases described herein.
  • the lipid nanoparticle can comprise a polynucleotide (e.g., mRNA) comprising a first nucleic acid sequence encoding a first engineered meganuclease described herein having specificity for the CNR 1-2 recognition sequence, and a second nucleic acid sequence encoding a second engineered meganuclease described herein having specificity for the CNR 21-22 recognition sequence.
  • a pair of engineered meganucleases in the same cell e.g., a CNS cell, such as a neuron cell or neuroglia cell
  • a CNS cell such as a neuron cell or neuroglia cell
  • lipid nanoparticles contemplated for use in the disclosure comprise at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid.
  • lipid nanoparticles can comprise from about 50 mol % to about 85 mol % of a cationic lipid, from about 13 mol % to about 49.5 mol % of a non-cationic lipid, and from about 0.5 mol % to about 10 mol % of a lipid conjugate, and are produced in such a manner as to have a non- lamellar (i.e., non-bilayer) morphology.
  • lipid nanoparticles can comprise from about 40 mol % to about 85 mol % of a cationic lipid, from about 13 mol % to about 49.5 mol % of a non-cationic lipid, and from about 0.5 mol % to about 10 mol % of a lipid conjugate and are produced in such a manner as to have a non-lamellar (i.e., non-bilayer) morphology.
  • Cationic lipids can include, for example, one or more of the following: palmitoyi-oleoyl- nor-arginine (PONA), MPDACA, GUADACA, ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate) (MC3), LenMC3, CP-LenMC3, y-LenMC3, CP-y-LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Amide, Pan-MC3, Pan-MC4 and Pan MC5, 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), 2, 2-dilinoleyl-4-(2-dimethylamin
  • the cationic lipid can also be DLinDMA, DLin-K-C2-DMA (“XTC2”), MC3, LenMC3, CP-LenMC3, y-LenMC3, CP-y-LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Amide, Pan-MC3, Pan-MC4, Pan MC5, or mixtures thereof.
  • XTC2 DLin-K-C2-DMA
  • the cationic lipid may comprise from about 50 mol % to about 90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, or from about 50 mol % to about 60 mol % of the total lipid present in the particle.
  • the cationic lipid may comprise from about 40 mol % to about 90 mol %, from about 40 mol % to about 85 mol %, from about 40 mol % to about 80 mol %, from about 40 mol % to about 75 mol %, from about 40 mol % to about 70 mol %, from about 40 mol % to about 65 mol %, or from about 40 mol % to about 60 mol % of the total lipid present in the particle.
  • the non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids.
  • the non-cationic lipid comprises one of the following neutral lipid components: (1) cholesterol or a derivative thereof; (2) a phospholipid; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.
  • Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'- hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.
  • the phospholipid may be a neutral lipid including, but not limited to, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidy
  • the non-cationic lipid may comprise from about 10 mol % to about 60 mol %, from about 15 mol % to about 60 mol %, from about 20 mol % to about 60 mol %, from about 25 mol % to about 60 mol %, from about 30 mol % to about 60 mol %, from about 10 mol % to about 55 mol %, from about 15 mol % to about 55 mol %, from about 20 mol % to about 55 mol %, from about 25 mol % to about 55 mol %, from about 30 mol % to about 55 mol %, from about 13 mol % to about 50 mol %, from about 15 mol % to about 50 mol % or from about 20 mol % to about 50 mol % of the total lipid present in the particle.
  • the non-cationic lipid is a mixture of a phospholipid and
  • the conjugated lipid that inhibits aggregation of particles may comprise, e.g., one or more of the following: a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, a cationic-polymer-lipid conjugates (CPLs), or mixtures thereof.
  • the nucleic acid-lipid particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate.
  • the PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL.
  • the conjugated lipid that inhibits aggregation of particles may comprise a PEG-lipid including, e.g., a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG- phospholipid, a PEG-ceramide (Cer), or mixtures thereof.
  • the PEG-DAA conjugate may be PEG- di lauryl oxy propyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (Cl 8), or mixtures thereof.
  • Additional PEG-lipid conjugates suitable for use in the disclosure include, but are not limited to, mPEG2000-l,2-di-0-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG).
  • PEG-C-DOMG mPEG2000-l,2-di-0-alkyl-sn3-carbomoylglyceride
  • the synthesis of PEG-C-DOMG is described in WO 2009/086558.
  • Yet additional PEG-lipid conjugates suitable for use in the disclosure include, without limitation, l-[8'-(l,2-dimyristoyl-3-propanoxy)-carboxamido- 3',6'-dioxaoctanyl]carbamoyl-co-methyl-poly(ethylene glycol) (2KPEG-DMG).
  • 2KPEG-DMG The synthesis of 2KPEG-DMG is described in U.S. Pat. No. 7,404
  • the conjugated lipid that inhibits aggregation of particles may comprise from about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % to about 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to about 1.7 mol %, from about 1 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol % to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol %, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % (or any fraction thereof
  • the PEG moiety has an average molecular weight of about 2,000 Daltons.
