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WO2025213138A1 - Crispr/rna-guided nuclease related methods and compositions for treating primary open angle glaucoma - Google Patents

Crispr/rna-guided nuclease related methods and compositions for treating primary open angle glaucoma

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Publication number
WO2025213138A1
WO2025213138A1 PCT/US2025/023321 US2025023321W WO2025213138A1 WO 2025213138 A1 WO2025213138 A1 WO 2025213138A1 US 2025023321 W US2025023321 W US 2025023321W WO 2025213138 A1 WO2025213138 A1 WO 2025213138A1
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WO
WIPO (PCT)
Prior art keywords
molecule
nucleic acid
rna
casl2a
grna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/023321
Other languages
French (fr)
Inventor
Paul Joseph WRIGHTON
Heather Dawn MACLEOD
Georgia Giannoukos
Meng Wu
Eugenio Marco Rubio
Chrysa M. LATRICK
Mark Thomas JONES
Xiaoqi Sun
John Anthony Zuris
Christopher Wilson
Steve Bottega
Mariacarmela ALLOCCA
Shrutokirti DE
Jiwon YOM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Editas Medicine Inc
Original Assignee
Editas Medicine Inc
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Publication date
Application filed by Editas Medicine Inc filed Critical Editas Medicine Inc
Publication of WO2025213138A1 publication Critical patent/WO2025213138A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • Glaucoma is the second leading cause of blindness in the world.
  • POAG is the leading cause of glaucoma, representing more than 50% of glaucoma in the United States (Quigley 1997). POAG affects over 3 million subjects in the United States (Glaucoma Research Foundation: www.glaucoma.org; accessed December 14, 2021). Approximately 1% of subjects ages 40-89 have POAG.
  • Methods and compositions discussed herein are designed to allow for the correction of disorders of the eye, e.g., disorders that affect trabecular meshwork cells, photoreceptor cells and any other cells in the eye, including those of the iris, ciliary body, optic nerve or aqueous humor.
  • disorders of the eye e.g., disorders that affect trabecular meshwork cells, photoreceptor cells and any other cells in the eye, including those of the iris, ciliary body, optic nerve or aqueous humor.
  • methods and compositions discussed herein provide for treating or delaying the onset or progression of POAG.
  • POAG is a common form of glaucoma, characterized by damage to the optic nerve resulting in loss of vision. Degeneration of the trabecular meshwork leads to obstruction of the normal ability of aqueous humor to leave the eye without closure of the space (e.g., the '‘angle”) between the iris and cornea. This obstruction leads to increased IOP, which can result in progressive visual loss and blindness if not treated appropriately and in a timely fashion.
  • POAG is a progressive ophthalmologic disorder characterized by increased IOP.
  • compositions disclosed herein are broadly applicable to any mutation, e.g., a point mutation or a deletion, in the MYOC gene that gives rise to POAG.
  • methods and compositions discussed herein may be used to alter the MYOC gene to treat or prevent POAG by targeting the MYOC gene, e.g., the 5’ untranslated region (5’ UTR), exon 1, the exon 1/intron 1 border, intron 1, the intron 1/exon 2 border, exon 2, the exon 2/intron 2 border, intron 2, the intron 2/exon 3 border, exon 3, the exon 3/intron 3 border, the 3’ UTR or any combination thereof.
  • the 5’ untranslated region 5’ UTR
  • the methods and compositions discussed herein may be used to alter the MYOC gene to treat or prevent POAG by targeting the coding sequence of the MYOC gene.
  • the gene e.g., the coding sequence of the MYOC gene
  • is targeted to knockout the gene e.g., to eliminate expression of the gene, e.g., to knockout both alleles of the MYOC gene, e.g., by induction of an alteration comprising a deletion or mutation in the MYOC gene.
  • the method provides an alteration that comprises an insertion or deletion.
  • a targeted knockout approach is mediated by non-homologous end joining (NHEJ) using a CRISPR/RNA-guided nuclease system comprising an RNA-guided nuclease (e.g., Casl2a molecule).
  • NHEJ non-homologous end joining
  • the alteration may comprise an indel.
  • the alteration may comprise a frameshift indel that results in decreased expression of the MYOC gene.
  • a coding region, e.g., an early coding region, of the MYOC gene is targeted to knockout the MYOC gene.
  • targeting affects both alleles of the MYOC gene.
  • a targeted knockout approach reduces or eliminates expression of functional MYOC gene product.
  • the method provides an alteration that comprises an insertion or deletion.
  • the methods and compositions discussed herein may be used to alter the MYOC gene to treat or prevent POAG by targeting a non-coding sequence of the MYOC gene, e.g., promoter, an enhancer, an intron, 5’UTR, 3’UTR, and/or poly adenylation signal.
  • the gene e.g., the non-coding sequence of the MYOC gene
  • the method provides an alteration that comprises an insertion or deletion.
  • the 5’ UTR of exon 1, exon 1, the exon 1/intron 1 border, intron 1, the intron 1/exon 2 border, exon 2, the exon 2/intron 2 border, intron 2, the intron 2/exon 3 border, exon 3, the exon 3/intron 3 border, the 3’ UTR or any combination thereof of the MYOC gene is targeted for alteration of the MYOC gene.
  • the 5’ UTR region is targeted for alteration of the MYOC gene.
  • exon 1 of the MYOC gene is targeted for alteration of the MYOC gene.
  • the exon 1/intron 1 border of the MYOC gene is targeted for alteration of the MYOC gene.
  • the targeting domains of the first and second gRNA molecules are configured such that a cleavage event, e.g., a double strand or single strand break, is positioned, independently for each of the gRNA molecules, within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of the target position.
  • the breaks e.g., double strand or single strand breaks, are positioned on both sides of a nucleotide of a MYOC target position in the MYOC gene.
  • the breaks, e.g., double strand or single strand breaks are positioned on one side, e.g., upstream or downstream, of a nucleotide of a MYOC target position in the MYOC gene.
  • a single strand break is accompanied by an additional single strand break, positioned by a second gRNA molecule, as discussed below.
  • the targeting domains are configured such that a cleavage event, e.g., the two single strand breaks, are positioned within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of a MYOC target position.
  • the first and second gRNA molecules are configured such that, when guiding an RNA-guided nuclease nickase molecule (e.g., a Casl2a nickase), a single strand break will be accompanied by an additional single strand break, positioned by a second gRNA molecule, sufficiently close to one another to result in alteration of a MYOC target position in the MYOC gene.
  • an RNA-guided nuclease nickase molecule e.g., a Casl2a nickase
  • a double strand break can be accompanied by an additional double strand break, positioned by a second gRNA molecule, as is discussed below.
  • the targeting domain of a first gRNA molecule is configured such that a double strand break is positioned upstream of a MYOC target position in the MYOC gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of the target position; and the targeting domain of a second gRNA molecule is configured such that a double strand break is positioned downstream of a MYOC target position in the MFOC gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of the target position.
  • a first and second single strand breaks can be accompanied by two additional single strand breaks positioned by a third gRNA molecule and a fourth gRNA molecule.
  • the targeting domain of a first and second gRNA molecule are configured such that two single strand breaks are positioned upstream of a MYOC target position in the MYOC gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of the target position; and the targeting domains of a third and fourth gRNA molecule are configured such that two single strand breaks are positioned downstream of a MYOC target position in the MYOC gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of the target position.
  • multiple gRNAs when multiple gRNAs are used to generate (1) two single stranded breaks in close proximity, (2) two double stranded breaks, e.g., flanking a MYOC target position, e.g., a mutation (e.g., to remove a piece of DNA, e.g., a insertion mutation), or to create more than one indel in the MYOC gene, (3) one double stranded break and two paired nicks flanking a MYOC target position, e.g., a mutation (e.g., to remove a piece of DNA, e.g., a insertion mutation) or (4) four single stranded breaks, two on each side of a mutation, that they are targeting the same MYOC target position. It is further contemplated herein that multiple gRNAs may be used to target more than one MYOC target position in the same gene.
  • the targeting domain of the first gRNA molecule and the targeting domain of the second gRNA molecules are complementary to opposite strands of the target nucleic acid molecule.
  • the gRNA molecule and the second gRNA molecule are configured such that the PAMs are oriented outward.
  • the targeting domain of a gRNA molecule is configured to avoid unwanted target chromosome elements, such as repeat elements, e.g., Alu repeats, in the target domain.
  • the gRNA molecule may be a first, second, third and/or fourth gRNA molecule, as described herein.
  • the targeting domain of a gRNA molecule comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence set forth in Table 10.
  • the targeting domain of a gRNA molecule comprises a sequence that is the same as, or differs by no more than 3 nucleotides from, a sequence of a targeting domain selected from the group consisting of a Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8 targeting domain (Table 10).
  • the targeting domain of the gRNA molecule is configured to target an RNA-guided nuclease (e.g., Casl2a molecule), sufficiently close to a MYOC target position to reduce, decrease or repress expression of the MYOC gene.
  • an RNA-guided nuclease e.g., Casl2a molecule
  • the targeting domain which is complementary with a target domain from the MYOC gene, is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides or more in length.
  • the targeting domain is 16 nucleotides in length.
  • the targeting domain is 17 nucleotides in length.
  • the targeting domain is 18 nucleotides in length.
  • the targeting domain is 19 nucleotides in length.
  • the targeting domain is 20 nucleotides in length.
  • the targeting domain is 21 nucleotides in length.
  • the targeting domain is 22 nucleotides in length.
  • the targeting domain is 23 nucleotides in length.
  • the targeting domain is 24 nucleotides in length.
  • the targeting domain is 25 nucleotides in length.
  • the targeting domain is 26 nucleotides in length.
  • the targeting domain comprises 16 nucleotides. In an embodiment, the targeting domain comprises 17 nucleotides. In an embodiment, the targeting domain comprises 18 nucleotides. In an embodiment, the targeting domain comprises 19 nucleotides. In an embodiment, the targeting domain comprises 20 nucleotides. In an embodiment, the targeting domain comprises 21 nucleotides. In an embodiment, the targeting domain comprises 22 nucleotides. In an embodiment, the targeting domain comprises 23 nucleotides. In an embodiment, the targeting domain comprises 24 nucleotides. In an embodiment, the targeting domain comprises 25 nucleotides. In an embodiment, the targeting domain comprises 26 nucleotides.
  • a cleavage event e.g., a double strand or single strand break
  • an RNA-guided nuclease molecule e.g., Casl2a molecule
  • the RNA-guided nuclease molecule e.g., Casl2a molecule
  • the RNA-guided nuclease molecule may form a double strand break in a target nucleic acid or a single strand break in a target nucleic acid (e.g., a nickase molecule).
  • the RNA-guided nuclease molecule e.g., Casl2a molecule
  • a single strand break may be formed in the strand of the target nucleic acid to which the targeting domain of the gRNA molecule is complementary. In another embodiment, a single strand break may be formed in the strand of the target nucleic acid other than the strand to which the targeting domain of the gRNA molecule is complementary.
  • the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain configured to, in combination with a corresponding RNA-guided nuclease, provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a MYOC target position in the MYOC gene to allow alteration, e.g., alteration associated with NHEI, of a MYOC target position in the MYOC gene.
  • a gRNA molecule e.g., a first gRNA molecule
  • a targeting domain configured to, in combination with a corresponding RNA-guided nuclease, provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a MYOC target position in the MYOC gene to allow alteration, e.g., alteration associated with NHEI, of a M
  • the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain configured to target an RNA-guided nuclease molecule (e.g., Casl2a molecule), sufficiently close to a MYOC target position to reduce, decrease or repress expression of the MYOC gene.
  • a gRNA molecule e.g., a first gRNA molecule
  • a targeting domain configured to target an RNA-guided nuclease molecule (e.g., Casl2a molecule)
  • the nucleic acid may encode a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain comprising 16 nucleotides or more in length.
  • the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 16 nucleotides in length.
  • the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 17 nucleotides in length.
  • the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 18 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 19 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 20 nucleotides in length.
  • the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 21 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 22 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 23 nucleotides in length.
  • the targeting domain comprises 16 nucleotides. In an embodiment, the targeting domain comprises 17 nucleotides. In an embodiment, the targeting domain comprises 18 nucleotides. In an embodiment, the targeting domain comprises 19 nucleotides. In an embodiment, the targeting domain comprises 20 nucleotides. In an embodiment, the targeting domain comprises 21 nucleotides. In an embodiment, the targeting domain comprises 22 nucleotides. In an embodiment, the targeting domain comprises 23 nucleotides. In an embodiment, the targeting domain comprises 24 nucleotides. In an embodiment, the targeting domain comprises 25 nucleotides. In an embodiment, the targeting domain comprises 26 nucleotides.
  • a nucleic acid comprises (a) a sequence that encodes a gRNA molecule e.g., the first gRNA molecule, comprising a targeting domain that is complementary with a target domain in the MYOC gene as disclosed herein, and further comprising (b) a sequence that encodes an RNA-guided nuclease molecule (e.g., Casl2a molecule).
  • a gRNA molecule e.g., the first gRNA molecule
  • a targeting domain that is complementary with a target domain in the MYOC gene as disclosed herein
  • an RNA-guided nuclease molecule e.g., Casl2a molecule
  • the RNA-guided nuclease molecule may form a double strand break in a target nucleic acid or a single strand break in a target nucleic acid (e.g., a nickase molecule).
  • a single strand break is formed in the strand of the target nucleic acid to which the targeting domain of the gRNA molecule is complementary. In another embodiment, a single strand break is formed in the strand of the target nucleic acid other than the strand to which to which the targeting domain of the gRNA molecule is complementary.
  • the RNA-guided nuclease molecule e.g., Casl2a molecule catalyzes a double strand break.
  • a nucleic acid disclosed herein may comprise (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the MYOC gene as disclosed herein; and/or (b) a sequence that encodes an RNA-guided nuclease molecule (e.g., Casl2a molecule).
  • a nucleic acid disclosed herein may comprise (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the MYOC gene as disclosed herein; (b) a sequence that encodes an RNA-guided nuclease molecule (e.g., Casl2a molecule); and further may comprise (c)(i) a sequence that encodes a second gRNA molecule described herein having a targeting domain that is complementary to a second target domain of the MYOC gene, and optionally, (c)(ii) a sequence that encodes a third gRNA molecule described herein having a targeting domain that is complementary to a third target domain of the MYOC gene; and optionally, (c)(iii) a sequence that encodes a fourth gRNA molecule described herein having a targeting domain that is complementary to a fourth target domain of the MYOC gene.
  • RNA-guided nuclease molecule e.g.,
  • a nucleic acid encodes a second gRNA molecule comprising a targeting domain configured to, in combination with a corresponding RNA-guided nuclease, provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a MYOC target position in the MYOC gene, to allow alteration, e.g., alteration associated with NHEJ, of a MYOC target position in the MYOC gene, either alone or in combination with the break positioned by the first gRNA molecule.
  • a cleavage event e.g., a double strand break or a single strand break
  • a nucleic acid encodes a third gRNA molecule comprising a targeting domain configured to, in combination with a corresponding RNA-guided nuclease, provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a MYOC target position in the MYOC gene to allow alteration, e.g., alteration associated with NHEJ, of a MYOC target position in the MYOC gene, either alone or in combination with the break positioned by the first and/or second gRNA molecule.
  • a cleavage event e.g., a double strand break or a single strand break
  • a nucleic acid encodes a fourth gRNA molecule comprising a targeting domain configured to, in combination with a corresponding RNA-guided nuclease, provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a MYOC target position in the MYOC gene to allow alteration, e.g., alteration associated with NHEJ, of a MYOC target position in the MYOC gene, either alone or in combination with the break positioned by the first gRNA molecule, the second gRNA molecule and/or the third gRNA molecule.
  • a cleavage event e.g., a double strand break or a single strand break
  • the nucleic acid encodes a second gRNA molecule.
  • the second gRNA molecule is selected to target the same MYOC target position as the first gRNA molecule.
  • the nucleic acid may encode a third gRNA molecule, and further optionally, the nucleic acid may encode a fourth gRNA molecule.
  • the third gRNA molecule and the fourth gRNA molecule are selected to target the same MYOC target position as the first and second gRNA molecules.
  • the nucleic acid encodes a second gRNA molecule comprising a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Table 10.
  • the nucleic acid encodes a second gRNA molecule comprising a targeting domain selected from a targeting domain sequence from Table 10.
  • the third and fourth gRNA molecules may independently comprise a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Table 10.
  • the third and fourth gRNA molecules may independently comprise a targeting domain sequence from Table 10.
  • the nucleic acid encodes a second gRNA molecule which is a modular gRNA molecule, e.g., wherein one or more nucleic acid molecules encode a modular gRNA molecule.
  • the nucleic acid encodes a second gRNA molecule, which is a chimeric gRNA molecule.
  • the third and fourth gRNA molecule may be a modular gRNA molecule or a chimeric gRNA molecule. When multiple gRNA molecules are used, any combination of modular or chimeric gRNA molecules may be used.
  • a nucleic acid may encode a second, a third, and/or a fourth gRNA molecule, each independently comprising a targeting domain comprising 16 nucleotides or more in length.
  • the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 16 nucleotides in length.
  • the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 17 nucleotides in length.
  • the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 18 nucleotides in length.
  • the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 19 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 20 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 21 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 22 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 23 nucleotides in length.
  • the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 24 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 25 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 26 nucleotides in length.
  • first nucleic acid molecule e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector
  • second nucleic acid molecule e.g., a second vector, e.g., a second viral vector, e.g., a second AAV vector.
  • the first and second nucleic acid molecules may be AAV vectors.
  • the nucleic acid comprises (a), (b) and (c)(i). In an embodiment, the nucleic acid comprises (a), (b), (c)(i) and (c)(ii). In an embodiment, the nucleic acid comprises (a), (b), (c)(i), (c)(ii) and (c)(iii). Each of (a) and (c)(i), (c)(ii) and/or (c)(iii) may be present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., the same adeno- associated virus (AAV) vector. In an embodiment, the nucleic acid molecule is an AAV vector.
  • (a) and (c)(i) are on different vectors.
  • (a) may be present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (c)(i) may be present on a second nucleic acid molecule, e.g., a second vector, e.g., a second viral vector, e.g., a second AAV vector.
  • the first and second nucleic acid molecules are AAV vectors.
  • each of (a), (b), and (c)(i) are present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., an AAV vector.
  • the nucleic acid molecule is an AAV vector.
  • one of (a), (b), and (c)(i) is encoded on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and a second and third of (a), (b), and (c)(i) is encoded on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector.
  • the first and second nucleic acid molecule may be AAV vectors.
  • first nucleic acid molecule e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector
  • second nucleic acid molecule e.g., a second vector, e.g., a second viral vector, e.g., a second AAV vector.
  • the first and second nucleic acid molecule may be AAV vectors.
  • each of (a), (b) and (c)(i) are present on different nucleic acid molecules, e.g., different vectors, e.g., different viral vectors, e.g., different AAV vector.
  • vectors e.g., different viral vectors, e.g., different AAV vector.
  • (a) may be on a first nucleic acid molecule
  • (c)(i) on a third nucleic acid molecule may be AAV vectors.
  • each of (a), (b), (c)(i), (c)(ii) and (c)(iii) may be present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., an AAV vector.
  • the nucleic acid molecule is an AAV vector.
  • the promoter may comprise a sequence selected from the group consisting of the promoter sequences set forth in Table 6 (DCN #1, DCN #2, ID3, MT2A, SERPINF1, SERPING1 #1, SERPING1 #2, TEMPI, CP, MT1X, OLFML3, PCOLCE, PDGFRA, PTN, RARRE SI, RBP4 #1, RBP4 #2, RBP4 #3, PDPN, APOD, B2M, PTGDS, EEF1 Al , ANGPTL7, MGP, RPS24, PLA2G2A, CHI3L1 , FTL, TAGLN, PCP4, MYL 9, MYOC, CMV, and mini-CMV promoter).
  • Table 6 DCN #1, DCN #2, ID3, MT2A, SERPINF1, SERPING1 #1, SERPING1 #2, TEMPI, CP, MT1X, OLFML3, PCOLCE, PDGFRA, PTN, RARRE SI, R
  • an isolated nucleic acid comprising a promoter comprising a sequence selected from the group consisting of the sequences set forth in Table 6 (DCN #1, DCN #2, ID3, MT2A, SERPINF1, SERPING1 #1, SERPING1 #2, TIMP1, CP, MT1X, OLFML3, PCOLCE, PDGFRA, PTN, RARRE SI, RBP4 #1, RBP4 #2, RBP4 #3, PDPN, APOD, B2M, PTGDS, EEF1A1, ANGPTL7, MGP, RPS24, PLA2G2A, CHI3L1, FTL, TAGLN, PCP4, MYL 9, MYOC, CMV, and mini-CMV promoter).
  • Table 6 DCN #1, DCN #2, ID3, MT2A, SERPINF1, SERPING1 #1, SERPING1 #2, TIMP1, CP, MT1X, OLFML3, PCOLCE, PDGFRA, PTN, RARRE SI
  • the promoter is operably linked to a trans gene.
  • a vector comprising any of the foregoing nucleic acids.
  • the vector is for use in expressing the transgene in a cell, e.g., a trabecular meshwork (TM) cell.
  • TM trabecular meshwork
  • a method of expressing a transgene in a cell e.g., a TM cell, the method comprising contacting the cell with any of the foregoing vectors.
  • a gRNA molecule comprising a targeting domain that may bind a target domain of a MYOC gene.
  • the targeting domain may be complementary to the target domain of the MYOC gene.
  • the targeting domain may be configured to, in combination with an RNA- guided nuclease, provide a cleavage event selected from a double strand break and a single strand break, within 500, 400, 300, 200, 100, 50, 25, or 10 nucleotides of a MYOC target position.
  • the MYOC target position may be in a region selected from the group consisting of a 5’ untranslated region (“UTR”), exon 1, an exon 1/intron 1 border, intron 1, an intron 1/exon 2 border, exon 2, an exon 2/intron 2 border, intron 2, an intron 2/exon 3 border, exon 3, an exon 3/intron 3 border, and 3’ UTR of the MYOC gene.
  • the targeting domain may comprise a sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence from Table 10.
  • the targeting domain may comprise a sequence that is the same as a targeting domain sequence from Table 10.
  • the targeting domain comprises a sequence selected from the group consisting of a targeting domain sequence in Table 10.
  • the targeting domain may comprise a sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain selected from the group consisting of a Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8 targeting domain (Table 10).
  • the targeting domain may comprise a sequence selected from the group consisting of a Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8 targeting domain (Table 10).
  • the targeting domain may be a length selected from the group consisting of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26 nucleotides or more in length.
  • the gRNA molecule may comprise a targeting domain and a crRNA direct repeat extension.
  • the crRNA direct repeat extension may comprise a sequence that is the same as, or differs by no more than 3, 4, 5, or 6 nucleotides from, a crRNA direct repeat extension sequence of UAAUUUCUACUCUUGUAGAU (SEQ ID NO:49).
  • the crRNA direct repeat extension sequence may be UAAUUUCUACUCUUGUAGAU (SEQ ID NO:49).
  • the Cas 12a molecule may be encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Cas 12a polynucleotide sequences).
  • the nucleic acid may comprise a promoter operably linked to the sequence that encodes the Casl2a molecule of (b).
  • first nucleic acid molecule may be present on a first nucleic acid molecule; and (b) may be present on a second nucleic acid molecule.
  • first and second nucleic acid molecules may be AAV vectors.
  • first nucleic acid molecule may be other than an AAV vector and the second nucleic acid molecule may be an AAV vector.
  • the nucleic acid may comprise a promoter operably linked to the sequence that encodes the gRNA molecule of (a).
  • the Cas 12a molecule may be encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Casl2a polynucleotide sequences).
  • the composition may further comprise (c) a second gRNA molecule, a third gRNA molecule, and/or a fourth gRNA molecule.
  • a composition of (a), (b) and optionally (c) a second, third and/or fourth gRNA molecule may further comprise (d) a template nucleic acid.
  • the gRNA molecule may be encapsulated in any of the LNPs disclosed herein.
  • the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease may be encapsulated in any of the LNPs disclosed herein.
  • the nucleic acid encoding the RNA-guided nuclease may be mRNA.
  • the composition may be a pharmaceutical composition.
  • the compositions described herein, e.g., pharmaceutical compositions described herein, can be used in the treatment or prevention of POAG in a subject, e.g., in accordance with a method disclosed herein.
  • a method of altering a cell comprising contacting the cell with: (a) a gRNA molecule that targets the MYOC gene, e.g., a gRNA molecule as described herein; (b) an RNA-guided nuclease molecule or a nucleic acid encoding the RNA- guided nucleic molecule, e.g., a Casl2a molecule as described herein; and optionally, (c) a second, third and/or fourth gRNA molecule that targets MYOC gene, e.g., a second third and/or fourth gRNA molecule as described herein; and optionally, (d) a template nucleic acid.
  • a gRNA molecule that targets the MYOC gene e.g., a gRNA molecule as described herein
  • an RNA-guided nuclease molecule or a nucleic acid encoding the RNA- guided nucleic molecule e.g., a Ca
  • the gRNA molecule may be encapsulated in any of the LNPs as described herein.
  • disclosed herein is a method of altering a cell comprising contacting the cell with (a) a gRNA molecule disclosed herein; (b) an RNA-guided nuclease molecule or a nucleic acid encoding the RNA-guided nuclease molecule; and optionally, (c) a second gRNA molecule disclosed herein.
  • the RNA-guided nuclease may be a Casl2a molecule.
  • the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease may be encapsulated in any of the LNPs as described herein.
  • the Casl2a molecule may comprise any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Casl2a polypeptide sequences).
  • the Cas 12a molecule may be encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Casl2a polynucleotide sequences).
  • the contacting step may comprise delivering to the cell the RNA- guided nuclease molecule of (b) a nucleic acid which encodes the gRNA molecule of (a), and optionally a nucleic acid which encodes the second gRNA molecule of (c). In certain embodiments, the contacting step may comprise delivering to the cell the RNA-guided nuclease molecule of (b), the gRNA molecule of (a) and optionally the second gRNA molecule of (c).
  • the contacting step may comprise delivering to the cell a nucleic acid that encodes the RNA-guided nuclease molecule of (b), the gRNA molecule of (a), and optionally the second gRNA molecule of (c).
  • the method comprises contacting the cell with (a) and (b).
  • the method comprises contacting the cell with (a), (b), and (c).
  • the method comprises contacting the cell with (a), (b), (c) and (d).
  • the gRNA molecule of (a) and optionally (c) may comprise a sequence selected from a sequence set forth in Table 10, or a gRNA molecule that differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence set forth in Table 10.
  • the method comprises contacting a cell from a subject suffering from or likely to develop POAG.
  • the cell may be from a subject having a mutation at a in the MYOC gene (e.g., a mutation MYOC target position in the MYOC gene).
  • the cell being contacted in the disclosed method is a target cell from the eye of the subject.
  • the cell may be a trabecular meshwork cell, retinal pigment epithelial cell, a retinal cell, an iris cell, a ciliary body cell and/or the optic nerve.
  • the contacting may be performed ex vivo and the contacted cell may be returned to the subject’s body after the contacting step. In other embodiments, the contacting step may be performed in vivo.
  • the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses at least one of (a), (b), and (c).
  • the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses each of (a), (b), and (c).
  • the contacting step of the method comprises delivering to the cell an RNA-guided nuclease molecule (e.g., Casl2a molecule) of (b) and a nucleic acid which encodes a gRNA molecule (a) and optionally, a second gRNA molecule (c)(i) (and further optionally, a third gRNA molecule (c)(ii) and/or fourth gRNA molecule (c)(iii)).
  • an RNA-guided nuclease molecule e.g., Casl2a molecule
  • a nucleic acid which encodes a gRNA molecule (a) and optionally, a second gRNA molecule (c)(i) (and further optionally, a third gRNA molecule (c)(ii) and/or fourth gRNA molecule (c)(iii)).
  • the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses at least one of (a), (b), (c) and (d).
  • the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses each of (a), (b), and (c).
  • contacting comprises delivering to the cell an RNA-guided nuclease molecule or a nucleic acid encoding the RNA-guided nuclease molecule (e.g., Casl2a molecule) of (b), as a protein or a messenger RNA (mRNA), and a nucleic acid which encodes a gRNA molecule of (a) and optionally a second, third and/or fourth gRNA molecule (c).
  • RNA-guided nuclease molecule or a nucleic acid encoding the RNA-guided nuclease molecule e.g., Casl2a molecule
  • mRNA messenger RNA
  • a method of treating a subject suffering from or likely to develop POAG e.g., by altering the structure, e.g., sequence, of a target nucleic acid of the subject, comprising contacting the subject (or a cell from the subject) with:
  • gRNA molecule that targets the MYOC gene e.g., a gRNA molecule disclosed herein;
  • a method of treating a subject comprising contacting a subject (or a cell from the subject) with:
  • the method may further comprise contacting the subject with a third gRNA molecule. In certain embodiments, the method may further comprise contacting the subject with a fourth gRNA molecule. In certain embodiments, the method may comprise contacting the subject with (a), (b), and (c). In certain embodiments, the subject may be suffering from POAG. In certain embodiments, the subject may have a mutation in the MYOC gene. In certain embodiments, the contacting step may be performed in vivo. In certain embodiments, the contacting step may comprise subretinal delivery. In certain embodiments, the contacting step may comprise subretinal injection. In certain embodiments, the contacting step may comprise intravitreal delivery. In certain embodiments, the contacting step may comprise intravitreal injection.
  • the contacting step may comprise delivering to the subject the RNA-guided nuclease molecule of (b), the gRNA molecule of (a) and optionally the second gRNA molecule of (c).
  • the contacting step may comprise delivering to the subject a nucleic acid that encodes the RNA-guided nuclease molecule of (b), the gRNA molecule of (a), and optionally the second gRNA molecule of (c).
  • the gRNA molecule may be encapsulated in an LNP described herein.
  • contacting comprises contacting with (a) and (b). In some embodiments, contacting comprises contacting with (a), (b), and (c)(i). In some embodiments, contacting comprises contacting with (a), (b), (c)(i) and (c)(ii). In some embodiments, contacting comprises contacting with (a), (b), (c)(i), (c)(ii) and (c)(iii). In some embodiments, contacting comprises contacting with (a), (b), (c)(i) and (d). In some embodiments, contacting comprises contacting with (a), (b), (c)(i), (c)(ii) and (d). In some embodiments, contacting comprises contacting with (a), (b), (c)(i), (c)(ii) and (d). In some embodiments, contacting comprises contacting with (a), (b), (c)(i), (c)(ii), (c)(iii) and (d).
  • a cell of the subject is contacted ex vivo with (a), (b), (d) and optionally (c).
  • the cell is returned to the subject’s body.
  • a cell of the subject is contacted is in vivo with (a), (b) (d) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
  • the cell of the subject is contacted in vivo by subretinal delivery of (a), (b), (d) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
  • the contacting step comprises contacting the subject with a nucleic acid, e.g., a vector, e.g., an AAV vector, described herein, e.g., a nucleic acid that encodes at least one of (a), (b), (d) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
  • a nucleic acid e.g., a vector, e.g., an AAV vector, described herein, e.g., a nucleic acid that encodes at least one of (a), (b), (d) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
  • the contacting step comprises delivering to the subject the RNA- guided nuclease molecule, e.g., a Casl2a molecule of (b), as a protein or mRNA, the gRNA molecule of (a), as an RNA, a nucleic acid of (d) and optionally the second gRNA molecule of (c)(i), further optionally the third gRNA molecule of (c)(ii), and still further optionally the fourth gRNA molecule of (c)(iii), as an RNA.
  • the RNA- guided nuclease molecule e.g., a Casl2a molecule of (b), as a protein or mRNA
  • the gRNA molecule of (a) as an RNA
  • a nucleic acid of (d) and optionally the second gRNA molecule of (c)(i)
  • the third gRNA molecule of (c)(ii) further optionally the third gRNA molecule of (c)
  • the contacting step comprises delivering to the subject the gRNA molecule of (a), as an RNA, optionally the second gRNA molecule of (c)(i), further optionally the third gRNA molecule of (c)(ii), and still further optionally the fourth gRNA molecule of (c)(iii), as an RNA, a nucleic acid that encodes the RNA-guided nuclease molecule, e.g., a Casl2a molecule of (b), and a nucleic acid of (d).
  • a cell of the subject is contacted ex vivo with (a), (b) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
  • the cell is returned to the subject’s body.
  • the contacting step comprises delivering to the subject the RNA- guided nuclease molecule, e.g., a Casl2a molecule of (b), as a protein or mRNA, and a nucleic acid which encodes (a) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
  • the RNA- guided nuclease molecule e.g., a Casl2a molecule of (b), as a protein or mRNA
  • a nucleic acid which encodes (a) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
  • the contacting step comprises delivering to the subject the RNA- guided nuclease molecule, e.g., a Casl2a molecule of (b), as a protein or mRNA, the gRNA molecule of (a), as an RNA, and optionally the second gRNA molecule of (c)(i), further optionally the third gRNA molecule of (c)(ii), and still further optionally the fourth gRNA molecule of (c)(iii), as an RNA.
  • the RNA- guided nuclease molecule e.g., a Casl2a molecule of (b), as a protein or mRNA
  • the gRNA molecule of (a) as an RNA
  • the second gRNA molecule of (c)(i) further optionally the third gRNA molecule of (c)(ii)
  • the fourth gRNA molecule of (c)(iii) as an RNA.
  • compositions as disclosed herein for use in treating POAG in a subject.
  • Headings including numeric and alphabetical headings and subheadings, are for organization and presentation and are not intended to be limiting.
  • Fig. 1 shows data from the screening assay in T cells testing the editing activity of a series of SaCas9 RNPs comprising S. aureus Cas9 CS'aCas9) enzyme and an .S' «Cas9 gRNA (Table 9). Gray triangles represent results showing the percentage of frameshift indels introduced into the MYOC gene by each RNP. Black dots represent the percentage of indels introduced into the MYOC gene by each RNP. All samples were run in duplicate. [0122] Figs.
  • FIG. 2A-2B show dose response data from T cells using increasing concentrations (0.0891 pM, 0.178 pM, 0.356 pM, 0.7125 pM, 1.425 pM, 2.85 pM, 5.7 pM, and 11.4 pM) of .S'aCas9 RNP comprising SaCas9 enzyme and A/Cas9 gRNA (Cas9-1, Cas9-5, Cas9-6, Cas9-24, Cas9-56, Cas9-57, Cas9-59 (Table 9)).
  • Fig. 2A shows the percentage of indels introduced into the MYOC gene by RNP concentration for each RNP tested.
  • Fig. 2B shows the percentage of frameshift indels introduced into the MYOC gene by RNP concentration for each RNP tested. All samples were run in duplicate.
  • Figs. 4A-4B show dose response data from T cells using increasing concentrations (0.010 pM, 0.030 pM, 0.080 pM, 0.25 pM, 0.80 pM, 2.53 pM, and 8.0 pM) of AvCas l 2a RNP comprising AsCasl2a enzyme and an AsCasl2a gRNA (Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, or Casl2a-8 (Table 10)).
  • Fig. 4A shows the percentage of indels introduced into the MYOC gene by RNP concentration for each RNP tested.
  • Fig. 4B shows the percentage of frameshift indels introduced into the MYOC gene by RNP concentration for each RNP tested. All samples were run in duplicate.
  • Figs. 5A-5B show dose response data in human trabecular meshwork cells (HTMCs) using increasing concentrations (0.010 pM, 0.030 pM, 0.080 pM, 0.25 pM, 0.80 pM, 2.53 pM, and 8.0 pM) of RNP comprising AsCasl2a enzyme and an AsCasl2a gRNA (Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, or Casl2a-8 (Table 10)) or RNP comprising SaCas9 enzyme complexed with an SaCasO gRNA (i.e., Cas9-1 , Cas9-5, Cas9-6, Cas9-24 (Table 9)).
  • RNP comprising AsCasl2a enzyme and an AsCasl2a gRNA
  • SaCasO gRNA i.e., Cas9-1 , Ca
  • Fig. 5A shows the indel rate (%) in HTMCs with increasing concentration of RNP comprising .S'aCas9 enzyme complexed with .S' «Cas9 gRNAs or RNP comprising AsCas 12a enzyme complexed with AsCas 12a gRNAs.
  • Fig. 5B shows the percentage of frameshift indels in HTMCs with increasing concentration of RNP comprising S «Cas9 enzyme complexed with .Sc/Cas9 gRNAs or RNP comprising AsCasl2a enzyme complexed with AsCasl2a gRNAs. [0126] Figs.
  • FIG. 6A and 6B show expression of green fluorescent protein (GFP) in HTMCs using various promoters (promoter sequences are set forth in Table 6).
  • Fig. 6A shows the percentage of GFP+ HTMCs after nucleofection with plasmids containing the indicated putative promoter driving expression of GFP.
  • Fig. 6B shows the mean fluorescence intensity of GFP+ HTMCs after nucleofection with plasmids containing the indicated putative promoter driving expression of GFP.
  • Figs. 7A- 7F show histology data from in vivo screening of LNPs formulated with different ionizable lipids, for transfection of the mouse trabecular meshwork (TM).
  • LNPs (50% ionizable lipid, 38.5% cholesterol, 10% DSPC, and 1.5% DMG-PEG2k) encapsulating GFP mRNA and irrelevant gRNA were formulated with different ionizable lipids and injected intracamerally into wildtype mice with 1 pl of 500 pg/mL LNP-GFP mRNA (i.e., GFP mRNA encapsulated in LNPs). Twenty-four hours post injection eyes were collected and analyzed for GFP expression using immunohistochemistry.
  • Figs. 8A and 8B show transfection of human and non-human primate (NHP) trabecular meshwork tissue with two different ionizable lipids.
  • Fig. 8A shows the percentage (%) of GFP+ cells in human TM region for control (Ctrl) and 0.5 mg/mL and 0.05 mg/mL LNP-GFP mRNA (i.e., GFP mRNA encapsulated in LNPs) of LNPs formed with MC3 (MC3) and LNPs formed with ALC-0315 (ALC-0315).
  • Fig. 8A shows the percentage (%) of GFP+ cells in human TM region for control (Ctrl) and 0.5 mg/mL and 0.05 mg/mL LNP-GFP mRNA (i.e., GFP mRNA encapsulated in LNPs) of LNPs formed with MC3 (MC3) and LNPs formed with ALC-0315 (ALC-0315).
  • the Y-axis represents GFP expression using the following scoring scale for GFP expression in the TM region: - 0: no staining; 1 : sparse GFP + staining; 2: more GFP + staining; 3: GFP + staining level between 2 and 4; and 4: highest level of GFP + staining.
  • the score was averaged over four TM regions in two eye sections. The grade for each group was the average of the GFP expression score in all mice within that group.
  • Fig. 9B shows histology data for mouse TM transfected with Vehicle.
  • Fig. 9C shows histology data for mouse TM transfected with LNPs containing 1.5% DMG-PEG.
  • FIG. 11B shows results of transfection with LNP1.
  • Fig. 11C shows results of transfection with LNP8.
  • Fig. 11D shows results of transfection with LNP14.
  • Fig. HE shows results of transfection with LNP15.
  • Fig. HF shows results of transfection with LNP 19.
  • Arrows show representative location of TM cells with GFP expression.
  • Fig. 13 shows the genomic DNA editing (% indel) observed in the NHP TM tissue. Ill-seq analysis was performed to determine AsCasl2a-mediated editing at the MYOC locus (see Table 16 for primers used).
  • Fig. 16A shows the percentage of indels introduced into the MYOC gene determined by Ill-Seq (see Table 16 for primers used). Data are presented as mean with SD analyzed with one-way ANOVA followed by Tukey’s test. Fig. 16B shows the percentage of myocilin mRNA remaining determined by RT-ddPCR. Data are presented as mean with standard deviation (SD) and analyzed with one-way ANOVA followed by Tukey’s multiple comparison test compared to vehicle. Vehicle is a tris buffered salt (TBS) solution plus 0.167 part 60% sucrose.
  • TBS tris buffered salt
  • LNP-AsCasl2a mRNA i.e., AsCasl2a mRNA encapsulated in LNPs.
  • anterior chambers were collected followed by extraction of mRNA and gDNA.
  • 18B shows the percentage of indels introduced into the MYOC gene as determined by Ill- Seq after dosing with LNPs with various concentrations of LNP-AsCasl2a mRNA (25 pg/mL, 100 pg/mL, and 500 pg/mL) (see Table 16 for primers used). Data are presented as mean with standard deviation (SD) and analyzed with one-way ANOVA followed by Tukey’s test. *, P ⁇ 0.05; **, P ⁇ 0.005; ***, P ⁇ 0.0005; ****, P ⁇ 0.0001. Vehicle (“Veh”) is PBS. Note that gDNA is from a mixed population of cells from the anterior chamber and not exclusively the TM tissue. Fig.
  • 18C shows the percentage of myocilin mRNA determined by RT-ddPCR after dosing with LNPs with various concentrations of LNP-AsCasl2a mRNA (25 pg/mL, 100 pg/mL, and 500 pg/mL). Data are presented as mean with standard deviation (SD) and analyzed with one-way ANOVA with Tukey’s multi comparison test compared to Vehicle. Vehicle (“Veh”) is PBS. Fig.
  • FIG. 18D shows the percentage of indels introduced into the MYOC gene in primary TM cells as determined by Ill-Seq with LNPs with various concentrations of LNP-AsCasl2a mRNA: LNP stock (circles), 25 pg/mL (downward pointing triangles), 100 pg/mL (upward pointing triangles), and 500 pg/mL (squares) (see Table 16 for primers used).
  • Primary TM cells were treated with increasing concentrations (based on AsCasl2a mRNA) of LNP encapsulating AsCasl2a mRNA plus gRNA.
  • Fig. 18D provides in vitro confirmation that LNPs that were tested in vivo also edit primary TM cells in vitro.
  • mutant myocilin production in subjects with a mutation e.g., a mutation at T377 (e.g., T377R), a mutation at 1477 (e.g., I477N), or a mutation at P370 (e.g., P370L), or other mutations or other mutant MYOC alleles, through knock out of MYOC on ciliary body cells, iris cells, trabecular meshwork cells and retinal cells will prevent the production of the myocilin proteins.
  • a mutation at T377 e.g., T377R
  • a mutation at 1477 e.g., I477N
  • P370 e.g., P370L
  • other mutations or other mutant MYOC alleles through knock out of MYOC on ciliary body cells, iris cells, trabecular meshwork cells and retinal cells will prevent the production of the myocilin proteins.
  • POAG does not progress or has delayed progression compared to a subject who has not received the therapy.
  • Described herein are methods for treating or delaying the onset or progression of POAG caused by mutations in the MYOC gene including but not limited to mutations in exon 3, e.g., a mutation at T377 (e.g., T377R), a mutation at 1477 (e.g., I477N), or a mutation at P370 (e.g., P370L).
  • the disclosed methods for treating or delaying the onset or progression of POAG alter the MYOC gene by genome editing using a gRNA that is complementary to a target domain of the MYOC gene and an RNA-guided nuclease (e.g., Casl2a enzyme).
  • the MYOC gene is targeted as a targeted knockout.
  • a knockout of the MYOC gene may offer a benefit to subjects with POAG who have a mutation in the MYOC gene as well as subjects with POAG without a known MYOC mutation.
  • MYOC mutations are gain of function mutations leading to altered TM function and the development of IOP.
  • patients with heterozygous early truncating mutations (Arg46stop) do not develop disease.
  • MYOC knock-out mice do not develop POAG and have no detected eye abnormalities. Further, a few patients have been identified who express no myocilin in the eye and have no phenotype. Without wishing to be bound by theory, it is contemplated herein that a knockout of MYOC gene in the eye prevents the development of POAG.
  • Alteration of one or both MYOC alleles may be performed prior to disease onset or after disease onset, but preferably early in the disease course.
  • treatment is initiated after onset of the disease, but early in the course of disease progression (e.g., prior to vision loss, a decrease in visual acuity and/or an increase in IOP).
  • treatment is initiated in a subject who has no MYOC mutation but has increased intraocular pressure. In an embodiment, treatment is initiated in a subject at onset of an increase in intraocular pressure. In an embodiment, treatment is initiated in a subject after onset of an increase in intraocular pressure.
  • a subject’s vision can be evaluated, e.g., prior to treatment, or after treatment, e.g., to monitor the progress of the treatment.
  • the subject’s vision is evaluated prior to treatment, e.g., to determine the need for treatment.
  • the subject’s vision is evaluated after treatment has been initiated, e.g., to assess the effectiveness of the treatment.
  • Genome editing systems can be implemented (e.g., administered or delivered to a cell or a subject) in a variety of ways, and different implementations may be suitable for distinct applications.
  • a genome editing system is implemented, in certain embodiments, as a protein/RNA complex (a ribonucleoprotein, or RNP), which can be included in a pharmaceutical composition that optionally includes a pharmaceutically acceptable carrier and/or an encapsulating agent, such as, without limitation, a lipid or polymer micro- or nanoparticle, micelle, or liposome.
  • a protein/RNA complex a ribonucleoprotein, or RNP
  • RNP ribonucleoprotein
  • an encapsulating agent such as, without limitation, a lipid or polymer micro- or nanoparticle, micelle, or liposome.
  • Maeder which is incorporated by reference herein, describes a genome editing system for correcting a point mutation (C.2991+1655A to G) in the human CEP290 gene that results in the creation of a cryptic splice site, which in turn reduces or eliminates the function of the gene.
  • the genome editing system of Maeder utilizes two guide RNAs targeted to sequences on either side of (i.e., flanking) the point mutation, and forms DSBs that flank the mutation. This, in turn, promotes deletion of the intervening sequence, including the mutation, thereby eliminating the cryptic splice site and restoring normal gene function.
  • genome editing systems operate by forming DSBs
  • such systems optionally include one or more components that promote or facilitate a particular mode of double-strand break repair or a particular repair outcome.
  • Cotta-Ramusino also describes genome editing systems in which a single stranded oligonucleotide “donor template” is added; the donor template is incorporated into a target region of cellular DNA that is cleaved by the genome editing system, and can result in a change in the target sequence.
  • genome editing systems modify a target sequence, or modify expression of a gene in or near the target sequence, without causing single- or double-strand breaks.
  • a genome editing system may include an RNA-guided nuclease fused to a functional domain that acts on DNA, thereby modifying the target sequence or its expression.
  • an RNA-guided nuclease can be connected to (e.g., fused to) a cytidine deaminase functional domain, and may operate by generating targeted C-to-A substitutions. Exemplary nuclease/deaminase fusions are described in Komor 2016, which is incorporated by reference herein.
  • a genome editing system may utilize a cleavage-inactivated (i.e., a “dead”) nuclease, such as a dead Cas9 (dCas9) or dead Casl2a (dCasl 2a), and may operate by forming stable complexes on one or more targeted regions of cellular DNA, thereby interfering with functions involving the targeted region(s) including, without limitation, mRNA transcription, chromatin remodeling, etc.
  • a genome editing system may include an RNA-guided helicase that unwinds DNA within or proximal to the target sequence, without causing single- or double- stranded breaks.
  • Guide RNAs include a “targeting domain” that is fully or partially complementary to a target domain within a target sequence, such as a DNA sequence in the genome of a cell where editing is desired.
  • Targeting domains are referred to by various names in the literature, including without limitation “guide sequences” (Hsu 2013, incorporated by reference herein), “complementarity regions” (Cotta-Ramusino), “spacers” (Briner 2014) and generically as “crRNAs” (Jiang).
  • targeting domains are typically 10-30 nucleotides in length, and in certain embodiments are 16-26 nucleotides in length (for instance, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length), and are at or near the 5’ terminus of in the case of a Cas9 gRNA, and at or near the 3’ terminus in the case of a Casl2a gRNA.
  • the Casl2a gRNA may include a crRNA direct repeat extension sequence.
  • an exemplary crRNA direct repeat extension sequence may be UAAUUUCUACUCUUGUAGAU (SEQ ID NO: 49) (corresponding DNA sequence is TAATTTCTACTCTTGTAGAT (SEQ ID NO: 50)).
  • gRNAs can be defined, in broad terms, by their targeting domain sequences, and skilled artisans will appreciate that a given targeting domain sequence can be incorporated in any suitable gRNA, including a unimolecular or chimeric gRNA, or a gRNA that includes one or more chemical modifications and/or sequential modifications (substitutions, additional nucleotides, truncations, etc.). Thus, for economy of presentation in this disclosure, gRNAs may be described solely in terms of their targeting domain sequences.
  • gRNA should be understood to encompass any suitable gRNA that can be used with any RNA- guided nuclease, and not only those gRNAs that are compatible with a particular species of Casl2a.
  • the term gRNA can, in certain embodiments, include a gRNA for use with any RNA-guided nuclease occurring in a Class 2 CRISPR system, such as a type II or type V or CRISPR system, or an RNA-guided nuclease derived or adapted therefrom.
  • a unimolecular, or chimeric, gRNA may comprise, a crRNA direct repeat extension sequence and a targeting domain (which is complementary to a target nucleic acid in the MYOC gene, e.g., a targeting domain from Table 10).
  • the targeting domain is 16 nucleotides in length. In an embodiment, the targeting domain is 17 nucleotides in length. In an embodiment, the targeting domain is 18 nucleotides in length. In an embodiment, the targeting domain is 19 nucleotides in length. In an embodiment, the targeting domain is 20 nucleotides in length. In an embodiment, the targeting domain is 21 nucleotides in length. In an embodiment, the targeting domain is 22 nucleotides in length. In an embodiment, the targeting domain is 23 nucleotides in length. In an embodiment, the targeting domain is 24 nucleotides in length. In an embodiment, the targeting domain is 25 nucleotides in length. In an embodiment, the targeting domain is 26 nucleotides in length.
  • the targeting domain comprises 16 nucleotides. In an embodiment, the targeting domain comprises 17 nucleotides. In an embodiment, the targeting domain comprises 18 nucleotides. In an embodiment, the targeting domain comprises 19 nucleotides. In an embodiment, the targeting domain comprises 20 nucleotides. In an embodiment, the targeting domain comprises 21 nucleotides. In an embodiment, the targeting domain comprises 22 nucleotides. In an embodiment, the targeting domain comprises 23 nucleotides. In an embodiment, the targeting domain comprises 24 nucleotides. In an embodiment, the targeting domain comprises 25 nucleotides. In an embodiment, the targeting domain comprises 26 nucleotides.
  • the targeting domain of the gRNA molecule has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a targeting domain nucleotide sequence disclosed herein, e.g., a targeting domain nucleotide sequence set forth in Table 10, e.g., a targeting domain nucleotide sequence from the group consisting of a Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8.
  • the targeting domain of the gRNA molecule has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations relative to a targeting domain nucleotide sequence disclosed herein, e.g., a targeting domain nucleotide sequence set forth in Table 10, e.g., a targeting domain nucleotide sequence from the group consisting of a Casl2a-l, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8.
  • Certain exemplary modifications discussed in this section can be included at any position within a gRNA sequence including, without limitation at or near the 5’ end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5’ end) and/or at or near the 3’ end (e.g., within 1- 10, 1-5, or 1-2 nucleotides of the 3’ end).
  • modifications are positioned within functional motifs, such as the repeat-anti-repeat duplex of a Cas9 gRNA, a stem loop structure of a Cas9 or Casl2a gRNA, and/or a targeting domain of a gRNA.
  • a gRNA whether transcribed in vivo from a DNA vector, or in vitro transcribed gRNA, can include either or both of a 5’ cap structure or cap analog and a 3’ polyA tract.
  • any suitable moiety can be used to provide such bridges, include without limitation methylene, propylene, ether, or amino bridges; 0-amino (wherein amino can be, e.g., NHz; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy or O(CH2)n-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or poly amino).
  • a gRNA comprises a 4’- S, 4’-Se or a 4’-C-aminomethyl-2’-O-Me modification.
  • the modified gRNA comprises a IxPSOMe modification on a 5’ end and/or a 3’ end; in certain embodiments, the modified gRNA comprises a 3xPSOMe modification on a 5’ end and/or a 3’ end.
  • the hairpin region of the modified gRNA comprises 5’ inverted dT modifications. In certain embodiments, the hairpin region of the modified gRNA comprises 3’ inverted dT modifications. In certain embodiments, the hairpin region of the modified gRNA comprises a 3’ or 4’ pseudoknot. In certain embodiments, the hairpin region of the modified gRNA comprises a 3’ pseudoknot.
  • the hairpin region of the modified gRNA comprises a locked nucleic acid (LNA). In certain embodiments, the hairpin region of the modified gRNA comprises a LNA with a 5’ extension.
  • LNA locked nucleic acid
  • the targeting domain of the gRNA comprises modifications on nucleotides 1, 2, 3, 7, 8, 9, 10, 11, 12, 14, 15, 17, 19 and optionally 20 and 21.
  • the targeting domain of the gRNA comprises one or more of 2’Fluorine modifications, 2’O-methyl modifications, 5’ inverted dT modifications, or 3’ inverted dT modifications.
  • a gRNA can comprise both a conservative hairpin modification pattern and a conservative targeting region modification pattern. In any of the above embodiments, a gRNA can comprise either of a conservative hairpin modification pattern and a conservative targeting region modification pattern. In any of the above embodiments, a gRNA can comprise neither a conservative hairpin modification pattern nor a conservative targeting region modification pattern.