  • the conjugated lipid that inhibits aggregation of particles may comprise from about 5.0 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the PEG moiety has an average molecular weight of about 750 Daltons.
  • the composition may comprise amphoteric liposomes, which contain at least one positive and at least one negative charge carrier, which differs from the positive one, the isoelectric point of the liposomes being between 4 and 8. This objective is accomplished owing to the fact that liposomes are prepared with a pH-dependent, changing charge.
  • Liposomal structures with the desired properties are formed, for example, when the amount of membrane-forming or membrane-based cationic charge carriers exceeds that of the anionic charge carriers at a low pH and the ratio is reversed at a higher pH. This is always the case when the ionizable components have a pKa value between 4 and 9. As the pH of the medium drops, all cationic charge carriers are charged more and all anionic charge carriers lose their charge.
  • weakly cationic compounds include, without limitation: His-Chol (histaminyl- cholesterol hemi succinate), Mo-Chol (morpholine-N-ethylamino-cholesterol hemi succinate), or histidinyl-PE.
  • neutral compounds include, without limitation: cholesterol, ceramides, phosphatidyl cholines, phosphatidyl ethanolamines, tetraether lipids, or diacyl glycerols.
  • Anionic compounds useful for amphoteric liposomes include those non-cationic compounds previously described herein.
  • examples of weakly anionic compounds can include: CHEMS (cholesterol hemi succinate), alkyl carboxylic acids with 8 to 25 carbon atoms, or diacyl glycerol hemisuccinate.
  • Additional weakly anionic compounds can include the amides of aspartic acid, or glutamic acid and PE as well as PS and its amides with glycine, alanine, glutamine, asparagine, serine, cysteine, threonine, tyrosine, glutamic acid, aspartic acid or other amino acids or aminodicarboxylic acids.
  • the esters of hydroxycarboxylic acids or hydroxy dicarboxylic acids and PS are also weakly anionic compounds.
  • amphoteric liposomes may contain a conjugated lipid, such as those described herein above.
  • conjugated lipids include, without limitation, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3 -amines.
  • PEG-modified diacylglycerols and dialkylglycerols are particularly examples.
  • the conjugated lipid that inhibits aggregation of particles may comprise from about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % to about 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to about 1.7 mol %, from about 1 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol % to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol %, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % (or any fraction thereof
  • the PEG moiety has an average molecular weight of about 2,000 Daltons.
  • the conjugated lipid that inhibits aggregation of particles may comprise from about 5.0 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the PEG moiety has an average molecular weight of about 750 Daltons.
  • the remaining balance of the amphoteric liposome can comprise a mixture of cationic compounds and anionic compounds formulated at various ratios.
  • the ratio of cationic to anionic lipid may selected in order to achieve the desired properties of nucleic acid encapsulation, zeta potential, pKa, or other physicochemical property that is at least in part dependent on the presence of charged lipid components.
  • the lipid nanoparticles have a composition, which specifically enhances delivery and uptake in the liver, and specifically within hepatocytes.
  • compositions of the disclosure can further comprise one or more additional agents useful in the treatment of FTD and/or ALS in the subject.
  • the present disclosure also provides engineered meganucleases described herein, or polynucleotides described herein encoding the same, or cells described herein expressing engineered meganucleases described herein for use as a medicament.
  • the present disclosure further provides the use of engineered meganucleases described herein, or polynucleotides disclosed herein encoding the same, or cells described herein expressing engineered meganucleases described herein in the manufacture of a medicament for treating FTD and/or ALS, for increasing levels of a modified C90rf72 gene or RNA (i.e., lacking the non-coding sequences of the hexanucleotide repeat region and exon lb of the C90rf72 gene), or reducing the symptoms associated with FTD and/or ALS.
  • a modified C90rf72 gene or RNA i.e., lacking the non-coding sequences of the hexanucleotide repeat region and exon lb of the C

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Abstract

La présente divulgation concerne des méganucléases modifiées qui lient et clivent des séquences de reconnaissance dans un gène C9Orf72. La présente divulgation concerne également les procédés d'utilisation de ces méganucléases modifiées pour fabriquer des cellules génétiquement modifiées. En outre, la divulgation concerne des compositions pharmaceutiques comprenant des protéines de méganucléases modifiées ou des polynucléotides codant des méganucléases modifiées de la divulgation, ainsi que l'utilisation de telles compositions pour la modification d'un gène C9Orf72 chez un sujet ou pour le traitement de la sclérose latérale amyotrophique (SLA) et/ou de la démence fronto-temporale (DFT).
PCT/IB2025/057836 2024-08-01 2025-07-31 Méganucléases modifiées ayant une spécificité pour les séquences de reconnaissance dans le gène c9orf72 Pending WO2026028165A1 (fr)

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