  • RNA-guided nuclease molecules include, but are not limited to, naturally-occurring Class 2 CRISPR nucleases such as Casl2a (also referred to as Cpfl), and Cas9, as well as other nucleases derived or obtained therefrom.
  • Class 2 CRISPR nucleases such as Casl2a (also referred to as Cpfl), and Cas9, as well as other nucleases derived or obtained therefrom.
  • RNA-guided nuclease may be Cas- ⁇ D (Pausch 2020).
  • RNA-guided nucleases may require different sequential relationships between PAMs and protospacers.
  • Cas9s recognize PAM sequences that are 3’ of the protospacer.
  • Cast 2a on the other hand, generally recognizes PAM sequences that are 5’ of the protospacer.
  • RNA-guided nucleases can also recognize specific PAM sequences. 5. aureus Cas9, for instance, recognizes a PAM sequence of NNGRRT or NNGRRV, wherein the N residues are immediately 3’ of the region recognized by the gRNA targeting domain. .S’.
  • PAMs examples include, without limitation, the nucleotide sequences set forth in SEQ ID NOs:l-7 (NGGNG (SEQ ID NO:1), NNAGAAW (SEQ ID NO:2), NAAR (SEQ ID NO:3), NNGRR (SEQ ID NO:4), NNGRRN (SEQ ID NO:5), NNGRRT (SEQ ID NO:6), and NNGRRV (SEQ ID NO:7)).
  • RNA-guided nucleases can be characterized by their DNA cleavage activity: naturally-occurring RNA-guided nucleases typically form DSBs in target nucleic acids, but engineered variants have been produced that generate only SSBs (discussed above and in Ran & Hsu 2013, incorporated by reference herein), or that do not cut at all.
  • Casl 2a (also known as Cpfl) in complex with crRNA and a double-stranded (ds) DNA target including a TTTN PAM sequence has been solved by Yamano 2016 (incorporated by reference herein).
  • Casl2a like Cas9, has two lobes: a REC (recognition) lobe, and a NUC (nuclease) lobe.
  • the REC lobe includes RECI and REC2 domains, which lack similarity to any known protein structures.
  • the NUC lobe meanwhile, includes three RuvC domains (RuvC-I, -II and -III) and a BH domain.
  • a Casl2a molecule may be a modified Casl2a molecule.
  • a modified Casl2a molecule may include one or more modifications.
  • the modifications may be, without limitation, one or more mutations in a Casl2a nucleotide sequence or Casl2a polypeptide sequence, one or more additional sequences such as a His tag or a nuclear localization signal (NLS), or a combination thereof.
  • the Casl2a molecule may be derived from a Casl2a molecule selected from the group consisting of Acidaminococcus sp. strain BV3L6 Cpfl protein (AsCasl2a or AsCpfl), Lachnospiraceae bacterium ND2006 Cpfl protein (LbCpfl), and Lachnospiraceae bacterium MA2020 (Lb2 Cpfl).
  • the Casl2a molecule may comprise a sequence selected from the group consisting of SEQ ID NOs: 18- 20, having the codon-optimized nucleic acid sequences of SEQ ID NOs:21-23, respectively.
  • nNLS refers to the nucleoplasmin NLS
  • sNLS refers to the SV40 NLS. Additional permutations of the identity and C-terminal positions of NLS sequences, e.g., appending two or more nNLS sequences or combinations of nNLS and sNLS sequences (or other NLS sequences), as well as sequences with and without purification sequences, e.g., six -histidine sequences, are within the scope of the instantly disclosed subject matter.
  • the NLS sequence of the modified Casl2a molecule may be positioned at or near the N-terminus of the Casl2a protein sequence.
  • the modified Casl2a protein may be selected from the following: His- sNLS-AsCpfl (SEQ ID NO: 11), His-sNLS-sNLS-AsCpfl (SEQ ID NO: 12), and sNLS- sNLS-AsCpfl (SEQ ID NO: 13).
  • the modified Casl2a molecule may comprise NLS sequences positioned at or near both the N-terminus and C-terminus of the Casl2a protein sequence.
  • the modified Casl2a protein may be selected from the following: His-sNLS-AsCpfl-sNLS (SEQ ID NO: 14) and His-sNLS-sNLS-AsCpfl- sNLS-sNLS (SEQ ID NO: 15). Additional permutations of the identity and N-terminal/C- terminal positions of NLS sequences, e.g., appending two or more nNLS sequences or combinations of nNLS and sNLS sequences (or other NLS sequences) to either the N- terminal/C-terminal positions, as well as sequences with and without purification sequences, e.g., six-histidine sequences, are within the scope of the instantly disclosed subject matter.
  • a modified Casl2a molecule is used herein, wherein the modified Casl 2a molecule comprises mutations at one or more residues of AsCasl 2a (Acidaminococcus sp. BV3L6) selected from the group consisting of 11, 12, 13, 14, 15, 16, 17, 34, 36, 39, 40, 43, 46, 47, 50, 54, 57, 58, 111, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,
  • a modified Casl2a molecule as used herein may include any of the Casl2a (referred to also as Cpfl) proteins described in International Publication Number WO 2017/184768 Al by Zhang et al. (“’768 Publication”), which is incorporated by reference herein.
  • Cpfl Casl2a
  • any of the Casl2a molecules or modified Casl2a molecules disclosed herein may be complexed with one or more gRNA comprising the targeting domain set forth in Table 10 to alter the MYOC gene.
  • any of the Casl2a molecules or modified Casl2a molecules disclosed herein may be complexed with one or more gRNA comprising a sequence set forth in Table 10.
  • the modified Casl2a molecule may be His- AsCpfl -nNLS (SEQ ID NO:8) or His-AsCpfl-sNLS- sNLS (SEQ ID NO:9).
  • a modified Casl2a molecule used herein may comprise any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (exemplary Casl2a polypeptide sequences) or be encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (exemplary Casl2a polynucleotide sequences).
  • the modified Casl2a molecule may include a modified Casl2a (i.e., Cpfl) molecule described in Kleinstiver 2019.
  • the modified Casl2a molecule may be enAsCpfl.
  • the modified Casl2a molecule may cleave target sites with a TTTV PAM.
  • the modified Casl2a molecule may cleave target sites with a NWYN PAM.
  • Cpfl Casl2a
  • an RNA-guided nuclease has at least 80%, at least 85%, at least 86%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity relative to a wild-type RNA-guided nuclease and/or an RNA-guided nuclease disclosed herein (e.g., an RNA-guided nuclease comprising an amino acid sequence depicted in Table 14 or comprising any of SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48).
  • an RNA-guided nuclease has less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations relative to a wild-type RNA- guided nuclease and/or an RNA-guided nuclease disclosed herein (e.g., an RNA-guided nuclease comprising an amino acid sequence depicted in Table 14 or comprising any of SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48).
  • a naturally occurring Cas9 protein comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which comprise particular structural and/or functional domains.
  • the REC lobe comprises an arginine-rich bridge helix (BH) domain, and at least one REC domain (e.g., a RECI domain and, optionally, a REC2 domain).
  • the REC lobe does not share structural similarity with other known proteins, indicating that it is a unique functional domain.
  • the BH domain appears to play a role in gRNA:DNA recognition, while the REC domain is thought to interact with the repeat: anti-repeat duplex of the gRNA and to mediate the formation of the Cas9/gRNA complex.
  • the NUC lobe comprises a RuvC domain, an HNH domain, and a PAM-interacting (PI) domain.
  • the RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves the non-complementary (i.e., bottom) strand of the target nucleic acid. It may be formed from two or more split RuvC motifs (such as RuvC I, RuvCII, and RuvCIII in 5. pyogenes and S. aureus ⁇ .
  • the HNH domain meanwhile, is structurally similar to HNN endonuclease motifs, and cleaves the complementary (i.e., top) strand of the target nucleic acid.
  • the PI domain as its name suggests, contributes to PAM specificity.
  • RNA-guided nucleases suitable for use in the context of the methods, strategies, and treatment modalities provided herein are listed in Table 3 below, and the methods, compositions, and treatment modalities disclosed herein can, in some embodiments, make use of any combination of RNA-guided nucleases disclosed herein, or known to those of ordinary skill in the art. Table 3.
  • RNA-guided nucleases described above have activities and properties that can be useful in a variety of applications, but the skilled artisan will appreciate that RNA-guided nucleases can also be modified in certain instances, to alter cleavage activity, PAM specificity, or other structural or functional features.
  • Exemplary mutations that may be made in the RuvC domains, in the Cas9 HNH domain, or in the Casl2a Nuc domain are described in Ran & Hsu 2013 and Yamano 2016, as well as in Cotta-Ramusino.
  • mutations that reduce or eliminate activity in one of the two nuclease domains result in RNA-guided nucleases with nickase activity, but it should be noted that the type of nickase activity varies depending on which domain is inactivated.
  • inactivation of a RuvC domain of a Cas9 will result in a nickase that cleaves the complementary or top strand as shown below (where C denotes the site of cleavage).
  • RNA-guided nucleases have been split into two or more parts, as described by Zetsche 2015 and Fine 2015 (both incorporated by reference herein).
  • RNA-guided nucleases can be, in certain embodiments, size-optimized or truncated, for instance via one or more deletions that reduce the size of the nuclease while still retaining gRNA association, target and PAM recognition, and cleavage activities.
  • RNA guided nucleases are bound, covalently or non-covalently, to another polypeptide, nucleotide, or other structure, optionally by means of a linker. Exemplary bound nucleases and linkers are described by Guilinger 2014, incorporated by reference herein for all purposes.
  • RNA-guided nucleases also optionally include a tag, such as, but not limited to, a nuclear localization signal to facilitate movement of RNA-guided nuclease protein into the nucleus.
  • a tag such as, but not limited to, a nuclear localization signal to facilitate movement of RNA-guided nuclease protein into the nucleus.
  • the RNA-guided nuclease can incorporate C- and/or N- terminal nuclear localization signals. Nuclear localization sequences are known in the art and are described in Maeder and elsewhere.
  • a nucleic acid encoding an RNA-guided nuclease can be a synthetic nucleic acid sequence.
  • the synthetic nucleic acid molecule can be chemically modified.
  • an mRNA encoding an RNA-guided nuclease will have one or more (e.g., all) of the following properties: it can be capped; polyadenylated; and substituted with 5 -methylcytidine and/or pseudouridine.
  • Synthetic nucleic acid sequences can also be codon optimized, e.g., at least one noncommon codon or less-common codon has been replaced by a common codon.
  • the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., described herein. Examples of codon optimized Cas9 coding sequences are presented in Cotta-Ramusino.
  • a nucleic acid encoding an RNA-guided nuclease may comprise a nuclear localization sequence (NLS).
  • NLS nuclear localization sequences are known in the art.
  • thermostability of ribonucleoprotein (RNP) complexes comprising gRNAs and RNA-guided nucleases can be measured via DSF.
  • the DSF technique measures the thermostability of a protein, which can increase under favorable conditions such as the addition of a binding RNA molecule, e.g., a gRNA.
  • Gene conversion and gene correction are facilitated, in some cases, by the formation of one or more nicks in or around the targeted region, as described in Ran & Hsu 2013 and Cotta-Ramusino.
  • a dual-nickase strategy is used to form two offset SSBs that, in turn, form a single DSB having an overhang (e.g., a 5’ overhang).
  • NHEJ NHEJ pathway
  • Alt-NHEJ NHEJ
  • NHEJ is referred to as an “error prone” repair pathway because of its association with indel mutations.
  • a DSB is repaired by NHEJ without alteration of the sequence around it (a so-called “perfect” or “scarless” repair); this generally requires the two ends of the DSB to be perfectly ligated. Indels, meanwhile, are thought to arise from enzymatic processing of free DNA ends before they are ligated that adds and/or removes nucleotides from either or both strands of either or both free ends.
  • indels can be characterized by (a) their relative and absolute frequencies in the genomes of cells contacted with genome editing systems and (b) the distribution of numerical differences relative to the unedited sequence, e.g., ⁇ 1, ⁇ 2, ⁇ 3, etc.
  • multiple gRNAs can be screened to identify those gRNAs that most efficiently drive cutting at a target site based on an indel readout under controlled conditions.
  • Guides that produce indels at or above a threshold frequency, or that produce a particular distribution of indels, can be selected for further study and development.
  • Indel frequency and distribution can also be useful as a readout for evaluating different genome editing system implementations or formulations and delivery methods, for instance by keeping the gRNA constant and varying certain other reaction conditions or delivery methods.
  • Genome editing systems may also be employed for multiplex gene editing to generate two or more DSBs, either in the same locus or in different loci.
  • Any of the RNA-guided nucleases and gRNAs disclosed herein may be used in genome editing systems for multiplex gene editing. Strategies for editing that involve the formation of multiple DSBs, or SSBs, are described in, for instance, Cotta-Ramusino.
  • multiple gRNAs and an RNA-guided nuclease may be used in genome editing systems to introduce alterations (e.g., deletions, insertions) into the MYOC gene.
  • the RNA-guided nuclease may be Casl2a.
  • Donor template design is described in detail in the literature, for instance in Cotta- Ramusino.
  • DNA oligomer donor templates oligodeoxynucleotides or ODNs
  • ssODNs single stranded
  • dsODNs double-stranded
  • the homology arms can have any suitable length (including 0 nucleotides if only one homology arm is used), and 3’ and 5’ homology arms can have the same length, or can differ in length.
  • the selection of appropriate homology arm lengths can be influenced by a variety of factors, such as the desire to avoid homologies or microhomologies with certain sequences such as Alu repeats or other very common elements.
  • a 5’ homology arm can be shortened to avoid a sequence repeat element.
  • a 3’ homology arm can be shortened to avoid a sequence repeat element.
  • both the 5’ and the 3’ homology arms can be shortened to avoid including certain sequence repeat elements.
  • homology arm designs can improve the efficiency of editing or increase the frequency of a desired repair outcome.
  • a replacement sequence in donor templates have been described elsewhere, including in Cotta-Ramusino.
  • a replacement sequence can be any suitable length (including zero nucleotides, where the desired repair outcome is a deletion), and typically includes one, two, three or more sequence modifications relative to the naturally-occurring sequence within a cell in which editing is desired.
  • One common sequence modification involves the alteration of the naturally-occurring sequence to repair a mutation that is related to a disease or condition of which treatment is desired.
  • Another common sequence modification involves the alteration of one or more sequences that are complementary to, or then, the PAM sequence of the RNA-guided nuclease or the targeting domain of the gRNA(s) being used to generate an SSB or DSB, to reduce or eliminate repeated cleavage of the target site after the replacement sequence has been incorporated into the target site.
  • a linear ssODN can be configured to (i) anneal to the nicked strand of the target nucleic acid, (ii) anneal to the intact strand of the target nucleic acid, (iii) anneal to the plus strand of the target nucleic acid, and/or (iv) anneal to the minus strand of the target nucleic acid.
  • An ssODN may have any suitable length, e.g., about, at least, or no more than 80-200 nucleotides (e.g., 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides).
  • the donor template can be adjacent to, or flanked by, target sites recognized by one or more gRNAs, to facilitate the formation of free DSBs on one or both ends of the donor template that can participate in repair of corresponding SSBs or DSBs formed in cellular DNA using the same gRNAs.
  • exemplary nucleic acid vectors suitable for use as donor templates are described in Cotta- Ramusino, which is incorporated by reference. [0283] Whatever format is used, a template nucleic acid can be designed to avoid undesirable sequences. In certain embodiments, one or both homology arms can be shortened to avoid overlap with certain sequence repeat elements, e.g., Alu repeats, LINE elements, etc.
  • silent, non-pathogenic SNPs may be included in the ssODN donor template to allow for identification of a gene editing event.
  • the cells are induced pluripotent stem cells (iPS) cells or cells derived from iPS cells, e.g., iPS cells from the subject, modified to alter the gene and differentiated into trabecular meshwork cells, retinal progenitor cells or retinal cells, e.g., retinal photoreceptors, and injected into the eye of the subject, e.g., into the trabecular meshwork, or, e.g., subretinally, e.g., in the submacular region of the retina.
  • iPS induced pluripotent stem cells
  • the cells are targeted in vivo, e.g., by delivery of the components, e.g., a Casl2a molecule and gRNA molecules, to the target cells.
  • the target cells are trabecular meshwork cells, retinal pigment epithelium or photoreceptor cells.
  • AAV is used to transduce the target cells.
  • the genome editing systems of this disclosure can be implemented in any suitable manner, meaning that the components of such systems, including without limitation the RNA-guided nuclease, gRNA, and optional donor template nucleic acid, can be delivered, formulated, or administered in any suitable form or combination of forms that results in the transduction, expression or introduction of a genome editing system and/or causes a desired repair outcome in a cell, tissue or subject.
  • Tables 4 and 5 set forth several, non-limiting examples of genome editing system implementations. Those of skill in the art will appreciate, however, that these listings are not comprehensive, and that other implementations are possible. With reference to Table 4 in particular, the table lists several exemplary implementations of a genome editing system comprising a single gRNA and an optional donor template.
  • genome editing systems can incorporate multiple gRNAs, multiple RNA-guided nucleases, and other components such as proteins, and a variety of implementations will be evident to the skilled artisan based on the principles illustrated in the table.
  • [N/A] indicates that the genome editing system does not include the indicated component.
  • Nucleic acids encoding genome editing systems or components thereof can be delivered directly to cells as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by the target cells (e.g., erythrocytes, HSCs).
  • Nucleic acid vectors such as the vectors summarized in Table 5, can also be used.
  • Nucleic acid vectors can comprise one or more sequences encoding genome editing system components, such as an RNA-guided nuclease, a gRNA and/or a donor template.
  • a vector can also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), associated with (e.g., inserted into or fused to) a sequence coding for a protein.
  • a nucleic acid vector can include a Cas9 coding sequence that includes one or more nuclear localization sequences (e.g., a nuclear localization sequence from S V40).
  • the nucleic acid vector can also include any suitable number of regulatory /control elements, e.g., promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRES). These elements are well known in the art, and are described in Cotta-Ramusino.
  • regulatory /control elements e.g., promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRES). These elements are well known in the art, and are described in Cotta-Ramusino.
  • Nucleic acid vectors according to this disclosure include recombinant viral vectors. Exemplary viral vectors are set forth in Table 5, and additional suitable viral vectors and their use and production are described in Cotta-Ramusino. Other viral vectors known in the art can also be used.
  • viral particles can be used to deliver genome editing system components in nucleic acid and/or peptide form. For example, “empty” viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to alter target tissue specificity.
  • non-viral vectors can be used to deliver nucleic acids encoding genome editing systems according to the present disclosure.
  • One important category of non-viral nucleic acid vectors are nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art, and are summarized in Cotta-Ramusino. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. For instance, organic (e.g., lipid and/or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of this disclosure.
  • the vector is a lipid nanoparticle (LNP).
  • the LNP can have a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. In some embodiments, the LNP has a size ranging from about 1 nm to about 1000 nm. In some embodiments, the LNP has a size ranging from about 1 nm to about 500 nm. In some embodiments, the LNP has a size ranging from about 1 nm to about 250 nm. In some embodiments, the LNP has a size ranging from about 25 nm to about 200 nm.
  • the LNP has a size ranging from about 25 nm to about 100 nm. In some embodiments, the LNP has a size ranging from about 35 nm to about 75 nm. In some embodiments, the LNP has a size ranging from about 25 nm to about 60 nm.
  • LNPs can be made from ionizable lipids (e.g., cationic lipids), neutral lipids, structural lipids, helper lipids, and PEGylated lipids, or a combination of these.
  • fusogenic phospholipids e.g. DOPE
  • sterols e.g. cholesterol
  • Exemplary lipids for use in nanoparticle formulations, and/or gene transfer are shown in Table 7, and Table 8 lists exemplary polymers for use in gene transfer and/or nanoparticle formulations.
  • the non-viral vectors may be lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • Ionizable lipids may include, but are not limited to, MC3 (also called Dlin-MC3-DMA), SM-102, ALC-0315, 5A2-SC8, and BAMEA-O16B.
  • an ionizable lipid may include C12-200.
  • LNPs may comprise about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% ionizable lipid.
  • LNPs may further comprise cholesterol.
  • LNPs may further comprise a phospholipid, e.g., distearoylphosphatidylcholine (DSPC).
  • LNPs may further comprise a PEG lipid, e.g., dimethylglycine (DMG)-PEG2k.
  • the LNPs of the present disclosure comprise a cationic lipid, e.g., those cationic lipids disclosed in Table 7.
  • the cationic lipids that find use in the LNPs, compositions, and methods of the present disclosure include, but are not limited to: l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA); 1,2- dilinolenyloxyN, N-dimethylaminopropane (DLenDMA); 2,2-dilinoleyl-4-(2-dimethyl- aminoethyl)-[l,3]dioxolane (DLin-K-C2-DMA.; '"XTC2"'); 2,2-dilinoleyl-4-(3-dimethyl- aminoopropyl)- [1,3] -dioxolane (DLin-K-C3-DMA); 2,2-dilinoieyl
  • the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: LP-01 or (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate or a pharmaceutically acceptable salt thereof.
  • the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: (9Z,9'Z,12Z,12'Z)-2-(((3-(4-methylpiperazin-l- yl)propanoyl)oxy)methyl)propane-l,3-diyl bis(octadeca-9, 12-dienoate); (9Z,9'Z,12Z,12'Z)-2- (((4-(pyrrolidin-l-yl)butanoyl)oxy)methyl)propane-l,3-diylbis(octadeca-9, 12-dienoate); (9Z,9'Z, 12Z, 12'Z)-2-(((4-(piperidin- 1 -yl)butanoyl)oxy)methyl)propane- 1 , 3 -diyl bis(octadeca-
  • the cationic lipids that find use in the LNPs, compositions, and methods of the present disclosure include, but are not limited to: 6,6'- (Methylazanediyl)Bis(N,N-Dioctylhexanamide); 6,6'-(Octylazanediyl)Bis(N,N- Dioctylhexan-amide); 6,6'-(Hexylazanediyl)Bis(N,N-Dioctylhexanamide); 10, 10'- (Methylazanediyl)Bis (N,N-Dioctyldecanamide); 8,8'-(Methylazanediyl)Bis(N,N- Didecyloctanamide); 6,6'-(Methylazanediyl)Bis(N,N-Didecylhexanamide); 6,6'- (Methylazanediyl)
  • the LNPs of the present disclosure comprise a non-cationic lipid, e.g., those non-cationic lipids disclosed in Table 7.
  • the noncationic lipids that find use in the LNPs, compositions, and methods of the present disclosure include, but are not limited to lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dip
  • the PEGylated lipids comprise a PEG molecule with a molecular weight from about 200Da to about 5000Da. In certain embodiments, the PEGylated lipids comprise a PEG molecule with a molecular weight of 2000Da (2kDa).
  • the PEG-lipids can include, but are not limited to, those identified in Table 8.
  • the PEG-lipid can comprise PEG coupled to dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), methoxypolyethyleneglycol (PEG-DMG or PEG2000-DMG), PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides, PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof.
  • the PEG-lipid comprises detergent-like PEG lipids (e.g., PEG-DSPE).
  • the PEG moiety is conjugated directly to the lipid.
  • the PEG moiety is conjugated to the lipid via a linker moiety.
  • Any linker moiety suitable for conjugating the PEG to a lipid can be used including, but not limited to, ester-containing linker moieties and/or non-ester-containing linker moieties.
  • an ester-containing linker moiety is used to conjugate the PEG to the lipid.
  • Exemplary ester-containing linker moieties include, e.g., carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof.
  • non-ester containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-0-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety).
  • phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form the PEG-lipid conjugate.
  • phosphatidylethanolamines comprising saturated or unsaturated fatty acids with carbon chain lengths in the range of CIO to C20 are employed in connection with the LNPs, compositions, and methods disclosed herein.
  • phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used.
  • the phosphatidylethanolamines that find use in connection with the LNPs, compositions, and methods disclosed herein include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).
  • DMPE dimyristoylphosphatidylethanolamine
  • DPPE dipalmitoyl-phosphatidylethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • DSPE distearoyl-phosphatidylethanolamine
  • the PEG-DAA conjugate of the instant disclosure is a PEG- didecyloxypropyl (CIO) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG- dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16) conjugate, or a PEG- distearyloxypropyl (C18) conjugate.
  • the PEG moiety has an average molecular weight of about 750 or about 2,000 daltons.
  • the terminal hydroxyl group of the PEG moiety is substituted with a methyl group.
  • hydrophilic polymers can be used in place of PEG.
  • suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxy-propyl, methacrylamide, polymethacrylamide, and polydimethylacrylamide, polylactic acid, poly glycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
  • the LNP comprises a PEG-lipid where the PEG-lipid comprises at least 0. 1 mol% of the total lipid.
  • the PEG-lipid component can comprise from about 0. 1 mol% to 5 mol% of the total lipid.
  • the LNP comprises a cationic lipid where the cationic lipid component comprises at least 10 mol% of the total lipid.
  • the cationic lipid component can comprise from about 10 mol% to 70 mol% of the total lipid.
  • the cationic lipid component can comprise from about 10 mol% to 60 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 50 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 40 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 30 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 20 mol% of the total lipid. In certain embodiments, the LNP comprises cholesterol where cholesterol comprises at least 10 mol% of the total lipid.
  • cholesterol can comprise from about 10 mol% to 70 mol% of the total lipid.
  • the cholesterol can comprise from about 10 mol% to 60 mol% of the total lipid.
  • the cholesterol can comprise from about 10 mol% to 50 mol% of the total lipid.
  • the cholesterol can comprise from about 10 mol% to 40 mol% of the total lipid.
  • the cholesterol can comprise from about 10 mol% to 30 mol% of the total lipid.
  • the cholesterol can comprise from about 10 mol% to 20 mol% of the total lipid.
  • the LNP comprises a non-cationic lipid where the non-cationic lipid comprises at least 10 mol% of the total lipid.
  • the noncationic lipid can comprise from about 10 mol% to 70 mol% of the total lipid.
  • the non-cationic lipid component can comprise from about 10 mol% to 60 mol% of the total lipid.
  • the non-cationic lipid component can comprise from about 10 mol% to 50 mol% of the total lipid.
  • the non-cationic lipid component can comprise from about 10 mol% to 40 mol% of the total lipid.
  • the non-cationic lipid component can comprise from about 10 mol% to 30 mol% of the total lipid
  • the non-cationic lipid component can comprise from about 10 mol% to 20 mol% of the total lipid.
  • Non- viral vectors optionally include targeting modifications to improve uptake and/or selectively target certain cell types. These targeting modifications can include e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars (e.g., N-acetylgalactosamine (GalNAc)), and cell penetrating peptides.
  • Such vectors also optionally use fusogenic and endosome-destabilizing peptides/polymers, undergo acid- triggered conformational changes (e.g., to accelerate endosomal escape of the cargo), and/or incorporate a stimuli-cleavable polymer, e.g., for release in a cellular compartment.
  • a stimuli-cleavable polymer e.g., for release in a cellular compartment.
  • disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.
  • nucleic acid molecules other than the components of a genome editing system, e.g., the RNA-guided nuclease molecule component and/or the gRNA molecule component described herein, are delivered.
  • the nucleic acid molecule is delivered at the same time as one or more of the components of the genome editing system.
  • the nucleic acid molecule is delivered before or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the genome editing system are delivered.
  • the nucleic acid molecule is delivered by a different means than one or more of the components of the genome editing system, e.g., the RNA-guided nuclease molecule component and/or the gRNA molecule component, are delivered.
  • the nucleic acid molecule can be delivered by any of the delivery methods described herein.
  • the nucleic acid molecule can be delivered by a viral vector, e.g., an integration-deficient lentivirus, and the RNA-guided nuclease molecule component and/or the gRNA molecule component can be delivered by electroporation, e.g., such that the toxicity caused by nucleic acids (e.g., DNAs) can be reduced.
  • the nucleic acid molecule encodes a therapeutic protein, e.g., a protein described herein. In certain embodiments, the nucleic acid molecule encodes an RNA molecule, e.g., an RNA molecule described herein.
  • RNPs complexes of gRNAs and RNA-guided nucleases
  • RNAs encoding RNA-guided nucleases and/or gRNAs can be delivered into cells or administered to subjects by art-known methods, some of which are described in Cotta-Ramusino.
  • RNA- guided nuclease-encoding and/or gRNA-encoding RNA can be delivered, e.g., by microinjection, electroporation, transient cell compression or squeezing (see, e.g., Lee 2012).
  • Lipid-mediated transfection, peptide-mediated delivery, GalNAc- or other conjugate- mediated delivery, and combinations thereof, can also be used for delivery in vitro and in vivo.
  • a protective, interactive, non-condensing (PINC) system may be used for delivery.
  • In vitro delivery via electroporation comprises mixing the cells with the RNA encoding RNA-guided nucleases and/or gRNAs, with or without donor template nucleic acid molecules, in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude.
  • Systems and protocols for electroporation are known in the art, and any suitable electroporation tool and/or protocol can be used in connection with the various embodiments of this disclosure.
  • aureus Cas9 (5aCas9) guide RNAs (gRNAs) were designed to target the UTR and coding sequence of the MYOC gene using the PAM NNGRRT (see Table 9 for the sequences of the .S'aCas9 gRNA targeting domains) to identify ribonucleoproteins (RNPs) that introduce frameshift indels likely to disrupt expression of functional MYOC.
  • RNP 0.178 pM, 0.356 pM, 0.7125 pM, 1.425 pM, 2.85 pM, 5.7 pM, and 11.4 pM) of RNP comprising an .ShCas9 gRNA complexed with wildtype 5aCas9 enzyme in a 2: 1 ratio.
  • the genomic DNA was extracted four days post nucleofection and NGS was performed (see Table 15 for NGS primers used) on the MYOC PCR products to assess the total percentage of indels (Fig. 2A) and the percentage of frameshift indels (Fig. 2B).
  • RNPs comprising AvCas 12a gRNAs (Table 10) complexed with an AvCas 12a (AsCasl2a polypeptide sequence is set forth in SEQ ID NO:33) in a 2:1 ratio (guide RNA/Casl2a) were screened in a T cell assay to analyze the RNP gene editing.
  • AvCas 12a gRNAs that introduced greater than 80% indels and targeted the 5’UTR or exon 1 of the MYOC gene were selected for further assessment (Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, Casl2a-8 (Table 10)). Briefly, primary T cells were nucleofected with increasing concentrations (0.010 pM, 0.030 pM, 0.080 pM, 0.25 pM, 0.80 pM, 2.53 pM, and 8.0 pM) of RNPs.
  • the genomic DNA was extracted four days post nucleofection and NGS (see Table 16 for NGS primers used) was performed on the MYOC PCR products to assess the total percentage of indels (Fig. 4A) and percentage of frameshift indels (Fig. 4B).
  • Example 3 Acidaminococcus sp. Casl2a guide RNAs targeting the MYOC gene provide an increase in gene editing in human trabecular meshwork cells
  • HTMCs human trabecular meshwork cells
  • RNPs comprising an .S'aCas9 gRNA (Cas9-1 , Cas9-5, Cas9-6, Cas9-24 (Table 9)) complexed with S «Cas9 CS «Cas9 polypeptide sequence is set forth in SEQ ID NO:47) or an sCas l 2a gRNA (Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, or Casl2a-8 (Table 10)) complexed with AsCas 12a (A Casl2a polypeptide sequence is set forth in SEQ ID NO:33).
  • the genomic DNA was extracted four days post nucleofection and NGS (see Tables 15 and 16 for NGS primers used) was performed on the MYOC PCR products to assess the percentage of total indels (Fig. 5A) and the percentage of frameshift indels (Fig. 5B).
  • Example 4 Promoters for driving expression in human trabecular meshwork cells
  • HTMCs Various putative promoters were tested to identify promoters capable of driving expression in HTMCs. Based on single cell RNAseq data (Van Zyl 2020), genes that are strongly expressed in human trabecular meshwork cell populations were identified. The UCSC Browser and Eukaryotic Promoter Database were used to select the potential promoter and enhancer sequences that were then synthesized and cloned into plasmids. Briefly, HTMCs were nucleofected with 4.7E+10 copies (-200 ng/grl J of plasmid containing a putative promoter driving expression of enhanced green fluorescent protein (GFP or EGFP).
  • GFP enhanced green fluorescent protein
  • the promoters tested were DCN #1, DCN #2, ID3, MT2A, SERPINF1, SERPING1 #1, SERPING1 #2, TIMP1, CP, MT IX, OLFML3, PCOLCE, PDGFRA, PTN, RARRE SI, RBP4 #1, RBP4 #2, RBP4 #3, PDPN, APOD, B2M, PTGDS, EEF1A1, ANGPTL7, MGP, RPS24, PLA2G2A, CHI3L1, FTL, TAGLN, PCP4, MYL 9, MYOC, CMV, and mini-CMV (see Table 6 for the sequences of the promoters).
  • the following day cells were subjected to flow cytometry to determine the percentage of GFP+ cells and mean fluorescence intensity of GFP+ cells (Figs. 6A and 6B, respectively).
  • Example 5 Screening ionizable lipids for LNP delivery
  • LNPs lipid nanoparticles
  • LNPs (MC3, SM- 102, ALC-0315, BAMEA-O16B) were formed with 50% ionizable lipid, 38.5% cholesterol, 10% distearoylphosphatidylcholine (DSPC), and 1.5% dimethylglycine (DMG)-PEG2k and encapsulated GFP messenger RNA (mRNA) and irrelevant gRNA.
  • 5A2-SC8 LNP was formed with 25% ionizable lipid, 48.5% cholesterol, 25% DSPC, and 1.5% DMG-PEG2k and encapsulated GFP mRNA and gRNA.
  • the lipid nanoparticle components ionizable lipid, cholesterol, DSPC, and DMG-PEG2k were dissolved in 100% ethanol and mixed in the indicated molar ratios.
  • the RNA cargo which included GFP mRNA and irrelevant gRNA (1:1 weight ratio (100% GFP mRNA:gRNA) was dissolved in 50 mM citrate buffer (pH 4.5), resulting in a concentration of RNA cargo of approximately 0.12 mg/mL.
  • LNPs were formed by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems Ignite or Spark Instrument, in accordance with the manufacturer’ s protocol.
  • LNPs were collected and ethanol was removed by one of the following two methods: 1) LNPs were diluted in PBS or TBS (1:40, vokvol) and loaded into AmiconTM Centrifugal Filter Units for ultrafiltration (MilliporeSigma, 30kD); or 2) LNPs were loaded into 10 kDa Slide-a-Lyzer G2 Dialysis Cassettes (ThermoFisher Scientific) for dialysis in TBS or PBS under gentle stirring (1 hour in room temperature and new buffer change, 3 hours in 4 °C and new buffer change, and overnight at 4 °C). The resultant mixture was concentrated to the target concentrations and then filtered using a 0.2-mm sterile filter. The filtrate was stored at 2 °C-8 °C for use within a week or stored at -80 °C for a longer time after addition of 10% sucrose.
  • FIG. 7A PBS (Vehicle)
  • 7B LNPs formed with MC3
  • 7C LNPs formed with SM-102
  • 7D LNPs formed with ALC-0315
  • 7E LNPs formed with 5A2-SC8
  • 7F LNPs formed with BAMEA-O16B
  • 7G LNPs formed with BAMEA- O16B
  • 7H LNPs formed with SM-102
  • Results showed that LNPs formed with MC3, SM-102, ALC-0315, and 5A2-SC8 ionizable lipids successfully mediated delivery of GFP mRNA to the trabecular meshwork (TM).
  • TM trabecular meshwork
  • MC3, ALC-0315, and 5A2-SC8 showed the highest specificity for the TM tissue, while SM-102 transfected multiple tissues within the anterior chamber.
  • Example 6 LNP ex vivo delivery to human and non-human primate corneal rims
  • LNPs encapsulating GFP mRNA and irrelevant gRNA were administered to human and non-human primate (NHP) corneal rims. Briefly, LNPs were formulated as described in Example 5 with either 50% MC3 or 50% ALC-0315 ionizable lipids (Table 11), 38.5% cholesterol, 10% DSPC, and 1.5% DMG-PEG2k and encapsulated GFP mRNA and irrelevant gRNA using the Ignite instrument.
  • LNPs were formulated as described in Example 5 using the Ignite instrument with two different PEG lipids at different percentages: DMG-PEG2k (1%, 1.5%, 2%, 3%) or DSPE- PEG2k (1%, 1.5%, 2%, 3%), each encapsulating GFP mRNA and irrelevant gRNA (50% ALC-0315, 10% DSPC, 39%, 38.5%, 38%, or 37% cholesterol (adjusted to the respective PEG ratio)).
  • LNPs encapsulating GFP mRNA and irrelevant gRNA formed with different percentages of ALC-0315, cholesterol, and DSPC with a fixed amount of 1.5% DMG-PEG2k were screened for transfection of primary human TM cells and HEK293T cells.
  • Table 12 shows the percent transfection of primary human TM cells and HEK293T cells, along with the biophysical properties of LNPs formulated with different ratios of ALC- 0315, cholesterol, and DSPC with a fixed 1.5% DMG-PEG2K (i.e., percentage of encapsulation of GFP-mRNA (Encap.
  • LNPs listed in Table 12 were formulated as described in Example 5. LNPs were formed by microfluidic mixing of the lipid and RNA solutions using the Spark Instrument from Precision NanoSystems, in accordance with the manufacturer’s protocol. Encapsulation efficiencies were determined by RiboGreen assay. Particle size and polydispersity were measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument.
  • DLS dynamic light scattering
  • LNP-GFP mRNA i.e., GFP mRNA encapsulated in LNPs
  • TM cells 0.05 pg/ml LNP-GFP mRNA (i.e., GFP mRNA encapsulated in LNPs)
  • TM cells 0.05 pg/ml LNP-GFP mRNA encapsulated in LNPs
  • HEK293T cells 0.005 pg/ml LNP-GFP mRNA for HEK293T cells. 24 hours later the percent GFP positive cells were determined by flow cytometry. Table 12. Biophysical Properties of LNPs and Transfection and Encapsulation
  • Figs. 10A and 10B show concentration dependent transfection of primary human TM cells and HEK293T cells, respectively, with LNPs (LNP1, LNP2, LNP5, LNP8, LNP14, LNP15, LNP17, LNP19 (Table 12)) with varying lipid ratios (ALC-0315, cholesterol, DSPC, and DMG-PEG2k).
  • LNPs LNP1, LNP2, LNP5, LNP8, LNP14, LNP15, LNP17, LNP19 (Table 12)
  • ALC-0315, cholesterol, DSPC, and DMG-PEG2k varying lipid ratios
  • Example 9 In vivo screening of LNPs for delivery to murine eyes
  • LNPs encapsulating GFP mRNA and irrelevant gRNA were generated as described in Example 5 and Example 8.
  • Wild-type C57BL/6J mice (aged 6-8 weeks) were dosed LNPs with 500 pg/mL of LNP-GFP mRNA (i.e., GFP mRNA encapsulated in LNPs) (5 mice per group for each LNP) via intracameral (i.c.) injections (1 pL in left eye only) and euthanized 24 hours later.
  • LNP-GFP mRNA i.e., GFP mRNA encapsulated in LNPs
  • 11A Vehicle, PBS
  • 11B LNP1
  • 11C LNP8
  • 11D LNP14
  • HE LNP15
  • HF LNP19
  • LNP1, LNP8, LNP14, LNP15, and LNP19 successfully mediated delivery of GFP mRNA to the TM.
  • Example 10 LNP delivered AsCasl2a mRNA and gRNA mediated ex-vivo editing of MYOC gene in human corneal rims
  • Fig. 12 shows results of editing using LNP encapsulating AsCasl2a mRNA and Casl2a-8 gRNA.
  • LNPs 50% ALC-0315, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k
  • mRNA encoding AsCasl2a SEQ ID NO:33
  • Casl2a-1 gRNA or Casl2a-2 gRNA (Table 10) were formulated as described in Example 5 using the Ignite instrument.
  • the RNA cargo concentration of AsCasl2a mRNA and gRNA are at a 1:1 ratio by weight in the LNPs.
  • Cynomolgus monkeys received intracameral injections of LNPs in both eyes (7.5 pg AsCasl2a mRNA/eye) and were euthanized one week later (Table 13).
  • LNPs 50% ALC-0315, 46% cholesterol, 12.5% DSPC, and 1.5% DMG-PEG2K
  • mRNA encoding AsCasl2a SEQ ID NO:33
  • Casl2a-1 gRNA Table 10
  • the RNA cargo concentration of AsCasl2a mRNA and gRNA are at a 1 : 1 ratio by weight in the LNPs.
  • mice Myoc mouse Myoc gene
  • human MYOC gene carrying the pathogenic Y437H mutation hMYOC Y437H
  • the knock- in gene includes the full-length human mutant MYOC Y437H gene in addition to 1.5 kb upstream, which contains a portion of the human promoter (Fig. 15A).
  • Intraocular pressure (IOP) was measured monthly on unanesthetized mice using a tonometer.
  • the MYO 1 -HOM mouse model (MYOC Y437H7Y437H ') presents with elevated IOP levels compared to wild-type control mice (Fig. 15B).
  • the mouse TM tissue is too small to dissect, therefore anterior chambers were collected followed by extraction of mRNA and gDNA.
  • the anterior chamber has a mixed cell population that includes the TM tissue, cornea, ciliary body, and other ancillary tissues.
  • Fig. 15B shows the percentage of myocilin mRNA remaining as determined by RT-ddPCR.
  • Fig. 15C shows the percentage of indels introduced into the MYOC gene determined by Ill-Seq (see Table 16 for NGS primers used).
  • Example 14 Non-human primate study to determine the dose dependency and ocular tolerability of editing using LNP delivery
  • LNPs 50% ALC0315, 46% cholesterol, 12.5% DSPC and 1.5% DMG-PEG2K encapsulating mRNA encoding AsCasl2a (SEQ ID NO:33) plus Casl2a-1 gRNA (Table 10) were formulated as described in Example 5 in TBS and 10% sucrose.
  • the RNA cargo concentration of AsCasl2a mRNA and gRNA are at a 1:1 ratio by weight in the LNPs.

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Abstract

Genome editing systems, guide RNAs, and CRISPR-mediated methods are provided for altering and/or modulating the expression of the MYOC gene and treating Primary Open Angle Glaucoma (POAG).

Description

CRISPR/RNA-GUIDED NUCLEASE RELATED METHODS AND COMPOSITIONS FOR TREATING PRIMARY OPEN ANGLE GLAUCOMA
PRIORITY CLAIM
[0001] This application claims the benefit of U.S. Provisional Application No. 63/575,569, filed April 5, 2024, and U.S. Provisional Application No. 63/643,039, filed May 6, 2024, both of which are incorporated herein by reference in their entirety, including the drawings.
SEQUENCE LISTING
[0002] This application contains a ST.26 compliant Sequence Listing, which was submitted in XML format via Patent Center, and is hereby incorporated by reference in its entirety. The XML copy, created on April 4, 2025, is named 1189458021WO00.xml and is 627,000 bytes in size.
FIELD
[0003] This disclosure relates to genome editing systems and methods for altering a target nucleic acid sequence, or modulating expression of a target nucleic acid sequence, and applications thereof in connection with the alteration or modulation of the MYOC gene and/or treatment of Primary Open Angle Glaucoma (POAG).
BACKGROUND
[0004] Glaucoma is the second leading cause of blindness in the world. POAG is the leading cause of glaucoma, representing more than 50% of glaucoma in the United States (Quigley 1997). POAG affects over 3 million subjects in the United States (Glaucoma Research Foundation: www.glaucoma.org; accessed December 14, 2021). Approximately 1% of subjects ages 40-89 have POAG.
[0005] POAG develops due to an imbalance between the production and outflow of aqueous humor within the eye. Aqueous humor (AH) is produced by the ciliary body located in the anterior chamber of the eye. The vast majority (80%) of AH drains through the trabecular meshwork (TM) to the episcleral venous system. The remainder (20%) of AH drains through the interstitium between the iris root and ciliary muscle (Feisal 2005). POAG is likely due to decreased drainage through the trabecular meshwork. Decreased outflow of AH results in increased intraocular pressure (IOP). IOP causes damage to the optic nerve and leads to progressive blindness. [0006] Mutations in the MYOC gene have been shown to be a leading genetic cause of POAG, accounting for 3% of POAG. Approximately 90,000 individuals in the United States have POAG that is caused by MYOC mutations. Many patients with MYOC mutations develop rapidly advancing disease and early-onset POAG, including juvenile-onset POAG.
[0007] MYOC mutations are inherited in an autosomal dominant fashion. Disease-causing mutations cluster in the olfactomedin domain of exon 3 of the MYOC gene. The most common MYOC mutation causing severe, early onset disease is a proline to leucine substitution at amino acid position 370 (P370L) (Waryah 2013). The most common MYOC mutation is a missense mutation at amino acid position 368 (Q368X). This mutation is associated with less severe disease, termed late-onset POAG.
[0008] Treatments that reduce IOP can slow the progression of POAG. Trabeculectomy surgery and eye drops are both effective in reducing IOP. Eye drops include alpha- adrenergic antagonists and beta-adrenergic antagonists. However, POAG is known as a silent cause of blindness, as it is painless and leads to progressive blindness if left untreated.
Despite advances in POAG therapies, there remains a need for the treatment and prevention of POAG. A one-time or several dose treatment that reduces IOP and prevents the progression of POAG would be beneficial in the treatment and prevention of POAG.
SUMM RY
[0009] Methods and compositions discussed herein are designed to allow for the correction of disorders of the eye, e.g., disorders that affect trabecular meshwork cells, photoreceptor cells and any other cells in the eye, including those of the iris, ciliary body, optic nerve or aqueous humor.
[0010] In one aspect, methods and compositions discussed herein provide for treating or delaying the onset or progression of POAG. POAG is a common form of glaucoma, characterized by damage to the optic nerve resulting in loss of vision. Degeneration of the trabecular meshwork leads to obstruction of the normal ability of aqueous humor to leave the eye without closure of the space (e.g., the '‘angle”) between the iris and cornea. This obstruction leads to increased IOP, which can result in progressive visual loss and blindness if not treated appropriately and in a timely fashion. POAG is a progressive ophthalmologic disorder characterized by increased IOP. [0011] Mutations in the MYOC gene (also known as GPOA, JO AG, TIGR, GLC1A, JOAG1 and myoci I in) have been shown to account for 3% of POAG. Certain mutations in MYOC lead to severe, early onset POAG. Mutations in the MYOC gene leading to POAG can be described based on the mutated amino acid residue(s) in the MY OC protein. Severe, early- onset POAG can be caused by mutations in the MYOC gene, including mutations in exon 3. Exemplary mutations include, but are not limited to, the mutations T377R, 1477, and P370L (Zhuo 2008).
[0012] The methods and compositions disclosed herein are broadly applicable to any mutation, e.g., a point mutation or a deletion, in the MYOC gene that gives rise to POAG.
[0013] In another aspect, methods and compositions discussed herein may be used to alter the MYOC gene to treat or prevent POAG by targeting the MYOC gene, e.g., the 5’ untranslated region (5’ UTR), exon 1, the exon 1/intron 1 border, intron 1, the intron 1/exon 2 border, exon 2, the exon 2/intron 2 border, intron 2, the intron 2/exon 3 border, exon 3, the exon 3/intron 3 border, the 3’ UTR or any combination thereof.
[0014] In another aspect, the methods and compositions discussed herein may be used to alter the MYOC gene to treat or prevent POAG by targeting the coding sequence of the MYOC gene. In one embodiment, the gene, e.g., the coding sequence of the MYOC gene, is targeted to knockout the gene, e.g., to eliminate expression of the gene, e.g., to knockout both alleles of the MYOC gene, e.g., by induction of an alteration comprising a deletion or mutation in the MYOC gene. In an embodiment, the method provides an alteration that comprises an insertion or deletion. While not wishing to be bound by theory, in an embodiment, a targeted knockout approach is mediated by non-homologous end joining (NHEJ) using a CRISPR/RNA-guided nuclease system comprising an RNA-guided nuclease (e.g., Casl2a molecule). In certain embodiments, the alteration may comprise an indel. In certain embodiments, the alteration may comprise a frameshift indel that results in decreased expression of the MYOC gene.
[0015] In one embodiment, a coding region, e.g., an early coding region, of the MYOC gene is targeted to knockout the MYOC gene. In an embodiment, targeting affects both alleles of the MYOC gene. In an embodiment, a targeted knockout approach reduces or eliminates expression of functional MYOC gene product. In an embodiment, the method provides an alteration that comprises an insertion or deletion. [0016] In another aspect, the methods and compositions discussed herein may be used to alter the MYOC gene to treat or prevent POAG by targeting a non-coding sequence of the MYOC gene, e.g., promoter, an enhancer, an intron, 5’UTR, 3’UTR, and/or poly adenylation signal. In one embodiment, the gene, e.g., the non-coding sequence of the MYOC gene, is targeted to knockout the gene, e.g., to eliminate expression of the gene, e.g., to knockout both alleles of the MYOC gene, e.g., by induction of an alteration comprising a deletion or mutation in the MYOC gene. In an embodiment, the method provides an alteration that comprises an insertion or deletion.
[0017] In certain embodiments, the 5’ UTR of exon 1, exon 1, the exon 1/intron 1 border, intron 1, the intron 1/exon 2 border, exon 2, the exon 2/intron 2 border, intron 2, the intron 2/exon 3 border, exon 3, the exon 3/intron 3 border, the 3’ UTR or any combination thereof of the MYOC gene is targeted for alteration of the MYOC gene. In certain embodiments, the 5’ UTR region is targeted for alteration of the MYOC gene. In certain embodiments, exon 1 of the MYOC gene is targeted for alteration of the MYOC gene. In certain embodiments, the exon 1/intron 1 border of the MYOC gene is targeted for alteration of the MYOC gene. In certain embodiments, intron 1 of the MYOC gene is targeted for alteration of the MYOC gene. In certain embodiments, the intron 1/exon 2 border of the MYOC gene is targeted for alteration of the MYOC gene. In certain embodiments, exon 2 of the MYOC gene is targeted for alteration of the MYOC gene. In certain embodiments, the exon 2/intron 2 border of the MYOC gene is targeted for alteration of the MYOC gene. In certain embodiments, intron 2 of the MYOC gene is targeted for alteration of the MYOC gene. In certain embodiments, the intron 2/exon 3 border of the MYOC gene is targeted for alteration of the MYOC gene. In certain embodiments, exon 3 border of the MYOC gene is targeted for alteration of the MYOC gene. In certain embodiments, the intron exon 3/intron 3 border of the MYOC gene is targeted for alteration of the MYOC gene. In certain embodiments, the 3’ UTR region is targeted for alteration of the MYOC gene.
[0018] In one aspect, disclosed herein is a gRNA molecule, e.g., an isolated or non- naturally occurring gRNA molecule, comprising a targeting domain that binds a target domain of a MYOC gene. In certain embodiments, the gRNA molecule may comprise a targeting domain which is complementary with a target domain from the MYOC gene.
[0019] In an embodiment, the gRNA molecule is configured to, in combination with a corresponding RNA-guided nuclease, provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a MYOC target position in the MYOC gene to allow alteration, e.g., alteration associated with HDR or NHEJ, of A MYOC target position in the MYOC gene. In an embodiment, the targeting domain is configured such that a cleavage event, e.g., a double strand or single strand break, is positioned within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of a MYOC target position. The break, e.g., a double strand or single strand break, can be positioned upstream or downstream of a MYOC target position in the MYOC gene.
[0020] In an embodiment, a second gRNA molecule comprising a second targeting domain is configured to, in combination with a corresponding RNA-guided nuclease, provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to the MYOC target position in the MYOC gene, to allow alteration, e.g., alteration associated with HDR or NHEJ, of the MYOC target position in the MYOC gene, either alone or in combination with the break positioned by the first gRNA molecule. In an embodiment, the targeting domains of the first and second gRNA molecules are configured such that a cleavage event, e.g., a double strand or single strand break, is positioned, independently for each of the gRNA molecules, within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of the target position. In an embodiment, the breaks, e.g., double strand or single strand breaks, are positioned on both sides of a nucleotide of a MYOC target position in the MYOC gene. In an embodiment, the breaks, e.g., double strand or single strand breaks, are positioned on one side, e.g., upstream or downstream, of a nucleotide of a MYOC target position in the MYOC gene.
[0021] In an embodiment, a single strand break is accompanied by an additional single strand break, positioned by a second gRNA molecule, as discussed below. For example, the targeting domains are configured such that a cleavage event, e.g., the two single strand breaks, are positioned within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of a MYOC target position. In an embodiment, the first and second gRNA molecules are configured such that, when guiding an RNA-guided nuclease nickase molecule (e.g., a Casl2a nickase), a single strand break will be accompanied by an additional single strand break, positioned by a second gRNA molecule, sufficiently close to one another to result in alteration of a MYOC target position in the MYOC gene. In an embodiment, the first and second gRNA molecules are configured such that a single strand break positioned by the second gRNA molecule is within 10, 20, 30, 40, or 50 nucleotides of the break positioned by the first gRNA molecule, e.g., when the RNA- guided nuclease is a Casl2a nickase. In an embodiment, the two gRNA molecules are configured to position cuts at the same position, or within a few nucleotides of one another, on different strands, e.g., essentially mimicking a double strand break.
[0022] In an embodiment, a double strand break can be accompanied by an additional double strand break, positioned by a second gRNA molecule, as is discussed below. For example, the targeting domain of a first gRNA molecule is configured such that a double strand break is positioned upstream of a MYOC target position in the MYOC gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of the target position; and the targeting domain of a second gRNA molecule is configured such that a double strand break is positioned downstream of a MYOC target position in the MFOC gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of the target position.
[0023] In an embodiment, a double strand break can be accompanied by two additional single strand breaks, positioned by a second gRNA molecule and a third gRNA molecule. For example, the targeting domain of a first gRNA molecule is configured such that a double strand break is positioned upstream of a MYOC target position in the MYOC gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of the target position; and the targeting domains of a second and third gRNA molecule are configured such that two single strand breaks are positioned downstream of a MYOC target position in the MYOC gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of the target position. In an embodiment, the targeting domain of the first, second and third gRNA molecules are configured such that a cleavage event, e.g., a double strand or single strand break, is positioned, independently for each of the gRNA molecules.
[0024] In an embodiment, a first and second single strand breaks can be accompanied by two additional single strand breaks positioned by a third gRNA molecule and a fourth gRNA molecule. For example, the targeting domain of a first and second gRNA molecule are configured such that two single strand breaks are positioned upstream of a MYOC target position in the MYOC gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of the target position; and the targeting domains of a third and fourth gRNA molecule are configured such that two single strand breaks are positioned downstream of a MYOC target position in the MYOC gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides of the target position.
[0025] It is contemplated herein that, in an embodiment, when multiple gRNAs are used to generate (1) two single stranded breaks in close proximity, (2) two double stranded breaks, e.g., flanking a MYOC target position, e.g., a mutation (e.g., to remove a piece of DNA, e.g., a insertion mutation), or to create more than one indel in the MYOC gene, (3) one double stranded break and two paired nicks flanking a MYOC target position, e.g., a mutation (e.g., to remove a piece of DNA, e.g., a insertion mutation) or (4) four single stranded breaks, two on each side of a mutation, that they are targeting the same MYOC target position. It is further contemplated herein that multiple gRNAs may be used to target more than one MYOC target position in the same gene.
[0026] In an embodiment, the targeting domain of the first gRNA molecule and the targeting domain of the second gRNA molecules are complementary to opposite strands of the target nucleic acid molecule. In an embodiment, the gRNA molecule and the second gRNA molecule are configured such that the PAMs are oriented outward.
[0027] In an embodiment, the targeting domain of a gRNA molecule is configured to avoid unwanted target chromosome elements, such as repeat elements, e.g., Alu repeats, in the target domain. The gRNA molecule may be a first, second, third and/or fourth gRNA molecule, as described herein.
[0028] In an embodiment, the targeting domain of a gRNA molecule comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence set forth in Table 10. In an embodiment, the targeting domain of a gRNA molecule comprises a sequence that is the same as, or differs by no more than 3 nucleotides from, a sequence of a targeting domain selected from the group consisting of a Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8 targeting domain (Table 10).
[0029] In an embodiment, when two or more gRNA molecules are used to position two or more breaks, e.g., two single stranded breaks, in the target nucleic acid sequence, each guide RNA molecule comprises a targeting domain sequence that is independently selected from any one of those in Table 10. In an embodiment, the targeting domain of a gRNA molecule comprises a sequence selected from the group consisting of a Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8 targeting domain (Table 10). [0030] In another embodiment, the 5’ UTR region, exon 1, the exon 1/intron 1 border, intron 1, the intron 1/exon 2 border, exon 2, the exon 2/intron 2 border, intron 2, the intron 2/exon 3 border, exon 3, the exon 3/intron 3 border, the 3’ UTR region or a combination thereof of the MYOC gene is targeted, e.g., for alteration. In an embodiment, the targeting domain comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence in Table 10. In an embodiment, the targeting domain is independently selected from those in Table 10.
[0031] In an embodiment, the targeting domain of the gRNA molecule is configured to target an RNA-guided nuclease (e.g., Casl2a molecule), sufficiently close to a MYOC target position to reduce, decrease or repress expression of the MYOC gene.
[0032] In an embodiment, the gRNA molecule, e.g., a gRNA molecule comprising a targeting domain, which is complementary with the MYOC gene, is a modular gRNA molecule. In other embodiments, the gRNA molecule is a unimolecular or chimeric gRNA molecule.
[0033] In an embodiment, the targeting domain, which is complementary with a target domain from the MYOC gene, is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides or more in length. In an embodiment, the targeting domain is 16 nucleotides in length. In an embodiment, the targeting domain is 17 nucleotides in length. In another embodiment, the targeting domain is 18 nucleotides in length. In still another embodiment, the targeting domain is 19 nucleotides in length. In still another embodiment, the targeting domain is 20 nucleotides in length. In still another embodiment, the targeting domain is 21 nucleotides in length. In still another embodiment, the targeting domain is 22 nucleotides in length. In still another embodiment, the targeting domain is 23 nucleotides in length. In still another embodiment, the targeting domain is 24 nucleotides in length. In still another embodiment, the targeting domain is 25 nucleotides in length. In still another embodiment, the targeting domain is 26 nucleotides in length.
[0034] In an embodiment, the targeting domain comprises 16 nucleotides. In an embodiment, the targeting domain comprises 17 nucleotides. In an embodiment, the targeting domain comprises 18 nucleotides. In an embodiment, the targeting domain comprises 19 nucleotides. In an embodiment, the targeting domain comprises 20 nucleotides. In an embodiment, the targeting domain comprises 21 nucleotides. In an embodiment, the targeting domain comprises 22 nucleotides. In an embodiment, the targeting domain comprises 23 nucleotides. In an embodiment, the targeting domain comprises 24 nucleotides. In an embodiment, the targeting domain comprises 25 nucleotides. In an embodiment, the targeting domain comprises 26 nucleotides.
[0035] A cleavage event, e.g., a double strand or single strand break, may be generated by an RNA-guided nuclease molecule (e.g., Casl2a molecule). The RNA-guided nuclease molecule (e.g., Casl2a molecule) may form a double strand break in a target nucleic acid or a single strand break in a target nucleic acid (e.g., a nickase molecule). In an embodiment, the RNA-guided nuclease molecule (e.g., Casl2a molecule) may catalyze a double strand break.
[0036] In an embodiment, a single strand break may be formed in the strand of the target nucleic acid to which the targeting domain of the gRNA molecule is complementary. In another embodiment, a single strand break may be formed in the strand of the target nucleic acid other than the strand to which the targeting domain of the gRNA molecule is complementary.
[0037] In another aspect, disclosed herein is a nucleic acid, e.g., an isolated or non-naturally occurring nucleic acid, e.g., DNA, that may comprise (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a MYOC target position in the MYOC gene as disclosed herein.
[0038] In an embodiment, the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain configured to, in combination with a corresponding RNA-guided nuclease, provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a MYOC target position in the MYOC gene to allow alteration, e.g., alteration associated with NHEI, of a MYOC target position in the MYOC gene.
[0039] In an embodiment, the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain configured to target an RNA-guided nuclease molecule (e.g., Casl2a molecule), sufficiently close to a MYOC target position to reduce, decrease or repress expression of the MYOC gene.
[0040] In an embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Table 10. In an embodiment, the nucleic acid encodes a gRNA molecule comprising a targeting domain that is selected from those in Table 10. [0041] In an embodiment, the nucleic acid encodes a modular gRNA molecule, e.g., one or more nucleic acids encode a modular gRNA molecule. In another embodiment, the nucleic acid encodes a chimeric gRNA molecule. The nucleic acid may encode a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain comprising 16 nucleotides or more in length. In an embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 16 nucleotides in length. In another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 17 nucleotides in length. In another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 18 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 19 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 20 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 21 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 22 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 23 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 24 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 25 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain that is 26 nucleotides in length.
[0042] In an embodiment, the targeting domain comprises 16 nucleotides. In an embodiment, the targeting domain comprises 17 nucleotides. In an embodiment, the targeting domain comprises 18 nucleotides. In an embodiment, the targeting domain comprises 19 nucleotides. In an embodiment, the targeting domain comprises 20 nucleotides. In an embodiment, the targeting domain comprises 21 nucleotides. In an embodiment, the targeting domain comprises 22 nucleotides. In an embodiment, the targeting domain comprises 23 nucleotides. In an embodiment, the targeting domain comprises 24 nucleotides. In an embodiment, the targeting domain comprises 25 nucleotides. In an embodiment, the targeting domain comprises 26 nucleotides.
[0043] In an embodiment, a nucleic acid comprises (a) a sequence that encodes a gRNA molecule e.g., the first gRNA molecule, comprising a targeting domain that is complementary with a target domain in the MYOC gene as disclosed herein, and further comprising (b) a sequence that encodes an RNA-guided nuclease molecule (e.g., Casl2a molecule).
[0044] The RNA-guided nuclease molecule (e.g., Casl2a molecule) may form a double strand break in a target nucleic acid or a single strand break in a target nucleic acid (e.g., a nickase molecule).
[0045] In an embodiment, a single strand break is formed in the strand of the target nucleic acid to which the targeting domain of the gRNA molecule is complementary. In another embodiment, a single strand break is formed in the strand of the target nucleic acid other than the strand to which to which the targeting domain of the gRNA molecule is complementary.
[0046] In an embodiment, the RNA-guided nuclease molecule (e.g., Casl2a molecule) catalyzes a double strand break.
[0047] A nucleic acid disclosed herein may comprise (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the MYOC gene as disclosed herein; and/or (b) a sequence that encodes an RNA-guided nuclease molecule (e.g., Casl2a molecule).
[0048] A nucleic acid disclosed herein may comprise (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the MYOC gene as disclosed herein; (b) a sequence that encodes an RNA-guided nuclease molecule (e.g., Casl2a molecule); and further may comprise (c)(i) a sequence that encodes a second gRNA molecule described herein having a targeting domain that is complementary to a second target domain of the MYOC gene, and optionally, (c)(ii) a sequence that encodes a third gRNA molecule described herein having a targeting domain that is complementary to a third target domain of the MYOC gene; and optionally, (c)(iii) a sequence that encodes a fourth gRNA molecule described herein having a targeting domain that is complementary to a fourth target domain of the MYOC gene. [0049] In an embodiment, a nucleic acid encodes a second gRNA molecule comprising a targeting domain configured to, in combination with a corresponding RNA-guided nuclease, provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a MYOC target position in the MYOC gene, to allow alteration, e.g., alteration associated with NHEJ, of a MYOC target position in the MYOC gene, either alone or in combination with the break positioned by the first gRNA molecule.
[0050] In an embodiment, a nucleic acid encodes a third gRNA molecule comprising a targeting domain configured to, in combination with a corresponding RNA-guided nuclease, provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a MYOC target position in the MYOC gene to allow alteration, e.g., alteration associated with NHEJ, of a MYOC target position in the MYOC gene, either alone or in combination with the break positioned by the first and/or second gRNA molecule.
[0051] In an embodiment, a nucleic acid encodes a fourth gRNA molecule comprising a targeting domain configured to, in combination with a corresponding RNA-guided nuclease, provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a MYOC target position in the MYOC gene to allow alteration, e.g., alteration associated with NHEJ, of a MYOC target position in the MYOC gene, either alone or in combination with the break positioned by the first gRNA molecule, the second gRNA molecule and/or the third gRNA molecule.
[0052] In an embodiment, the nucleic acid encodes a second gRNA molecule. The second gRNA molecule is selected to target the same MYOC target position as the first gRNA molecule. Optionally, the nucleic acid may encode a third gRNA molecule, and further optionally, the nucleic acid may encode a fourth gRNA molecule. The third gRNA molecule and the fourth gRNA molecule are selected to target the same MYOC target position as the first and second gRNA molecules.
[0053] In an embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Table 10. In an embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain selected from a targeting domain sequence from Table 10. In an embodiment, when a third or fourth gRNA molecule are present, the third and fourth gRNA molecules may independently comprise a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Table 10. In a further embodiment, when a third or fourth gRNA molecule are present, the third and fourth gRNA molecules may independently comprise a targeting domain sequence from Table 10.
[0054] In an embodiment, the nucleic acid encodes a second gRNA molecule which is a modular gRNA molecule, e.g., wherein one or more nucleic acid molecules encode a modular gRNA molecule. In another embodiment, the nucleic acid encodes a second gRNA molecule, which is a chimeric gRNA molecule. In another embodiment, when a nucleic acid encodes a third or fourth gRNA molecule, the third and fourth gRNA molecule may be a modular gRNA molecule or a chimeric gRNA molecule. When multiple gRNA molecules are used, any combination of modular or chimeric gRNA molecules may be used.
[0055] A nucleic acid may encode a second, a third, and/or a fourth gRNA molecule, each independently comprising a targeting domain comprising 16 nucleotides or more in length. In an embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 16 nucleotides in length. In an embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 17 nucleotides in length. In another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 18 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 19 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 20 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 21 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 22 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 23 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 24 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 25 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain that is 26 nucleotides in length.
[0056] In an embodiment, the targeting domain comprises 16 nucleotides. In an embodiment, the targeting domain comprises 17 nucleotides. In an embodiment, the targeting domain comprises 18 nucleotides. In an embodiment, the targeting domain comprises 19 nucleotides. In an embodiment, the targeting domain comprises 20 nucleotides. In an embodiment, the targeting domain comprises 21 nucleotides. In an embodiment, the targeting domain comprises 22 nucleotides. In an embodiment, the targeting domain comprises 23 nucleotides. In an embodiment, the targeting domain comprises 24 nucleotides. In an embodiment, the targeting domain comprises 25 nucleotides. In an embodiment, the targeting domain comprises 26 nucleotides.
[0057] In an embodiment, a nucleic acid encodes (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the MYOC gene as disclosed herein, and (b) a sequence that encodes an RNA-guided nuclease molecule (e.g., Casl2a molecule). In an embodiment, (a) and (b) are present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., the same adeno- associated virus (AAV) vector. In an embodiment, the nucleic acid molecule is an AAV vector. Exemplary AAV vectors that may be used in any of the described compositions and methods include an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV6 vector, a modified AAV6 vector, an AAV8 vector and an AAV9 vector.
[0058] In another embodiment, (a) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (b) is present on a second nucleic acid molecule, e.g., a second vector, e.g., a second viral vector, e.g., a second AAV vector. The first and second nucleic acid molecules may be AAV vectors.
[0059] In another embodiment, a nucleic acid encodes (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the MYOC gene as disclosed herein, and (b) a sequence that encodes an RNA-guided nuclease molecule (e.g., Casl2a molecule); and further comprise (c)(i) a sequence that encodes a second gRNA molecule as described herein and optionally, (c)(ii) a sequence that encodes a third gRNA molecule described herein having a targeting domain that is complementary to a third target domain of the MYOC gene; and optionally, (c)(iii) a sequence that encodes a fourth gRNA molecule described herein having a targeting domain that is complementary to a fourth target domain of the MYOC gene. In some embodiments, the nucleic acid comprises (a), (b) and (c)(i). In an embodiment, the nucleic acid comprises (a), (b), (c)(i) and (c)(ii). In an embodiment, the nucleic acid comprises (a), (b), (c)(i), (c)(ii) and (c)(iii). Each of (a) and (c)(i), (c)(ii) and/or (c)(iii) may be present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., the same adeno- associated virus (AAV) vector. In an embodiment, the nucleic acid molecule is an AAV vector.
[0060] In another embodiment, (a) and (c)(i) are on different vectors. For example, (a) may be present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (c)(i) may be present on a second nucleic acid molecule, e.g., a second vector, e.g., a second viral vector, e.g., a second AAV vector. In an embodiment, the first and second nucleic acid molecules are AAV vectors.
[0061] In another embodiment, each of (a), (b), and (c)(i) are present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., an AAV vector. In an embodiment, the nucleic acid molecule is an AAV vector. In an alternate embodiment, one of (a), (b), and (c)(i) is encoded on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and a second and third of (a), (b), and (c)(i) is encoded on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors.
[0062] In an embodiment, (a) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, a first AAV vector; and (b) and (c)(i) are present on a second nucleic acid molecule, e.g., a second vector, e.g., a second viral vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors.
[0063] In another embodiment, (b) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (a) and (c)(i) are present on a second nucleic acid molecule, e.g., a second vector, e.g., a second viral vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors.
[0064] In another embodiment, (c)(i) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (b) and (a) are present on a second nucleic acid molecule, e.g., a second vector, e.g., a second viral vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors.
[0065] In another embodiment, each of (a), (b) and (c)(i) are present on different nucleic acid molecules, e.g., different vectors, e.g., different viral vectors, e.g., different AAV vector. For example, (a) may be on a first nucleic acid molecule, (b) on a second nucleic acid molecule, and (c)(i) on a third nucleic acid molecule. The first, second and third nucleic acid molecule may be AAV vectors. [0066] In another embodiment, when a third and/or fourth gRNA molecule are present, each of (a), (b), (c)(i), (c)(ii) and (c)(iii) may be present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., an AAV vector. In an embodiment, the nucleic acid molecule is an AAV vector. In an alternate embodiment, each of (a), (b), (c)(i), (c)(ii) and (c)(iii) may be present on the different nucleic acid molecules, e.g., different vectors, e.g., the different viral vectors, e.g., different AAV vectors. In a further embodiment, each of (a), (b), (c)(i), (c)(ii) and (c)(iii) may be present on more than one nucleic acid molecule, but fewer than five nucleic acid molecules, e.g., AAV vectors.
[0067] In another embodiment, when (d) a template nucleic acid is present, each of (a), (b), and (d) may be present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., an AAV vector. In an embodiment, the nucleic acid molecule is an AAV vector. In an alternate embodiment, each of (a), (b), and (d) may be present on the different nucleic acid molecules, e.g., different vectors, e.g., the different viral vectors, e.g., different AAV vectors. In a further embodiment, each of (a), (b), and (d) may be present on more than one nucleic acid molecule, but fewer than three nucleic acid molecules, e.g., AAV vectors.
[0068] In another embodiment, when (d) a template nucleic acid is present, each of (a), (b), (c)(i) and (d) may be present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., an AAV vector. In an embodiment, the nucleic acid molecule is an AAV vector. In an alternate embodiment, each of (a), (b), (c)(i) and (d) may be present on the different nucleic acid molecules, e.g., different vectors, e.g., the different viral vectors, e.g., different AAV vectors. In a further embodiment, each of (a), (b), (c)(i) and (d) may be present on more than one nucleic acid molecule, but fewer than four nucleic acid molecules, e.g., AAV vectors.
[0069] In another embodiment, when (d) a template nucleic acid is present, each of (a), (b), (c)(i), (c)(ii) and (d) may be present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., an AAV vector. In an embodiment, the nucleic acid molecule is an AAV vector. In an alternate embodiment, each of (a), (b), (c)(i), (c)(ii) and (d) may be present on the different nucleic acid molecules, e.g., different vectors, e.g., the different viral vectors, e.g., different AAV vectors. In a further embodiment, each of (a), (b), (c)(i), (c)(ii) and (d) may be present on more than one nucleic acid molecule, but fewer than five nucleic acid molecules, e.g., AAV vectors. [0070] In another embodiment, when (d) a template nucleic acid is present, each of (a), (b), (c)(i), (c)(ii), (c)(iii) and (d) may be present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., an AAV vector. In an embodiment, the nucleic acid molecule is an AAV vector. In an alternate embodiment, each of (a), (b), (c)(i), (c)(ii), (c)(iii) and (d) may be present on the different nucleic acid molecules, e.g., different vectors, e.g., the different viral vectors, e.g., different AAV vectors. In a further embodiment, each of (a), (b), (c)(i), (c)(ii), (c)(iii) and (d) may be present on more than one nucleic acid molecule, but fewer than six nucleic acid molecules, e.g., AAV vectors.
[0071] The nucleic acids described herein may comprise a promoter operably linked to the sequence that encodes the gRNA molecule of (a), e.g., a promoter described herein. The nucleic acid may further comprise a second promoter operably linked to the sequence that encodes the second, third and/or fourth gRNA molecule of (c), e.g., a promoter described herein. In some embodiments, the promoter and second promoter differ from one another. In some embodiments, the promoter and second promoter are the same.
[0072] The nucleic acids described herein may further comprise a promoter operably linked to the sequence that encodes the RNA-guided nuclease molecule (e.g., Casl2a molecule) of (b), e.g., a promoter described herein. In certain embodiments, the promoter may comprise a sequence selected from the group consisting of the promoter sequences set forth in Table 6 (DCN #1, DCN #2, ID3, MT2A, SERPINF1, SERPING1 #1, SERPING1 #2, TEMPI, CP, MT1X, OLFML3, PCOLCE, PDGFRA, PTN, RARRE SI, RBP4 #1, RBP4 #2, RBP4 #3, PDPN, APOD, B2M, PTGDS, EEF1 Al , ANGPTL7, MGP, RPS24, PLA2G2A, CHI3L1 , FTL, TAGLN, PCP4, MYL 9, MYOC, CMV, and mini-CMV promoter).
[0073] In another aspect, disclosed herein is an isolated nucleic acid comprising a promoter comprising a sequence selected from the group consisting of the sequences set forth in Table 6 (DCN #1, DCN #2, ID3, MT2A, SERPINF1, SERPING1 #1, SERPING1 #2, TIMP1, CP, MT1X, OLFML3, PCOLCE, PDGFRA, PTN, RARRE SI, RBP4 #1, RBP4 #2, RBP4 #3, PDPN, APOD, B2M, PTGDS, EEF1A1, ANGPTL7, MGP, RPS24, PLA2G2A, CHI3L1, FTL, TAGLN, PCP4, MYL 9, MYOC, CMV, and mini-CMV promoter). In certain embodiments, the promoter is operably linked to a trans gene. In another aspect, disclosed herein is a vector comprising any of the foregoing nucleic acids. In certain embodiments, the vector is for use in expressing the transgene in a cell, e.g., a trabecular meshwork (TM) cell. In another aspect, disclosed herein is a method of expressing a transgene in a cell, e.g., a TM cell, the method comprising contacting the cell with any of the foregoing vectors.
[0074] In another aspect, disclosed herein is a gRNA molecule comprising a targeting domain that may bind a target domain of a MYOC gene. In certain embodiments, the targeting domain may be complementary to the target domain of the MYOC gene. In certain embodiments, the targeting domain may be configured to, in combination with an RNA- guided nuclease, provide a cleavage event selected from a double strand break and a single strand break, within 500, 400, 300, 200, 100, 50, 25, or 10 nucleotides of a MYOC target position. In certain embodiments, the MYOC target position may be in a region selected from the group consisting of a 5’ untranslated region (“UTR”), exon 1, an exon 1/intron 1 border, intron 1, an intron 1/exon 2 border, exon 2, an exon 2/intron 2 border, intron 2, an intron 2/exon 3 border, exon 3, an exon 3/intron 3 border, and 3’ UTR of the MYOC gene. In certain embodiments, the targeting domain may comprise a sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence from Table 10. In certain embodiments, the targeting domain may comprise a sequence that is the same as a targeting domain sequence from Table 10. In certain embodiments, the targeting domain comprises a sequence selected from the group consisting of a targeting domain sequence in Table 10. In certain embodiments, the targeting domain may comprise a sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain selected from the group consisting of a Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8 targeting domain (Table 10). In certain embodiments, the targeting domain may comprise a sequence selected from the group consisting of a Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8 targeting domain (Table 10). In certain embodiments, the targeting domain may be a length selected from the group consisting of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26 nucleotides or more in length. In certain embodiments, the gRNA molecule may comprise a targeting domain and a crRNA direct repeat extension. In certain embodiments, the crRNA direct repeat extension may comprise a sequence that is the same as, or differs by no more than 3, 4, 5, or 6 nucleotides from, a crRNA direct repeat extension sequence of UAAUUUCUACUCUUGUAGAU (SEQ ID NO:49). In certain embodiments, the crRNA direct repeat extension sequence may be UAAUUUCUACUCUUGUAGAU (SEQ ID NO:49). In certain embodiments, the gRNA molecule may be a modular gRNA molecule or a chimeric gRNA molecule. In certain embodiments, the gRNA molecule may comprise a sequence that is the same as, or differs by no more than 3 nucleotides from, a crRNA direct repeat extension sequence of UAAUUUCUACUCUUGUAGAU (SEQ ID NO:49).
[0075] In another aspect, provided herein is a nucleic acid that comprises: (a) a sequence that encodes any of the gRNA molecules disclosed herein. In certain embodiments, the nucleic acid may further comprise: (b) a sequence that encodes an RNA-guided nuclease molecule. In certain embodiments, the RNA-guided nuclease molecule may be a Casl2a molecule. In certain embodiments, the Casl2a molecule may comprise any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Cas 12a polypeptide sequences). In certain embodiments, the Cas 12a molecule may be encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Cas 12a polynucleotide sequences). In certain embodiments, the nucleic acid may comprise a promoter operably linked to the sequence that encodes the Casl2a molecule of (b). In certain embodiments, the promoter operably linked to the sequence that encodes the Cas 12a molecule of (b) may comprise a sequence selected from the group consisting of the sequences set forth in Table 6 (DCN #1, DCN #2, ID3, MT2A, SERPINF1, SERPING1 #1, SERPING1 #2, TIMP1, CP, MT1X, OLFML3, PCOLCE, PDGFRA, PTN, RARRE SI, RBP4 #1, RBP4 #2, RBP4 #3, PDPN, APOD, B2M, PTGDS, EEF1A1, ANGPTL7, MGP, RPS24, PLA2G2A, CHI3L1, FTL, TAGLN, PCP4, MYL 9, MYOC, CMV, and mini-CMV promoter). In certain embodiments, the nucleic acid may further comprise: (c) a sequence that encodes a second gRNA molecule having a targeting domain that is complementary to a second target domain of the MYOC gene. In certain embodiments, the second gRNA molecule may be any of the gRNA molecules disclosed herein. In certain embodiments, the nucleic acid may further comprise a third gRNA molecule. In certain embodiments, the nucleic acid may further comprise a fourth gRNA molecule. In certain embodiments, each of (a) and (b) may be present on the same nucleic acid molecule. In certain embodiments, the nucleic acid molecule may be an AAV vector. In certain embodiments, (a) may be present on a first nucleic acid molecule; and (b) may be present on a second nucleic acid molecule. In certain embodiments, the first and second nucleic acid molecules may be AAV vectors. In certain embodiments, the first nucleic acid molecule may be other than an AAV vector and the second nucleic acid molecule may be an AAV vector. In certain embodiments, the nucleic acid may comprise a promoter operably linked to the sequence that encodes the gRNA molecule of (a).
[0076] In another aspect, disclosed herein is a lipid nanoparticle (LNP) that may comprise one or more ionizable lipids. In certain embodiments, the LNP may encapsulate a gRNA molecule disclosed herein. In certain embodiments, the one or more ionizable lipids may be selected from the group consisting of MC3, SM-102, ALC-0315, 5A2-SC8, and BAMEA- O16B. In certain embodiments, the LNP may comprise cholesterol. In certain embodiments, the LNP may comprise distearoylphosphatidylcholine (DSPC). In certain embodiments, the LNP may comprise a PEG-lipid. In certain embodiments, the PEG-lipid may be dimethylglycine (DMG)-PEG2k. In certain embodiments, the LNP may encapsulate a nucleic acid encoding an RNA-guided nuclease molecule. In certain embodiments, the nucleic acid encoding the RNA-guided nuclease molecule may be messenger RNA (mRNA). In certain embodiments, the RNA-guided nuclease molecule may comprise any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Casl2a polypeptide sequences) or may be encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Cas 12a polynucleotide sequences).
[0077] In another aspect, disclosed herein is a composition comprising (a) a gRNA molecule described herein. In certain embodiments, the composition may further comprise (b) an RNA-guided nuclease molecule or nucleic acid encoding the RNA-guided nuclease molecule. In certain embodiments, the RNA-guided nuclease molecule may be a Casl2a molecule. In certain embodiments, the Cas 12a molecule may comprise any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Cas 12a polypeptide sequences). In certain embodiments, the Cas 12a molecule may be encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Casl2a polynucleotide sequences). In certain embodiments, the composition may further comprise (c) a second gRNA molecule, a third gRNA molecule, and/or a fourth gRNA molecule. In certain embodiments, a composition of (a), (b) and optionally (c) a second, third and/or fourth gRNA molecule, may further comprise (d) a template nucleic acid. In certain embodiments, the gRNA molecule may be encapsulated in any of the LNPs disclosed herein. In certain embodiments, the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease may be encapsulated in any of the LNPs disclosed herein. In certain embodiments, the nucleic acid encoding the RNA-guided nuclease may be mRNA. In an embodiment, the composition may be a pharmaceutical composition. The compositions described herein, e.g., pharmaceutical compositions described herein, can be used in the treatment or prevention of POAG in a subject, e.g., in accordance with a method disclosed herein.
[0078] In another aspect, disclosed herein is a method of altering a cell, e.g., altering the structure, e.g., altering the sequence, of a target nucleic acid of a cell, comprising contacting the cell with: (a) a gRNA molecule that targets the MYOC gene, e.g., a gRNA molecule as described herein; (b) an RNA-guided nuclease molecule or a nucleic acid encoding the RNA- guided nucleic molecule, e.g., a Casl2a molecule as described herein; and optionally, (c) a second, third and/or fourth gRNA molecule that targets MYOC gene, e.g., a second third and/or fourth gRNA molecule as described herein; and optionally, (d) a template nucleic acid. In an embodiment, the gRNA molecule may be encapsulated in any of the LNPs as described herein. In certain embodiments, disclosed herein is a method of altering a cell comprising contacting the cell with (a) a gRNA molecule disclosed herein; (b) an RNA-guided nuclease molecule or a nucleic acid encoding the RNA-guided nuclease molecule; and optionally, (c) a second gRNA molecule disclosed herein. In certain embodiments, the RNA-guided nuclease may be a Casl2a molecule. In an embodiment, the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease may be encapsulated in any of the LNPs as described herein. In certain embodiments, the Casl2a molecule may comprise any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Casl2a polypeptide sequences). In certain embodiments, the Cas 12a molecule may be encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Casl2a polynucleotide sequences). In certain embodiments, the nucleic acid encoding the RNA-guided nuclease may be mRNA. In certain embodiments, the cell may be from a subject suffering from POAG. In certain embodiments, the cell may be an ocular cell. In certain embodiments, the cell may be a trabecular meshwork cell. In certain embodiments, the contacting step may be performed in vivo. In certain embodiments, the contacting step may comprise contacting the cell with a nucleic acid that encodes at least one of (a), (b), and optionally (c). In certain embodiments, the contacting step may comprise contacting the cell with a nucleic acid described herein. In certain embodiments, the contacting step may comprise delivering to the cell the RNA- guided nuclease molecule of (b) a nucleic acid which encodes the gRNA molecule of (a), and optionally a nucleic acid which encodes the second gRNA molecule of (c). In certain embodiments, the contacting step may comprise delivering to the cell the RNA-guided nuclease molecule of (b), the gRNA molecule of (a) and optionally the second gRNA molecule of (c). In certain embodiments, the contacting step may comprise delivering to the cell a nucleic acid that encodes the RNA-guided nuclease molecule of (b), the gRNA molecule of (a), and optionally the second gRNA molecule of (c). [0079] In an embodiment, the method comprises contacting the cell with (a) and (b). In an embodiment, the method comprises contacting the cell with (a), (b), and (c). In an embodiment, the method comprises contacting the cell with (a), (b), (c) and (d).
[0080] The gRNA molecule of (a) and optionally (c) may comprise a sequence selected from a sequence set forth in Table 10, or a gRNA molecule that differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence set forth in Table 10.
[0081] In an embodiment, the method comprises contacting a cell from a subject suffering from or likely to develop POAG. The cell may be from a subject having a mutation at a in the MYOC gene (e.g., a mutation MYOC target position in the MYOC gene).
[0082] In an embodiment, the cell being contacted in the disclosed method is a target cell from the eye of the subject. The cell may be a trabecular meshwork cell, retinal pigment epithelial cell, a retinal cell, an iris cell, a ciliary body cell and/or the optic nerve. The contacting may be performed ex vivo and the contacted cell may be returned to the subject’s body after the contacting step. In other embodiments, the contacting step may be performed in vivo.
[0083] In an embodiment, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses at least one of (a), (b), and (c). In an embodiment, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses each of (a), (b), and (c). In another embodiment, the contacting step of the method comprises delivering to the cell an RNA-guided nuclease molecule (e.g., Casl2a molecule) of (b) and a nucleic acid which encodes a gRNA molecule (a) and optionally, a second gRNA molecule (c)(i) (and further optionally, a third gRNA molecule (c)(ii) and/or fourth gRNA molecule (c)(iii)).
[0084] In an embodiment, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses at least one of (a), (b), (c) and (d). In an embodiment, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses each of (a), (b), and (c). In another embodiment, the contacting step of the method comprises delivering to the cell an RNA-guided nuclease molecule (e.g., Casl2a molecule) of (b), a nucleic acid which encodes a gRNA molecule of (a) and a template nucleic acid of (d), and optionally, a second gRNA molecule (c)(1) (and further optionally, a third gRNA molecule (c)(ii) and/or fourth gRNA molecule (c)(iii).
[0085] In an embodiment, contacting comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, e.g., an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV6 vector, a modified AAV6 vector, an AAV8 vector or an AAV9 vector, as described herein.
[0086] In an embodiment, contacting comprises delivering to the cell an RNA-guided nuclease molecule or a nucleic acid encoding the RNA-guided nuclease molecule (e.g., Casl2a molecule) of (b), as a protein or a messenger RNA (mRNA), and a nucleic acid which encodes a gRNA molecule of (a) and optionally a second, third and/or fourth gRNA molecule (c).
[0087] In an embodiment, contacting comprises delivering to the cell an RNA-guided nuclease molecule (e.g., Casl2a molecule) of (b), as a protein or an mRNA, the gRNA molecule of (a), as an RNA, and optionally the second, third and/or fourth gRNA molecule of (c), as an RNA.
[0088] In an embodiment, contacting comprises delivering to the cell a gRNA molecule of (a) as an RNA, optionally the second, third and/or fourth gRNA molecule of (c) as an RNA, and a nucleic acid that encodes the RNA-guided nuclease molecule (e.g., Casl2a molecule) of (b).
[0089] In another aspect, disclosed herein is a method of treating a subject suffering from or likely to develop POAG, e.g., by altering the structure, e.g., sequence, of a target nucleic acid of the subject, comprising contacting the subject (or a cell from the subject) with:
(a) a gRNA molecule that targets the MYOC gene, e.g., a gRNA molecule disclosed herein;
(b) an RNA-guided nuclease molecule or nucleic acid encoding the RNA-guided nuclease molecule, e.g., a Casl2a molecule disclosed herein; and optionally, (c)(i) a second gRNA molecule that targets the MYOC gene, e.g., a second gRNA molecule disclosed herein, and further optionally, (c)(ii) a third gRNA molecule, and still further optionally, (c)(iii) a fourth gRNA molecule that target the MYOC gene, e.g., a third and fourth gRNA molecule disclosed herein.
[0090] In another aspect, disclosed herein is a method of treating a subject, comprising contacting a subject (or a cell from the subject) with:
(a) a gRNA molecule disclosed herein;
(b) an RNA-guided nuclease molecule or a nucleic acid encoding the RNA- guided nuclease molecule; and optionally, (c) a second gRNA molecule disclosed herein.
[0091] In certain embodiments, the method may further comprise contacting the subject with a third gRNA molecule. In certain embodiments, the method may further comprise contacting the subject with a fourth gRNA molecule. In certain embodiments, the method may comprise contacting the subject with (a), (b), and (c). In certain embodiments, the subject may be suffering from POAG. In certain embodiments, the subject may have a mutation in the MYOC gene. In certain embodiments, the contacting step may be performed in vivo. In certain embodiments, the contacting step may comprise subretinal delivery. In certain embodiments, the contacting step may comprise subretinal injection. In certain embodiments, the contacting step may comprise intravitreal delivery. In certain embodiments, the contacting step may comprise intravitreal injection. In certain embodiments, the contacting step may comprise contacting the subject with a nucleic acid that encodes at least one of (a), (b), and optionally (c). In certain embodiments, the contacting step may comprise contacting the subject with a nucleic acid described herein. In certain embodiments, the contacting step may comprise delivering to the subject the RNA- guided nuclease molecule of (b), a nucleic acid that encodes the gRNA molecule of (a), and optionally a nucleic acid that encodes the second gRNA molecule of (c). In certain embodiments, the contacting step may comprise delivering to the subject the RNA-guided nuclease molecule of (b), the gRNA molecule of (a) and optionally the second gRNA molecule of (c). In certain embodiments, the contacting step may comprise delivering to the subject a nucleic acid that encodes the RNA-guided nuclease molecule of (b), the gRNA molecule of (a), and optionally the second gRNA molecule of (c). In certain embodiments, the gRNA molecule may be encapsulated in an LNP described herein. In certain embodiments, the RNA-guided nuclease molecule or the nucleic acid encoding the RNA- guided nuclease molecule may be encapsulated in an LNP described herein. In certain embodiments, the nucleic acid encoding the RNA-guided nuclease may be mRNA. [0092] In some embodiments, the gRNA molecule may be encapsulated in any of the LNPs as described herein. In some embodiments, the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease may be encapsulated in any of the LNPs as described herein. In some embodiments, the nucleic acid encoding the RNA-guided nuclease may be mRNA.
[0093] In some embodiments, contacting comprises contacting with (a) and (b). In some embodiments, contacting comprises contacting with (a), (b), and (c)(i). In some embodiments, contacting comprises contacting with (a), (b), (c)(i) and (c)(ii). In some embodiments, contacting comprises contacting with (a), (b), (c)(i), (c)(ii) and (c)(iii). In some embodiments, contacting comprises contacting with (a), (b), (c)(i) and (d). In some embodiments, contacting comprises contacting with (a), (b), (c)(i), (c)(ii) and (d). In some embodiments, contacting comprises contacting with (a), (b), (c)(i), (c)(ii), (c)(iii) and (d).
[0094] The gRNA molecule of (a) or (c) (e.g., (c)(i), (c)(ii), or (c)(iii) may comprise a sequence set forth in Table 10, or a sequence that differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a sequence set forth in Table 10.
[0095] In an embodiment, a cell of the subject is contacted ex vivo with (a), (b), (d) and optionally (c). In an embodiment, the cell is returned to the subject’s body.
[0096] In an embodiment, a cell of the subject is contacted is in vivo with (a), (b) (d) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
[0097] In an embodiment, the cell of the subject is contacted in vivo by subretinal delivery of (a), (b), (d) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
[0098] In an embodiment, the contacting step comprises contacting the subject with a nucleic acid, e.g., a vector, e.g., an AAV vector, described herein, e.g., a nucleic acid that encodes at least one of (a), (b), (d) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
[0099] In an embodiment, the contacting step comprises delivering to the subject the RNA- guided nuclease molecule, e.g., a Casl2a molecule, of (b), as a protein or mRNA, and a nucleic acid which encodes (a), a nucleic acid of (d) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii). [0100] In an embodiment, the contacting step comprises delivering to the subject the RNA- guided nuclease molecule, e.g., a Casl2a molecule of (b), as a protein or mRNA, the gRNA molecule of (a), as an RNA, a nucleic acid of (d) and optionally the second gRNA molecule of (c)(i), further optionally the third gRNA molecule of (c)(ii), and still further optionally the fourth gRNA molecule of (c)(iii), as an RNA.
[0101] In an embodiment, the contacting step comprises delivering to the subject the gRNA molecule of (a), as an RNA, optionally the second gRNA molecule of (c)(i), further optionally the third gRNA molecule of (c)(ii), and still further optionally the fourth gRNA molecule of (c)(iii), as an RNA, a nucleic acid that encodes the RNA-guided nuclease molecule, e.g., a Casl2a molecule of (b), and a nucleic acid of (d).
[0102] In an embodiment, a cell of the subject is contacted ex vivo with (a), (b) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii). In an embodiment, the cell is returned to the subject’s body.
[0103] In an embodiment, a cell of the subject is contacted is in vivo with (a), (b) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii). In an embodiment, the cell of the subject is contacted in vivo by subretinal delivery of (a), (b) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
[0104] In an embodiment, the contacting step comprises contacting the subject with a nucleic acid, e.g., a vector, e.g., an AAV vector, described herein, e.g., a nucleic acid that encodes at least one of (a), (b), and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
[0105] In an embodiment, the contacting step comprises delivering to the subject the RNA- guided nuclease molecule, e.g., a Casl2a molecule of (b), as a protein or mRNA, and a nucleic acid which encodes (a) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
[0106] In an embodiment, the contacting step comprises delivering to the subject the RNA- guided nuclease molecule, e.g., a Casl2a molecule of (b), as a protein or mRNA, the gRNA molecule of (a), as an RNA, and optionally the second gRNA molecule of (c)(i), further optionally the third gRNA molecule of (c)(ii), and still further optionally the fourth gRNA molecule of (c)(iii), as an RNA. [0107] In an embodiment, the contacting step comprises delivering to the subject the gRNA molecule of (a), as an RNA, optionally the second gRNA molecule of (c)(i), further optionally the third gRNA molecule of (c)(ii), and still further optionally the fourth gRNA molecule of (c)(iii), as an RNA, and a nucleic acid that encodes the RNA-guided nuclease molecule, e.g., a Casl2a molecule of (b).
[0108] In another aspect, disclosed herein is a reaction mixture comprising a gRNA molecule, a nucleic acid, or a composition described herein, and a cell, e.g., a cell from a subject having, or likely to develop POAG, or a subject having a mutation in the MYOC gene.
[0109] In another aspect, disclosed herein is a kit comprising, (a) a gRNA molecule described herein, or nucleic acid that encodes the gRNA molecule, and one or more of the following:
(b) an RNA-guided nuclease molecule, e.g., a Casl2a molecule described herein, or a nucleic acid that encodes the RNA-guided nuclease molecule, e.g., a Casl2a molecule; and optionally
(c)(i) a second gRNA molecule, e.g., a second gRNA molecule described herein or a nucleic acid that encodes (c)(i); and/or
(e) a nucleic acid that encodes one or more of (b) and optionally (c). In certain embodiments, the kit may further comprise (c)(ii) a third gRNA molecule, e.g., a second gRNA molecule described herein or a nucleic acid that encodes (c)(ii). In certain embodiments, the kit may further comprise (c)(iii) a fourth gRNA molecule, e.g., a second gRNA molecule described herein or a nucleic acid that encodes (c)(iii). In certain embodiments, the kit may further comprise (d) a template nucleic acid, e.g., a template nucleic acid. In certain embodiments, the (b) RNA-guided nuclease molecule may be a Casl2a molecule. In certain embodiments, the Casl2a molecule may comprise any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Casl2a polypeptide sequences). In certain embodiments, the Casl2a molecule may be encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Casl2a polynucleotide sequences). In certain embodiments, the kit may comprise a nucleic acid that encodes one or more of (a), (b), and optionally (c). In certain embodiments, the kit may further comprise a third gRNA molecule targeting a MYOC target position. In certain embodiments, the kit may further comprise a fourth gRNA molecule targeting a MYOC target position. In an embodiment, the kit comprises nucleic acid, e.g., an AAV vector, that encodes one or more of (a), (b), (c)(i), (c)(ii), (c)(iii) and (d).
[0110] In another aspect, disclosed herein is non-naturally occurring template nucleic acid described herein.
[0111] In yet another aspect, disclosed herein is a gRNA molecule, e.g., a gRNA molecule described herein, for use in treating or preventing POAG in a subject, e.g., in accordance with a method of treating or preventing POAG as described herein. In certain embodiments, the gRNA molecule may be for use in treating POAG in a subject. In certain embodiments, the gRNA molecule may be used in combination with (b) an RNA-guided nuclease molecule or a nucleic acid molecule encoding the RNA-guided nuclease molecule. In certain embodiments, the RNA-guided nuclease molecule may be a Casl2a molecule. In certain embodiments, the Casl2a molecule may comprise any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Cas 12a polypeptide sequences). In certain embodiments, the Casl2a molecule may be encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Casl2a polynucleotide sequences). Additionally or alternatively, in an embodiment, the gRNA molecule may be used in combination with a second, third and/or fourth gRNA molecule, e.g., a second, third and/or fourth gRNA molecule described herein.
[0112] In still another aspect, disclosed herein is use of a gRNA molecule, e.g., a gRNA molecule described herein, in the manufacture of a medicament for treating or preventing POAG in a subject, e.g., in accordance with a method of treating or preventing POAG as described herein. In certain embodiments, the use of a gRNA molecule described herein may be for the manufacture of a medicament for treating POAG in a subject.
[0113] In an embodiment, the medicament comprises an RNA-guided nuclease molecule or a nucleic acid encoding the RNA-guided nuclease molecule, e.g., a Casl2a molecule, described herein. In certain embodiments, the Cas 12a molecule may comprise any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Casl2a polypeptide sequences). In certain embodiments, the Cas 12a molecule may be encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Casl2a polynucleotide sequences).
Additionally or alternatively, in an embodiment, the medicament comprises a second, third and/or fourth gRNA molecule, e.g., a second, third and/or fourth gRNA molecule described herein. [0114] In another aspect, disclosed herein is the use of a nucleic acid as disclosed herein, for use in treating POAG in a subject.
[0115] In another aspect, disclosed herein is the use of an LNP as disclosed herein, for use in treating POAG in a subject.
[0116] In another aspect, disclosed herein is the use of a composition as disclosed herein, for use in treating POAG in a subject.
[0117] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0118] Headings, including numeric and alphabetical headings and subheadings, are for organization and presentation and are not intended to be limiting.
[0119] Other features and advantages of the disclosure will be apparent from the detailed description, drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0120] The accompanying drawings are intended to provide illustrative, and schematic rather than comprehensive, examples of certain aspects and embodiments of the present disclosure. The drawings are not intended to be limiting or binding to any particular theory or model and are not necessarily to scale. Without limiting the foregoing, nucleic acids and polypeptides may be depicted as linear sequences, or as schematic two- or three-dimensional structures; these depictions are intended to be illustrative rather than limiting or binding to any particular model or theory regarding their structure.
[0121] Fig. 1 shows data from the screening assay in T cells testing the editing activity of a series of SaCas9 RNPs comprising S. aureus Cas9 CS'aCas9) enzyme and an .S'«Cas9 gRNA (Table 9). Gray triangles represent results showing the percentage of frameshift indels introduced into the MYOC gene by each RNP. Black dots represent the percentage of indels introduced into the MYOC gene by each RNP. All samples were run in duplicate. [0122] Figs. 2A-2B show dose response data from T cells using increasing concentrations (0.0891 pM, 0.178 pM, 0.356 pM, 0.7125 pM, 1.425 pM, 2.85 pM, 5.7 pM, and 11.4 pM) of .S'aCas9 RNP comprising SaCas9 enzyme and A/Cas9 gRNA (Cas9-1, Cas9-5, Cas9-6, Cas9-24, Cas9-56, Cas9-57, Cas9-59 (Table 9)). Fig. 2A shows the percentage of indels introduced into the MYOC gene by RNP concentration for each RNP tested. Fig. 2B shows the percentage of frameshift indels introduced into the MYOC gene by RNP concentration for each RNP tested. All samples were run in duplicate.
[0123] Fig. 3 shows data from the screening assay in T cells testing the editing activity of a series of AsCas 12a RNPs comprising Acidaminococcus sp. Casl2a (AsCas 12a) enzyme and an AsCas 12a gRNA (Table 10). Gray triangles represent results showing the percentage of frameshift indels introduced into the MYOC gene by each RNP. Black dots represent the percentage of indels introduced into the MYOC gene by each RNP. All samples were run in duplicate.
[0124] Figs. 4A-4B show dose response data from T cells using increasing concentrations (0.010 pM, 0.030 pM, 0.080 pM, 0.25 pM, 0.80 pM, 2.53 pM, and 8.0 pM) of AvCas l 2a RNP comprising AsCasl2a enzyme and an AsCasl2a gRNA (Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, or Casl2a-8 (Table 10)). Fig. 4A shows the percentage of indels introduced into the MYOC gene by RNP concentration for each RNP tested. Fig. 4B shows the percentage of frameshift indels introduced into the MYOC gene by RNP concentration for each RNP tested. All samples were run in duplicate.
[0125] Figs. 5A-5B show dose response data in human trabecular meshwork cells (HTMCs) using increasing concentrations (0.010 pM, 0.030 pM, 0.080 pM, 0.25 pM, 0.80 pM, 2.53 pM, and 8.0 pM) of RNP comprising AsCasl2a enzyme and an AsCasl2a gRNA (Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, or Casl2a-8 (Table 10)) or RNP comprising SaCas9 enzyme complexed with an SaCasO gRNA (i.e., Cas9-1 , Cas9-5, Cas9-6, Cas9-24 (Table 9)). Fig. 5A shows the indel rate (%) in HTMCs with increasing concentration of RNP comprising .S'aCas9 enzyme complexed with .S'«Cas9 gRNAs or RNP comprising AsCas 12a enzyme complexed with AsCas 12a gRNAs. Fig. 5B shows the percentage of frameshift indels in HTMCs with increasing concentration of RNP comprising S«Cas9 enzyme complexed with .Sc/Cas9 gRNAs or RNP comprising AsCasl2a enzyme complexed with AsCasl2a gRNAs. [0126] Figs. 6A and 6B show expression of green fluorescent protein (GFP) in HTMCs using various promoters (promoter sequences are set forth in Table 6). Fig. 6A shows the percentage of GFP+ HTMCs after nucleofection with plasmids containing the indicated putative promoter driving expression of GFP. Fig. 6B shows the mean fluorescence intensity of GFP+ HTMCs after nucleofection with plasmids containing the indicated putative promoter driving expression of GFP.
[0127] Figs. 7A- 7F show histology data from in vivo screening of LNPs formulated with different ionizable lipids, for transfection of the mouse trabecular meshwork (TM). LNPs (50% ionizable lipid, 38.5% cholesterol, 10% DSPC, and 1.5% DMG-PEG2k) encapsulating GFP mRNA and irrelevant gRNA were formulated with different ionizable lipids and injected intracamerally into wildtype mice with 1 pl of 500 pg/mL LNP-GFP mRNA (i.e., GFP mRNA encapsulated in LNPs). Twenty-four hours post injection eyes were collected and analyzed for GFP expression using immunohistochemistry. Fig. 7A shows results of transfection with PBS (Vehicle). Fig. 7B shows results of transfection with LNPs formed with MC3. Fig. 7C shows results of transfection with LNPs formed with SM-102. Fig. 7D shows results of transfection with LNPs formed with ALC-0315. Fig. 7E shows results of transfection with LNPs formed with 5A2-SC8. Fig. 7F shows results of transfection with LNPs formed with BAMEA-O16B. Fig. 7G shows results of transfection with LNPs formed with BAMEA-O16B. Fig. 7H shows results of transfection with LNPs formed with SM-102. Arrows in Figs. 7B-7E show representative location of TM cells with GFP expression. The TM area is outlined in Figs. 7G and 7H.
[0128] Figs. 8A and 8B show transfection of human and non-human primate (NHP) trabecular meshwork tissue with two different ionizable lipids. Fig. 8A shows the percentage (%) of GFP+ cells in human TM region for control (Ctrl) and 0.5 mg/mL and 0.05 mg/mL LNP-GFP mRNA (i.e., GFP mRNA encapsulated in LNPs) of LNPs formed with MC3 (MC3) and LNPs formed with ALC-0315 (ALC-0315). Fig. 8B shows the percentage (%) of GFP+ cells in non-human primate (NHP) TM region for control (Ctrl) and 0.5 mg/mL and 0.05 mg/mL of LNP-GFP mRNA encapsulated in LNPs formed with MC3 (MC3) and LNPs formed with ALC-0315 (ALC-0315).
[0129] Figs. 9A-9C show in vivo screening data from testing different percentages of DMG- PEG2k and DSPE-PEG2k, for transfection of the mouse TM. Fig. 9A shows the LNP-GFP expression with different PEG lipids (DMG (black bars) and DSPC (grey bars)) and ratios. Images were qualitatively analyzed and scored based on the strength and localization of GFP expression. The Y-axis represents GFP expression using the following scoring scale for GFP expression in the TM region: - 0: no staining; 1 : sparse GFP+ staining; 2: more GFP+ staining; 3: GFP+ staining level between 2 and 4; and 4: highest level of GFP+ staining. For each mouse, the score was averaged over four TM regions in two eye sections. The grade for each group was the average of the GFP expression score in all mice within that group. Fig. 9B shows histology data for mouse TM transfected with Vehicle. Fig. 9C shows histology data for mouse TM transfected with LNPs containing 1.5% DMG-PEG.
[0130] Figs. 10A and 10B show concentration dependent transfection of primary human TM cells and HEK293T cells with candidate lipid ratio LNPs (ALC-0315, cholesterol, DSPC, at different ratios and 1.5% DMG-PEG2k). Primary TM cells or HEK293T cells were treated with increasing concentrations of LNP encapsulating GFP mRNA and irrelevant gRNA for 72 hours followed by flow cytometry analysis for GFP expression. Fig. 10A shows the percentage (%) of GFP+ cells in primary human TM cells for LNP1, LNP2, LNP5, LNP8, LNP14, LNP15, LNP17, and LNP19 (Table 12). The ECso for the various LNPs is shown. Fig. 10B shows the percentage (%) of GFP+ cells in HEK293T cells for LNP1, LNP2, LNP5, LNP8, LNP14, LNP15, LNP17, and LNP19 (Table 12). The ECso for the various LNPs is shown.
[0131] Figs. 11A-11F show histology data from in vivo screening of LNPs formulated with different ratios of lipids for transfection of the mouse TM. LNPs (ALC-0315, cholesterol, DSPC, at different ratios and 1.5% DMG-PEG2k) encapsulating GFP mRNA and irrelevant gRNA were injected intracamerally into wildtype mice with 1 pl of 500 pg/mL LNP-GFP mRNA (i.e., GFP mRNA encapsulated in LNPs). Twenty-four hours post injection eyes were collected and analyzed for GFP expression using immunohistochemistry. Fig. 11A shows results of transfection with PBS (Vehicle). Fig. 11B shows results of transfection with LNP1. Fig. 11C shows results of transfection with LNP8. Fig. 11D shows results of transfection with LNP14. Fig. HE shows results of transfection with LNP15. Fig. HF shows results of transfection with LNP 19. Arrows show representative location of TM cells with GFP expression.
[0132] Fig. 12 shows the percentage of indels in the MYOC gene formed after treating human donor corneal rims with LNPs (50% MC3, 38.5% cholesterol, 10% DSPC, 1.5% DMG- PEG2k) encapsulating mRNA encoding AsCasl2a (SEQ ID NO:33) and Casl2a-8 gRNA (Table 10). Human donor corneal rims were treated with 0.5 mg/mL or 0.05 mg/mL LNP- AsCasl2a mRNA (i.e., AsCasl2a mRNA encapsulated in LNPs). Human donor corneal rims were cut into wedges and treated with 10 |iL of LNPs at the indicated concentration. Five days post-treatment the TM tissue was dissected, and genomic DNA extracted. The percentage of indels in the MYOC gene was determined using next generation Illumina RNA- sequencing (Ill-seq) (see Table 16 for primers used).
[0133] Fig. 13 shows genomic DNA editing data (% indel) from in vivo delivery of LNPs (50% ALC-0315, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k) encapsulating mRNA encoding AsCasl2a (SEQ ID NO:33) plus gRNA (Casl2a-1 gRNA or Casl2a-2 gRNA, Table 10) in NHP TM tissue. Four cynomolgus monkeys received intracameral injections of LNPs in both eyes (7.5 pg LNP-AsCasl2a mRNA/eye), while one cynomolgus monkey received vehicle injections in both eyes. One week post injection the eyes were collected, the TM tissue was dissected, and genomic DNA extracted. Fig. 13 shows the genomic DNA editing (% indel) observed in the NHP TM tissue. Ill-seq analysis was performed to determine AsCasl2a-mediated editing at the MYOC locus (see Table 16 for primers used).
[0134] Fig. 14 shows the percentage of indels introduced into the MYOC gene in primary human TM cells resulting from transfection with increasing concentrations of LNPs (40% ALC-0315, 46% cholesterol, 12.5% DSPC, and 1.5% DMG-PEG2k) encapsulating mRNA encoding AsCasl2a (SEQ ID NO:33) plus Casl2a-1 gRNA (Table 10). gDNA was isolated three days after treatment and the resulting percentage of indels introduced into the MYOC gene was determined by Ul-Seq (see Table 16 for primers used). The IC50 for Cas 12a- 1 is shown.
[0135] Figs. 15A-15D show the MY01-H0M mouse Y437H myocilin gene sequence, intraocular pressure (IOP) data, and in vivo data resulting from transfection with LNPs (40% ALC-0315, 46% cholesterol, 12.5% DSPC, and 1.5% DMG-PEG2K) encapsulating mRNA encoding AsCasl2a (SEQ ID NO:33) plus Cas 12a- 1 gRNA (Table 10). Fig. 15A shows the full length human myocilin gene (hMYOC) carrying the pathogenic Y437H mutation (hMYOCY477H ) inserted into the mouse chromosome of the humanized MYO 1 -HOM mouse model. The human myocilin gene sequence is shown in dark grey, and the mouse sequence is shown in light grey. The knock-in includes the full-length human myocilin gene (ATG- STOP codon), as well as 1.5 kb upstream of the ATG which contains a portion of the human promoter. Fig. 15B shows IOP data from the MYO 1 -HOM mouse model (“HOM”) compared to wild-type (“WT”) mice. IOP was measured monthly on unanesthetized mice using a tonometer. Data are presented as mean ± SEM and analyzed with 2- way ANOVA followed by Tukey’s test. *, P<0.05; **, P<0.005; ***, P<0.0005; ****, P<0.0001 compared to age-matched WT. N= 18 (2-month) and 32 (3- and 6-month) for WT mice; N = 28 (2- month), 40 (3-month) and 37 (6-month) for MY01-H0M mice. Fig. 15C shows the percentage of myocilin mRNA remaining determined by reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) (myocilin normalized to TM-specific gene, Meox2). Data presented as mean ± SD and analyzed by one-way ANOVA with Tukey’s multiple comparison test compared to “Untreated”. Fig. 15D shows the percentage of indels introduced into the MYOC gene determined by Ill-Seq (see Table 16 for primers used). It is important to note that editing is from a mixed population of cells from the anterior chamber and not exclusively the TM tissue.
[0136] Figs. 16A-16C show data from in vivo delivery of LNPs (40% ALC-0315, 46% cholesterol, 12.5% DSPC, 1.5% DMG-PEG2k) encapsulating mRNA encoding AsCasl2a (SEQ ID NO:33) plus Casl2a-1 gRNA (Table 10) in NHP TM tissue. Cynomolgus monkeys received intracameral injections of LNPs at increasing concentrations of LNP-AsCasl2a mRNA (100 pg/mL, 300 pg/mL, or 500 pg/mL mRNA) (i.e., AsCasl2a mRNA encapsulated in LNPs). One week post injection the eyes were collected, the TM tissue was dissected, and genomic DNA extracted. Fig. 16A shows the percentage of indels introduced into the MYOC gene determined by Ill-Seq (see Table 16 for primers used). Data are presented as mean with SD analyzed with one-way ANOVA followed by Tukey’s test. Fig. 16B shows the percentage of myocilin mRNA remaining determined by RT-ddPCR. Data are presented as mean with standard deviation (SD) and analyzed with one-way ANOVA followed by Tukey’s multiple comparison test compared to vehicle. Vehicle is a tris buffered salt (TBS) solution plus 0.167 part 60% sucrose.
[0137] Figs. 17A-17E show editing of the MYOC gene, reduction of myocilin mRNA and protein, and reduction in endoplasmic reticulum (ER) stress in an in vitro phenotypic assay resulting from transfection with LNPs (40% ALC-0315, 46% cholesterol, 12.5% DSPC, and 1.5% DMG-PEG2k) encapsulating mRNA encoding AsCasl2a (SEQ ID NO:33) plus Casl2a-1 (Table 10) gRNA. Fig. 17A shows results from a Jess Automated Western blot indicating that HEKT293T cells stably expressed either wildtype or Y437H mutant myocilin. Mutant myocilin protein is not secreted and builds up inside the cell resulting in elevated GRP78, a marker of ER stress. “UNT” represents untreated HEKT293T cells. For Figs. 17B-17E, HEK293T cells stably expressing either wildtype (circles) or Y437H mutant (squares) myocilin were treated with increasing concentrations (2.3 x 10 6 mg/mL, 6.9 x 10 6 mg/mL, 2.1 x IO-5 mg/mL, 6.2 x 10’5 mg/mL, 1.9 x 1 O'4 mg/mL, 5.6 x 10-4 mg/mL, 1.7 x 10’3 mg/mL, 5.0 x 10'3 mg/mL) of LNP-AsCasl2a mRNA (i.e., AsCasl2a mRNA encapsulated in LNPs). Fig. 17B shows the percentage of indels introduced into the MYOC gene as determined by Ill-Seq (see Table 16 for primers used). Fig. 17C shows the level of myocilin mRNA expression normalized to GAPDH as determined by RT-ddPCR. Fig. 17D shows the expression of myocilin protein (intracellular) normalized to GAPDH as determined by Jess Automated Western Blot. Fig. 17E shows the expression of GRP78 protein (marker of ER stress) normalized to GAPDH as determined by Jess Automated Western Blot.
[0138] Figs. 18A-18D show intraocular pressure (IOP), editing of the MYOC gene and mRNA expression levels in a MYO 1 -HOM mouse model following in vivo delivery of LNPs (40% ALC-0315, 46% cholesterol, 12.5% DSPC, 1.5% DMG-PEG2k) encapsulating AsCasl2a mRNA plus Casl2a-1 gRNA (Table 10), and in vitro editing confirmation. The mRNA sequence used encoded the AsCasl2a set forth in SEQ ID NO:48 with the addition of two C-terminal nuclear localization signals (NLS). MY01-H0M mice were injected intracamerally with LNPs with increasing concentrations (1 pl of 25 g/mL, 100 pg/mL, or 500 pg/mL) of LNP-AsCasl2a mRNA (i.e., AsCasl2a mRNA encapsulated in LNPs). At the end of the study the anterior chambers were collected followed by extraction of mRNA and gDNA. Fig. 18A shows the IOP (mmHg) results of mice before dosing with LNP and 4 and 6 weeks after dosing with LNPs with various concentrations of LNP-AsCasl 2a mRNA (25 pg/mL, 100 pg/mL, and 500 pg/mL). Vehicle (“Veh”) is phosphate buffered saline (PBS). The 95% confidence interval (CI) of wild type (WT) IOP is shown as a grey horizonal bar. Fig. 18B shows the percentage of indels introduced into the MYOC gene as determined by Ill- Seq after dosing with LNPs with various concentrations of LNP-AsCasl2a mRNA (25 pg/mL, 100 pg/mL, and 500 pg/mL) (see Table 16 for primers used). Data are presented as mean with standard deviation (SD) and analyzed with one-way ANOVA followed by Tukey’s test. *, P<0.05; **, P<0.005; ***, P<0.0005; ****, P<0.0001. Vehicle (“Veh”) is PBS. Note that gDNA is from a mixed population of cells from the anterior chamber and not exclusively the TM tissue. Fig. 18C shows the percentage of myocilin mRNA determined by RT-ddPCR after dosing with LNPs with various concentrations of LNP-AsCasl2a mRNA (25 pg/mL, 100 pg/mL, and 500 pg/mL). Data are presented as mean with standard deviation (SD) and analyzed with one-way ANOVA with Tukey’s multi comparison test compared to Vehicle. Vehicle (“Veh”) is PBS. Fig. 18D shows the percentage of indels introduced into the MYOC gene in primary TM cells as determined by Ill-Seq with LNPs with various concentrations of LNP-AsCasl2a mRNA: LNP stock (circles), 25 pg/mL (downward pointing triangles), 100 pg/mL (upward pointing triangles), and 500 pg/mL (squares) (see Table 16 for primers used). Primary TM cells were treated with increasing concentrations (based on AsCasl2a mRNA) of LNP encapsulating AsCasl2a mRNA plus gRNA. Fig. 18D provides in vitro confirmation that LNPs that were tested in vivo also edit primary TM cells in vitro.
DETAILED DESCRIPTION
Definitions
[0139] Unless otherwise specified, each of the following terms has the meaning associated with it in this section.
[0140] The indefinite articles “a” and “an” refer to at least one of the associated noun, and are used interchangeably with the terms “at least one” and “one or more.” For example, “a module” means at least one module, or one or more modules.
[0141] The conjunctions “or” and “and/or” are used interchangeably as non-exclusive disjunctions.
[0142] “Domain,” as used herein, is used to describe segments of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional property.
[0143] An “indel” is an insertion and/or deletion in a nucleic acid sequence. An indel may be the product of the repair of a DNA double strand break, such as a double strand break formed by a genome editing system of the present disclosure. An indel is most commonly formed when a break is repaired by an “error prone” repair pathway such as the NHEJ pathway described below.
[0144] “MYOC target position,” as used herein, refers to a target position, e.g., one or more nucleotides, in or near the MYOC gene, that are targeted for alteration using the methods described herein. In certain embodiments, alteration of the MYOC target position, e.g., by substitution, deletion, or insertion, may result in disruption (e.g., “knocking out”) of the MYOC gene. In certain embodiments, the MYOC target position may be located in the 5’ UTR, exon 1, the exon 1/intron 1 border, intron 1, the intron 1/exon 2 border, exon 2, the exon 2/intron 2 border, intron 2, the intron 2/exon 3 border, exon 3, the exon 3/intron 3 border, the 3’ UTR or a combination thereof of the MYOC gene.
[0145] “Subject,” as used herein, means a human, mouse, or non-human primate. A human subject can be any age (e.g., an infant, child, young adult, or adult), and may suffer from a disease, or may be in need of alteration of a gene.
[0146] “Treat,” “treating,” and “treatment” mean the treatment of a disease in a subject (e.g., a human subject), including one or more of inhibiting the disease, i.e., arresting or preventing its development or progression; relieving the disease, i.e., causing regression of the disease state; relieving one or more symptoms of the disease; and curing the disease.
[0147] “Prevent,” “preventing,” and “prevention” refer to the prevention of a disease in a subject, including (a) avoiding or precluding the disease; (b) affecting the predisposition toward the disease; or (c) preventing or delaying the onset of at least one symptom of the disease.
[0148] A “kit” refers to any collection of two or more components that together constitute a functional unit that can be employed for a specific purpose. By way of illustration (and not limitation), one kit according to this disclosure can include a guide RNA complexed or able to complex with an RNA-guided nuclease, and accompanied by (e.g., suspended in, or suspendable in) a pharmaceutically acceptable carrier. In certain embodiments, the kit may include a booster element. The kit can be used to introduce the complex into, for example, a cell or a subject, for the purpose of causing a desired genomic alteration in such cell or subject. The components of a kit can be packaged together, or they may be separately packaged. Kits according to this disclosure also optionally include directions for use (DFU) that describe the use of the kit e.g., according to a method of this disclosure. The DFU can be physically packaged with the kit, or it can be made available to a user of the kit, for instance by electronic means.
[0149] The terms “polynucleotide”, “nucleotide sequence”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, and “oligonucleotide” refer to a series of nucleotide bases (also called “nucleotides”), for example, in DNA or RNA, and mean any chain of two or more nucleotides. The polynucleotides, nucleotide sequences, nucleic acids, etc. can be chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded. They can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. A nucleotide sequence typically carries genetic information, including, but not limited to, the information used by cellular machinery to make proteins and enzymes. These terms include double- or single- stranded genomic DNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. These terms also include nucleic acids containing modified bases.
[0150] The terms “protein,” “peptide” and “polypeptide” are used interchangeably to refer to a sequential chain of amino acids linked together via peptide bonds. The terms include individual proteins, groups or complexes of proteins that associate together, as well as fragments or portions, variants, derivatives and analogs of such proteins. Peptide sequences are presented herein using conventional notation, beginning with the amino or N-terminus on the left, and proceeding to the carboxyl or C-terminus on the right. Standard one-letter or three- letter abbreviations can be used.
[0151] “X” as used herein in the context of an amino acid sequence, refers to any amino acid (e.g., any of the twenty natural amino acids) unless otherwise specified.
[0152] Conventional IUPAC notation is used in nucleotide sequences presented herein, as shown in Table 1, below (see also Cornish-Bowden 1985, incorporated by reference herein). It should be noted, however, that “T” denotes “Thymine or Uracil” in those instances where a sequence may be encoded by either DNA or RNA, for example in gRNA targeting domains.
Table 1: IUPAC nucleic add notation
[0153] The term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a gene if it affects the transcription of the gene. Operably linked nucleotide sequences are typically contiguous. However, as enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome.
[0154] Where ranges are provided herein, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.
Primary Open Angle Glaucoma (POAG)
[0155] Glaucoma is the second leading cause of blindness in the world. Primary Open Angle Glaucoma (POAG) is the leading cause of glaucoma and affects approximately 1% of patients ages 40-89.
[0156] POAG develops due to an imbalance between the production and outflow of aqueous humor within the eye. Aqueous humor (AH) is produced by the ciliary body located in the posterior chamber. The vast majority (approximately 80%) of AH drains through the trabecular meshwork (TM) to the episcleral venous system. A minority (approximately 20%) of AH drains through the interstitium between the iris root and ciliary muscle (Feisal 2005). POAG is likely due to decreased drainage through the trabecular meshwork; decreased outflow of AH results in increased intraocular pressure (IOP) and IOP causes damage to the optic nerve and leads to progressive blindness.
[0157] The etiology of POAG is multi-factorial and complex. However, mutations in the MYOC gene (also known as GLC1A, JOAG1 and TIGR) have been shown to be a leading genetic cause of POAG and of juvenile-onset POAG. Mutations in MYOC have been shown to account for 3% of POAG. Many patients with MYOC mutations develop rapidly advancing disease and/or earlier presentation of POAG, including juvenile-onset POAG.
[0158] The MYOC gene, also called the trabecular meshwork-induced glucocorticoid receptor (TIGR), encodes myocilin, a 504 amino acid protein encoded by 3 exons. Myocilin is found in the trabecular meshwork and plays a role in cytoskeletal function and in the regulation of IOP.
Methods to treat or prevent POAG
[0159] Methods and compositions described herein provide for a therapy, e.g., a one-time therapy, or a multi-dose therapy, that prevents or treats POAG. In an embodiment, a disclosed therapy prevents, inhibits, or reduces the production of mutant myocilin protein in cells of the anterior and posterior chamber of the eye in a subject who has POAG.
[0160] While not wishing to be bound by theory, in an embodiment, it is believed that knocking out MYOC on ciliary body cells, iris cells, trabecular meshwork cells, retinal cells, e.g., a rod photoreceptor cell, e.g., a cone photoreceptor cell, e.g., a retinal pigment epithelium cell, e.g., a horizontal cell, e.g., an amacrine cell, e.g., a ganglion cell, will prevent the progression of eye disease in subjects with POAG.
[0161] Myocilin is expressed in the eye, primarily by trabecular meshwork cells and the ciliary body. It is also expressed in the retina. Research indicates that MYOC mutations exert a toxic gain of function effect within trabecular meshwork cells. Mutant myocilin, especially mutants with missense or nonsense mutations in exon 3, e.g., a mutation at T377 (e.g., T377R), a mutation at 1477 (e.g., I477N), or a mutation at P370 (e.g., P370L), may misfold and aggregate in the endoplasmic reticulum (ER). Misfolding and aggregation within the ER elicits the ER stress and unfold protein response, which can lead to apoptosis and inflammation within trabecular meshwork cells. In addition, mutant myocilin protein may aggregate in the trabecular meshwork with other mutant proteins and/or with wild-type myocilin (in heterozygotes). Mutant myocilin aggregates may interfere with the outflow of aqueous humor to the episcleral venous system. Decreased aqueous humor outflow causes increased intraocular pressure, leading to POAG.
[0162] The elimination of mutant myocilin production in subjects with a mutation, e.g., a mutation at T377 (e.g., T377R), a mutation at 1477 (e.g., I477N), or a mutation at P370 (e.g., P370L), or other mutations or other mutant MYOC alleles, through knock out of MYOC on ciliary body cells, iris cells, trabecular meshwork cells and retinal cells will prevent the production of the myocilin proteins. In an embodiment, POAG does not progress or has delayed progression compared to a subject who has not received the therapy.
[0163] Described herein are methods for treating or delaying the onset or progression of POAG caused by mutations in the MYOC gene, including but not limited to mutations in exon 3, e.g., a mutation at T377 (e.g., T377R), a mutation at 1477 (e.g., I477N), or a mutation at P370 (e.g., P370L). The disclosed methods for treating or delaying the onset or progression of POAG alter the MYOC gene by genome editing using a gRNA that is complementary to a target domain of the MYOC gene and an RNA-guided nuclease (e.g., Casl2a enzyme).
[0164] Current treatments to prevent the progression of POAG include treatments that reduce IOP. For example, trabeculectomy surgery and eye drops, including alpha-adrenergic antagonists and beta-adrenergic antagonists, are both effective in preventing POAG progression. However, further treatments are needed to reduce IOP and prevent progression of POAG. Disclosed herein are methods that knockout a MYOC gene. Targeted knockout of the MYOC gene includes targeting one or both alleles of the MYOC gene. The disclosed methods may be useful to permanently decrease IOP and prevent the progressive visual loss of POAG. Further, the disclosed methods are more convenient than taking daily eye drops or having surgery.
[0165] Disclosed herein are multiple approaches to altering or modifying, i.e., knocking out, the MYOC gene, using the CRISPR/RNA-guided nuclease system to treat POAG.
[0166] In one embodiment, the MYOC gene is targeted as a targeted knockout. A knockout of the MYOC gene may offer a benefit to subjects with POAG who have a mutation in the MYOC gene as well as subjects with POAG without a known MYOC mutation. There is evidence that MYOC mutations are gain of function mutations leading to altered TM function and the development of IOP. There is further evidence that patients with heterozygous early truncating mutations (Arg46stop) do not develop disease. MYOC knock-out mice do not develop POAG and have no detected eye abnormalities. Further, a few patients have been identified who express no myocilin in the eye and have no phenotype. Without wishing to be bound by theory, it is contemplated herein that a knockout of MYOC gene in the eye prevents the development of POAG.
[0167] There is also evidence to support a dominant negative effect of certain heterozygous mutations on the wild-type allele (Kuchtey 2013). Without wishing to be bound by theory, it is contemplated herein that a knockout of both alleles reverses the dominant negative effect and is beneficial for patients.
[0168] Alteration of one or both MYOC alleles may be performed prior to disease onset or after disease onset, but preferably early in the disease course.
[0169] In an embodiment, treatment is initiated prior to onset of the disease.
[0170] In an embodiment, treatment is initiated after onset of the disease, but early in the course of disease progression (e.g., prior to vision loss, a decrease in visual acuity and/or an increase in IOP).
[0171] In an embodiment, treatment is initiated after onset of the disease, but prior to a measurable increase in IOP. In an embodiment, treatment is initiated prior to loss of visual acuity. In an embodiment, treatment is initiated at onset of loss of visual acuity. In an embodiment, treatment is initiated after onset of loss of visual acuity.
[0172] In an embodiment, treatment is initiated in a subject who has tested positive for a mutation in the MYOC gene, e.g., prior to disease onset or in the earliest stages of disease.
[0173] In an embodiment, a subject has a family member that has been diagnosed with POAG. For example, the subject has a family member that has been diagnosed with POAG, and the subject demonstrates a symptom or sign of the disease or has been found to have a mutation in the MYOC gene.
[0174] In an embodiment, treatment is initiated in a subject who has no MYOC mutation but has increased intraocular pressure. In an embodiment, treatment is initiated in a subject at onset of an increase in intraocular pressure. In an embodiment, treatment is initiated in a subject after onset of an increase in intraocular pressure. [0175] In an embodiment, treatment is initiated in a subject with signs consistent with POAG on ophthalmologic exam, including but not limited to: increased intraocular pressure; cupping of the optic nerve on slit lamp exam, stereobiomicroscopy or ophthalmoscopy; pallor of the optic disk; thinning or notching of the optic disk rim; hemorrhages of the optic disc; vertical cup-to-disk ratio of >0.6 or cup-to-disk asymmetry between eyes of greater than 0.2; peripapillary atrophy.
[0176] A subject’s vision can be evaluated, e.g., prior to treatment, or after treatment, e.g., to monitor the progress of the treatment. In an embodiment, the subject’s vision is evaluated prior to treatment, e.g., to determine the need for treatment. In an embodiment, the subject’s vision is evaluated after treatment has been initiated, e.g., to assess the effectiveness of the treatment. Vision can be evaluated by one or more of: evaluation of increased IOP; evaluating changes in function relative to the contralateral eye, e.g., by utilizing retinal analytical techniques; by evaluating mean, median and distribution of change in best corrected visual acuity (BCVA); evaluation by Optical Coherence Tomography; evaluation of changes in visual field using perimetry; evaluation by full-field electroretinography (ERG); evaluation by slit lamp examination; evaluation of intraocular pressure; evaluation of autofluorescence, evaluation with fundoscopy; evaluation with fundus photography; evaluation with fluorescein angiography (FA); or evaluation of visual field sensitivity (FFST).
[0177] In other embodiments, a subject’s vision may be assessed by measuring the subject’s mobility, e.g., the subject’s ability to maneuver in space.
Methods of altering MYOC
[0178] As disclosed herein, a target site, e.g., MYOC gene, can be altered by gene editing, e.g., using CRISPR-RNA-guided nuclease (e.g., Casl2a) mediated methods as described herein.
[0179] An alteration of the MYOC gene can be mediated by any mechanism. Exemplary mechanisms that can be associated with an alteration of the MYOC gene include, but are not limited to, non-homologous end joining (e.g., classical or alternative), microhomology- mediated end joining (MMEJ), homology-directed repair (e.g., endogenous donor template mediated), SDSA (synthesis dependent strand annealing), single strand annealing or single strand invasion. [0180] In some embodiments, disclosed approaches give rise to alteration of the MYOC gene. In one embodiment, methods described herein introduce one or more breaks near a target site in at least one allele of the MYOC gene. In another embodiment, methods described herein introduce two or more breaks to flank a target site. The two or more breaks remove (e.g., delete) genomic sequence including the target site in the MYOC gene. In some embodiments, methods described herein result in alteration of the MYOC gene.
Genome editing systems
[0181] The term “genome editing system” refers to any system having RNA-guided DNA editing activity. Genome editing systems of the present disclosure include at least two components adapted from naturally occurring CRISPR systems: a guide RNA (gRNA) and an RNA-guided nuclease (e.g., Casl2a). These two components form a complex that is capable of associating with a specific nucleic acid sequence and editing the DNA in or around that nucleic acid sequence, for instance by making one or more of a single-strand break (an SSB or nick), a double-strand break (a DSB) and/or a point mutation.
[0182] Genome editing systems can be implemented (e.g., administered or delivered to a cell or a subject) in a variety of ways, and different implementations may be suitable for distinct applications. For instance, a genome editing system is implemented, in certain embodiments, as a protein/RNA complex (a ribonucleoprotein, or RNP), which can be included in a pharmaceutical composition that optionally includes a pharmaceutically acceptable carrier and/or an encapsulating agent, such as, without limitation, a lipid or polymer micro- or nanoparticle, micelle, or liposome. In certain embodiments, a genome editing system is implemented as one or more nucleic acids encoding the RNA-guided nuclease and guide RNA (optionally with one or more additional components); in certain embodiments, the genome editing system is implemented as one or more vectors comprising such nucleic acids, for instance a viral vector such as an adeno-associated virus (see section below under the heading “Delivery, formulations, and routes of administration”); and in certain embodiments, the genome editing system is implemented as a combination of any of the foregoing. Additional or modified implementations that operate according to the principles set forth herein will be apparent to the skilled artisan and are within the scope of this disclosure.
[0183] It should be noted that the genome editing systems of the present disclosure can be targeted to a single specific nucleotide sequence, or may be targeted to — and capable of editing in parallel — two or more specific nucleotide sequences through the use of two or more guide RNAs. The use of multiple gRNAs is referred to as “multiplexing” throughout this disclosure, and can be employed to target multiple, unrelated target sequences of interest, or to form multiple SSBs or DSBs within a single target domain and, in some cases, to generate specific edits within such target domain. For example, International Patent Publication No. WO 2015/138510 by Maeder et al. (“Maeder”), which is incorporated by reference herein, describes a genome editing system for correcting a point mutation (C.2991+1655A to G) in the human CEP290 gene that results in the creation of a cryptic splice site, which in turn reduces or eliminates the function of the gene. The genome editing system of Maeder utilizes two guide RNAs targeted to sequences on either side of (i.e., flanking) the point mutation, and forms DSBs that flank the mutation. This, in turn, promotes deletion of the intervening sequence, including the mutation, thereby eliminating the cryptic splice site and restoring normal gene function.
[0184] As another example, WO 2016/073990 by Cotta-Ramusino et al. (“Cotta-Ramusino”), which is incorporated by reference herein, describes a genome editing system that utilizes two gRNAs in combination with a Cas9 nickase (a Cas9 that makes a single strand nick such as S. pyogenes D10A), an arrangement termed a “dual-nickase system.” The dual-nickase system of Cotta-Ramusino is configured to make two nicks on opposite strands of a sequence of interest that are offset by one or more nucleotides, which nicks combine to create a double strand break having an overhang (5’ in the case of Cotta-Ramusino, though 3’ overhangs are also possible). The overhang, in turn, can facilitate homology directed repair events in some circumstances. And, as another example, WO 2015/070083 by Palestrant et al. (incorporated by reference herein) describes a gRNA targeted to a nucleotide sequence encoding Cas9 (referred to as a “governing RNA”), which can be included in a genome editing system comprising one or more additional gRNAs to permit transient expression of a Cas9 that might otherwise be constitutively expressed, for example in some virally transduced cells. These multiplexing applications are intended to be exemplary, rather than limiting, and the skilled artisan will appreciate that other applications of multiplexing are generally compatible with the genome editing systems described here.
[0185] As disclosed herein, in certain embodiments, genome editing systems may comprise multiple gRNAs that may be used to introduce mutations into the MYOC gene. In certain embodiments, genome editing systems disclosed herein may comprise multiple gRNAs used to introduce mutations into the MYOC gene. [0186] Genome editing systems can, in some instances, form double strand breaks that are repaired by cellular DNA double-strand break mechanisms such as NHEJ or HDR. These mechanisms are described throughout the literature (see, e.g., Davis & Maizels 2014 (describing Alt-HDR); Frit 2014 (describing Alt-NHEJ); lyama & Wilson 2013 (describing canonical HDR and NHEJ pathways generally)).
[0187] Where genome editing systems operate by forming DSBs, such systems optionally include one or more components that promote or facilitate a particular mode of double-strand break repair or a particular repair outcome. For instance, Cotta-Ramusino also describes genome editing systems in which a single stranded oligonucleotide “donor template” is added; the donor template is incorporated into a target region of cellular DNA that is cleaved by the genome editing system, and can result in a change in the target sequence.
[0188] In certain embodiments, genome editing systems modify a target sequence, or modify expression of a gene in or near the target sequence, without causing single- or double-strand breaks. For example, a genome editing system may include an RNA-guided nuclease fused to a functional domain that acts on DNA, thereby modifying the target sequence or its expression. As one example, an RNA-guided nuclease can be connected to (e.g., fused to) a cytidine deaminase functional domain, and may operate by generating targeted C-to-A substitutions. Exemplary nuclease/deaminase fusions are described in Komor 2016, which is incorporated by reference herein. Alternatively, a genome editing system may utilize a cleavage-inactivated (i.e., a “dead”) nuclease, such as a dead Cas9 (dCas9) or dead Casl2a (dCasl 2a), and may operate by forming stable complexes on one or more targeted regions of cellular DNA, thereby interfering with functions involving the targeted region(s) including, without limitation, mRNA transcription, chromatin remodeling, etc. In certain embodiments, a genome editing system may include an RNA-guided helicase that unwinds DNA within or proximal to the target sequence, without causing single- or double- stranded breaks. For example, a genome editing system may include an RNA-guided helicase configured to associate within or near the target sequence to unwind DNA and induce accessibility to the target sequence. In certain embodiments, the RNA-guided helicase may be complexed to a dead guide RNA that is configured to lack cleavage activity allowing for unwinding of the DNA without causing breaks in the DNA. Guide RNA (gRNA) molecules
[0189] The terms “guide molecule,” “guide RNA,” “gRNA,” “gRNA molecule,” refer to any nucleic acid that promotes the specific association (or “targeting”) of an RNA-guided nuclease such as Casl2a to a target sequence such as a genomic or episomal sequence in a cell. gRNAs can be unimolecular (comprising a single RNA molecule, and referred to alternatively as chimeric), or modular (comprising more than one, and typically two, separate RNA molecules, such as a crRNA and a tracrRNA, which are usually associated with one another, for instance by duplexing). gRNAs and their component parts are described throughout the literature (see, e.g., Briner 2014, which is incorporated by reference; Cotta- Ramusino). In certain embodiments, the guide molecule can be an RNA molecule. The guide molecule can also comprise one or more nucleotides other than RNA nucleotides, for example, the guide molecule can be a DNA/RNA hybrid molecule, and/or the guide molecule can comprise one or more modified nucleotides (including, but not limited to, one or more modified DNA or RNA nucleotides).
[0190] In bacteria and archea, type II CRISPR systems generally comprise an RNA-guided nuclease protein such as Cas9, a CRISPR RNA (crRNA) that includes a 5’ region that is complementary to a foreign sequence, and a trans-activating crRNA (tracrRNA) that includes a 5’ region that is complementary to, and forms a duplex with, a 3’ region of the crRNA. While not intending to be bound by any theory, it is thought that this duplex facilitates the formation of — and is necessary for the activity of — the Cas9/gRNA complex. As type II CRISPR systems were adapted for use in gene editing, it was discovered that the crRNA and tracrRNA could be joined into a single unimolecular or chimeric guide RNA, in one nonlimiting example, by means of a four nucleotide (e.g., GAAA) “tetraloop” or “linker” sequence bridging complementary regions of the crRNA (at its 3’ end) and the tracrRNA (at its 5’ end). (Mali 2013; Jiang 2013; Jinek 2012; all incorporated by reference herein).
[0191] Guide RNAs, whether unimolecular or modular, include a “targeting domain” that is fully or partially complementary to a target domain within a target sequence, such as a DNA sequence in the genome of a cell where editing is desired. Targeting domains are referred to by various names in the literature, including without limitation “guide sequences” (Hsu 2013, incorporated by reference herein), “complementarity regions” (Cotta-Ramusino), “spacers” (Briner 2014) and generically as “crRNAs” (Jiang). Irrespective of the names they are given, targeting domains are typically 10-30 nucleotides in length, and in certain embodiments are 16-26 nucleotides in length (for instance, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length), and are at or near the 5’ terminus of in the case of a Cas9 gRNA, and at or near the 3’ terminus in the case of a Casl2a gRNA.
[0192] In addition to the targeting domains, gRNAs typically (but not necessarily, as discussed below) include a plurality of domains that may influence the formation or activity of gRNA/RNA-guided nuclease complexes. For instance, as mentioned above, the duplexed structure formed by first and secondary complementarity domains of a gRNA (also referred to as a repeat: anti-repeat duplex) interacts with the recognition (REC) lobe of Cas9 and can mediate the formation of Cas9/gRNA complexes (Nishimasu 2014; Nishimasu 2015; both incorporated by reference herein). It should be noted that the first and/or second complementarity domains may contain one or more poly-A tracts, which can be recognized by RNA polymerases as a termination signal. The sequence of the first and second complementarity domains are, therefore, optionally modified to eliminate these tracts and promote the complete in vitro transcription of gRNAs, for instance through the use of A-G swaps as described in Briner 2014, or A-U swaps. These and other similar modifications to the first and second complementarity domains are within the scope of the present disclosure.
[0193] Along with the first and second complementarity domains, Cas9 gRNAs typically include two or more additional duplexed regions that are involved in nuclease activity in vivo but not necessarily in vitro. (Nishimasu 2015). A first stem-loop one near the 3’ portion of the second complementarity domain is referred to variously as the “proximal domain,” (Cotta-Ramusino) “stem loop 1” (Nishimasu 2014 and 2015) and the “nexus” (Briner 2014). One or more additional stem loop structures are generally present near the 3 ’ end of the gRNA, with the number varying by species: .S', pyogenes gRNAs typically include two 3’ stem loops (for a total of four stem loop structures including the repeat: anti-repeat duplex), while S. aureus and other species have only one (for a total of three stem loop structures). A description of conserved stem loop structures (and gRNA structures more generally) organized by species is provided in Briner 2014.
[0194] While the foregoing description has focused on gRNAs for use with Cas9, it should be appreciated that other RNA-guided nucleases exist which utilize gRNAs that differ in some ways from those described to this point. For instance, Cpfl (“CRISPR from Prevotella and Franciscella 1”), also referred to as Casl2a, is an RNA-guided nuclease that does not require a tracrRNA to function (Zetsche 2015, incorporated by reference herein). A gRNA for use in a Casl2a genome editing system may include a targeting domain and a complementarity domain (alternately referred to as a “handle”). It should also be noted that, in gRNAs for use with Cast 2a, the targeting domain is usually present at or near the 3’ end, rather than the 5’ end as described above in connection with Cas9 gRNAs (the handle is at or near the 5’ end of a Casl2a gRNA). Exemplary Casl2a gRNA targeting domains are set forth in Table 10.
[0195] In certain embodiments, the Casl2a gRNA may include a crRNA direct repeat extension sequence. In certain embodiments, an exemplary crRNA direct repeat extension sequence may be UAAUUUCUACUCUUGUAGAU (SEQ ID NO: 49) (corresponding DNA sequence is TAATTTCTACTCTTGTAGAT (SEQ ID NO: 50)).
[0196] Those of skill in the art will appreciate that although structural differences may exist between gRNAs from different prokaryotic species, or between Casl2a and Cas9 gRNAs, the principles by which gRNAs operate are generally consistent. Because of this consistency of operation, gRNAs can be defined, in broad terms, by their targeting domain sequences, and skilled artisans will appreciate that a given targeting domain sequence can be incorporated in any suitable gRNA, including a unimolecular or chimeric gRNA, or a gRNA that includes one or more chemical modifications and/or sequential modifications (substitutions, additional nucleotides, truncations, etc.). Thus, for economy of presentation in this disclosure, gRNAs may be described solely in terms of their targeting domain sequences.
[0197] More generally, skilled artisans will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using multiple RNA-guided nucleases. For this reason, unless otherwise specified, the term gRNA should be understood to encompass any suitable gRNA that can be used with any RNA- guided nuclease, and not only those gRNAs that are compatible with a particular species of Casl2a. By way of illustration, the term gRNA can, in certain embodiments, include a gRNA for use with any RNA-guided nuclease occurring in a Class 2 CRISPR system, such as a type II or type V or CRISPR system, or an RNA-guided nuclease derived or adapted therefrom.
[0198] In an embodiment, a unimolecular, or chimeric, gRNA may comprise, a crRNA direct repeat extension sequence and a targeting domain (which is complementary to a target nucleic acid in the MYOC gene, e.g., a targeting domain from Table 10).
In an embodiment, a modular gRNA comprises: a crRNA direct repeat extension sequence and a first strand comprising a targeting domain (which is complementary to a target nucleic acid in the MYOC gene, e.g., a targeting domain from Table 10).
[0199] In an embodiment, the targeting domain is 16 nucleotides in length. In an embodiment, the targeting domain is 17 nucleotides in length. In an embodiment, the targeting domain is 18 nucleotides in length. In an embodiment, the targeting domain is 19 nucleotides in length. In an embodiment, the targeting domain is 20 nucleotides in length. In an embodiment, the targeting domain is 21 nucleotides in length. In an embodiment, the targeting domain is 22 nucleotides in length. In an embodiment, the targeting domain is 23 nucleotides in length. In an embodiment, the targeting domain is 24 nucleotides in length. In an embodiment, the targeting domain is 25 nucleotides in length. In an embodiment, the targeting domain is 26 nucleotides in length.
[0200] In an embodiment, the targeting domain comprises 16 nucleotides. In an embodiment, the targeting domain comprises 17 nucleotides. In an embodiment, the targeting domain comprises 18 nucleotides. In an embodiment, the targeting domain comprises 19 nucleotides. In an embodiment, the targeting domain comprises 20 nucleotides. In an embodiment, the targeting domain comprises 21 nucleotides. In an embodiment, the targeting domain comprises 22 nucleotides. In an embodiment, the targeting domain comprises 23 nucleotides. In an embodiment, the targeting domain comprises 24 nucleotides. In an embodiment, the targeting domain comprises 25 nucleotides. In an embodiment, the targeting domain comprises 26 nucleotides.
[0201] In some embodiments, the targeting domain of the gRNA molecule has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a targeting domain nucleotide sequence disclosed herein, e.g., a targeting domain nucleotide sequence set forth in Table 10, e.g., a targeting domain nucleotide sequence from the group consisting of a Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8. In some embodiments, the targeting domain of the gRNA molecule has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations relative to a targeting domain nucleotide sequence disclosed herein, e.g., a targeting domain nucleotide sequence set forth in Table 10, e.g., a targeting domain nucleotide sequence from the group consisting of a Casl2a-l, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8. In some embodiments, the targeting domain of the gRNA molecule has less than 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations relative to a targeting domain nucleotide sequence disclosed herein, e.g., a targeting domain nucleotide sequence set forth in Table 10, e.g., a targeting domain nucleotide sequence from the group consisting of a Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8. gRNA modifications
[0202] The activity, stability, or other characteristics of gRNAs can be altered through the incorporation of certain modifications. As one example, transiently expressed or delivered nucleic acids can be prone to degradation by, e.g., cellular nucleases. Accordingly, the gRNAs described herein can contain one or more modified nucleosides or nucleotides which introduce stability toward nucleases. While not wishing to be bound by theory it is also believed that certain modified gRNAs described herein can exhibit a reduced innate immune response when introduced into cells. Those of skill in the art will be aware of certain cellular responses commonly observed in cells, e.g., mammalian cells, in response to exogenous nucleic acids, particularly those of viral or bacterial origin. Such responses, which can include induction of cytokine expression and release and cell death, may be reduced or eliminated altogether by the modifications presented herein.
[0203] Certain exemplary modifications discussed in this section can be included at any position within a gRNA sequence including, without limitation at or near the 5’ end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5’ end) and/or at or near the 3’ end (e.g., within 1- 10, 1-5, or 1-2 nucleotides of the 3’ end). In some cases, modifications are positioned within functional motifs, such as the repeat-anti-repeat duplex of a Cas9 gRNA, a stem loop structure of a Cas9 or Casl2a gRNA, and/or a targeting domain of a gRNA.
[0204] As one example, the 5’ end of a gRNA can include a eukaryotic mRNA cap structure or cap analog (e.g., a G(5’)ppp(5’)G cap analog, a m7G(5’)ppp(5’)G cap analog, or a 3’-O- Me-m7G(5’)ppp(5’)G anti reverse cap analog (ARCA)), as shown below:
The cap or cap analog can be included during either chemical synthesis or in vitro transcription of the gRNA.
[0205] Along similar lines, the 5’ end of the gRNA can lack a 5’ triphosphate group. For instance, in vitro transcribed gRNAs can be phosphatase-treated (e.g., using calf intestinal alkaline phosphatase) to remove a 5’ triphosphate group.
[0206] Another common modification involves the addition, at the 3 ’ end of a gRNA, of a plurality (e.g., 1-10, 10-20, or 25-200) of adenine (A) residues referred to as a polyA tract. The polyA tract can be added to a gRNA during chemical synthesis, following in vitro transcription using a polyadenosine polymerase (e.g., E. coli Poly(A)Polymerase), or in vivo by means of a polyadenylation sequence, as described in Maeder.
[0207] It should be noted that the modifications described herein can be combined in any suitable manner, e.g., a gRNA, whether transcribed in vivo from a DNA vector, or in vitro transcribed gRNA, can include either or both of a 5’ cap structure or cap analog and a 3’ polyA tract.
[0208] Guide RNAs can be modified at a 3’ terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as shown below: wherein “U” can be an unmodified or modified uridine. [0209] The 3’ terminal U ribose can be modified with a 2’3’ cyclic phosphate as shown below: wherein “U” can be an unmodified or modified uridine.
[0210] Guide RNAs can contain 3’ nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In certain embodiments, uridines can be replaced with modified uridines, e.g., 5-(2-amino)propyl uridine, and 5 -bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein.
[0211] In certain embodiments, sugar-modified ribonucleotides can be incorporated into the gRNA, e.g., wherein the 2’ OH-group is replaced by a group selected from H, -OR, -R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, -SH, -SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN). In certain embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphorothioate (PhTx) group. In certain embodiments, one or more of the nucleotides of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2’ -sugar modified, such as, 2’-O-methyl, 2’-O-methoxyethyl, or 2’-Fluoro modified including, e.g., 2’-F or 2’-O-methyl, adenosine (A), 2’-F or 2’-O-methyl, cytidine (C), 2’-F or 2’-O-methyl, uridine (U), 2’-F or 2’-O-methyl, thymidine (T), 2’-F or 2’-O- methyl, guanosine (G), 2 ’ -O-methoxyethyl-5 -methyluridine (Teo), 2’ 0- methoxyethyladenosine (Aeo), 2’-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof. [0212] Guide RNAs can also include “locked” nucleic acids (LNA) in which the 2’ OH- group can be connected, e.g., by a Cl-6 alkylene or Cl-6 heteroalkylene bridge, to the 4’ carbon of the same ribose sugar. Any suitable moiety can be used to provide such bridges, include without limitation methylene, propylene, ether, or amino bridges; 0-amino (wherein amino can be, e.g., NHz; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy or O(CH2)n-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or poly amino).
[0213] In certain embodiments, a gRNA can include a modified nucleotide which is multicyclic (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R- GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), or threose nucleic acid (TNA, where ribose is replaced with a-L-threofuranosyl-(3’^-2’ )).
[0214] Generally, gRNAs include the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary modified gRNAs can include, without limitation, replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphorami date backbone). Although the majority of sugar analog alterations are localized to the 2’ position, other sites are amenable to modification, including the 4’ position. In certain embodiments, a gRNA comprises a 4’- S, 4’-Se or a 4’-C-aminomethyl-2’-O-Me modification.
[0215] In certain embodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can be incorporated into the gRNA. In certain embodiments, O- and N-alkylated nucleotides, e.g., N6-methyl adenosine, can be incorporated into the gRNA. In certain embodiments, one or more or all of the nucleotides in a gRNA are deoxynucleotides.
[0216] In certain embodiments, gRNAs as used herein may be modified or unmodified gRNAs. In certain embodiments, a gRNA may include one or more modifications. In certain embodiments, the one or more modifications may include a phosphorothioate linkage modification, a phosphorodithioate (PS2) linkage modification, a 2’-O-methyl modification, or combinations thereof. In certain embodiments, the one or more modifications may be at the 5’ end of the gRNA, at the 3’ end of the gRNA, or combinations thereof.
[0217] In certain embodiments, a modified gRNA as used herein comprises one or more modifications in the crRNA direct repeat extension sequence (also referred to as the hairpin region), the targeting domain, or both. In certain embodiments, the hairpin region of the modified gRNA comprises 2’Fluorine. In certain embodiments, the hairpin region of the modified gRNA comprises a DNA extension 5’ to the hairpin region. In certain embodiments, the hairpin region of the modified gRNA comprises a 2’0-methyl modification. For example, in certain embodiments, the modified gRNA comprises a IxPSOMe modification on a 5’ end and/or a 3’ end; in certain embodiments, the modified gRNA comprises a 3xPSOMe modification on a 5’ end and/or a 3’ end. In certain embodiments, the hairpin region of the modified gRNA comprises 5’ inverted dT modifications. In certain embodiments, the hairpin region of the modified gRNA comprises 3’ inverted dT modifications. In certain embodiments, the hairpin region of the modified gRNA comprises a 3’ or 4’ pseudoknot. In certain embodiments, the hairpin region of the modified gRNA comprises a 3’ pseudoknot. In certain embodiments, the hairpin region of the modified gRNA comprises a locked nucleic acid (LNA). In certain embodiments, the hairpin region of the modified gRNA comprises a LNA with a 5’ extension.
[0218] In certain embodiments, the hairpin region of the modified gRNAs can comprise one or more of a 5’ extension, 2’Fluorine modification, 2’0-methyl modification, 5’ inverted dT modification, 3’ inverted dT modification, a pseudoknot, or an LNA. For example, in certain embodiments, the hairpin region of the modified gRNA comprises a 5’ extension and a IxPSOMe modification on the 5’ and 3’ ends. In certain embodiments, the hairpin region of the modified gRNA comprises a IxPSOMe modification on the 5 ’end and a 3’ pseudoknot. In certain embodiments, the hairpin region of the modified gRNA comprises a 5’ extension and IxPSOMe modification on the 3’end only. In certain embodiments, the hairpin region of the modified gRNA comprises a 5’ extension and a conservative 2’F hairpin. In certain embodiments, the hairpin region of the modified gRNA comprises a 5’ extension and an aggressive 2’F hairpin. In certain embodiments, the hairpin region of the modified gRNA comprises a 5’ extension and inverted dT modifications at the 5’ and 3’ ends. In certain embodiments, the hairpin region of the modified gRNA comprises a 5’ extension, inverted dT modifications at the 5’ and 3’ ends and an LNA. In certain embodiments, the hairpin region of the modified gRNA comprises a 5’ extension, IxPSOMe modification on 5’ and 3’ ends and an aggressive 2’F hairpin. In certain embodiments, the hairpin region of the modified gRNA comprises a 5’ extension, inverted dT modifications at the 5’ and 3’ ends and an aggressive 2’F hairpin. In certain embodiments, the hairpin region of the modified gRNA comprises a 5’ extension and a conservative 2’0Me modification. In certain embodiments, the hairpin region of the modified gRNA comprises a 5’ extension and an aggressive 2’0Me modification. Aggressive and conservative modification patterns are depicted in Table 2.
Table 2. gRNA Modification Pattern*
[0219] In certain embodiments, the targeting domain of the gRNA can comprise one or more of 5’ extensions, 2’Fluorine modifications, 2’0-methyl modifications, 5’ inverted dT modifications, or 3’ inverted dT modifications, a pseudoknot, or an LNA. In certain embodiments the targeting domain of the gRNA comprises modifications on nucleotides 1, 8,
9, 10, 11, 12, 17, 19 and optionally 20 and 21. In certain embodiments the targeting domain of the gRNA comprises modifications on nucleotides 1, 2, 3, 7, 8, 9, 10, 11, 12, 14, 15, 17, 19 and optionally 20 and 21. In certain embodiments, the targeting domain of the gRNA comprises one or more of 2’Fluorine modifications, 2’O-methyl modifications, 5’ inverted dT modifications, or 3’ inverted dT modifications.
[0220] In any of the above embodiments, a gRNA can comprise both a conservative hairpin modification pattern and a conservative targeting region modification pattern. In any of the above embodiments, a gRNA can comprise either of a conservative hairpin modification pattern and a conservative targeting region modification pattern. In any of the above embodiments, a gRNA can comprise neither a conservative hairpin modification pattern nor a conservative targeting region modification pattern.
[0221] In general, any combination of modifications may be used. In non-limiting examples, patterns of modification comprising, a combination of 5’ extension; 5’3’ idT; 2’F in an aggressive hairpin pattern; a combination of 5’3’ idT and 2’F in an aggressive hairpin pattern; or a combination of 5’3’ idT and 5’ extension combination can be used.
RNA-guided nuclease molecules
[0222] RNA-guided nuclease molecules according to the present disclosure include, but are not limited to, naturally-occurring Class 2 CRISPR nucleases such as Casl2a (also referred to as Cpfl), and Cas9, as well as other nucleases derived or obtained therefrom.
[0223] In functional terms, RNA-guided nucleases are defined as those nucleases that: (a) interact with (e.g., complex with) a gRNA; and (b) together with the gRNA, associate with, and optionally cleave or modify, a target region of a DNA that includes (i) a sequence complementary to the targeting domain of the gRNA and, optionally, (ii) an additional sequence referred to as a “protospacer adjacent motif,” or “PAM,” which is described in greater detail below. As the following examples will illustrate, RNA-guided nucleases can be defined, in broad terms, by their PAM specificity and cleavage activity, even though variations may exist between individual RNA-guided nucleases that share the same PAM specificity or cleavage activity. Skilled artisans will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using any suitable RNA-guided nuclease having a certain PAM specificity and/or cleavage activity. For this reason, unless otherwise specified, the term RNA-guided nuclease should be understood as a generic term, and not limited to any particular type (e.g., Cas9 vs.
Casl2a), species (e.g., 5. pyogenes vs. 5. aureus or variation (e.g., full-length vs. truncated or split; naturally-occurring PAM specificity vs. engineered PAM specificity, etc.) of RNA- guided nuclease. For example, in certain embodiments, the RNA-guided nuclease may be Cas-<D (Pausch 2020).
[0224] Various RNA-guided nucleases may require different sequential relationships between PAMs and protospacers. In general, Cas9s recognize PAM sequences that are 3’ of the protospacer. Cast 2a, on the other hand, generally recognizes PAM sequences that are 5’ of the protospacer. [0225] In addition to recognizing specific sequential orientations of PAMs and protospacers, RNA-guided nucleases can also recognize specific PAM sequences. 5. aureus Cas9, for instance, recognizes a PAM sequence of NNGRRT or NNGRRV, wherein the N residues are immediately 3’ of the region recognized by the gRNA targeting domain. .S’. pyogenes Cas9 recognizes NGG PAM sequences. And F. novicida Casl2a recognizes a TTN PAM sequence. PAM sequences have been identified for a variety of RNA-guided nucleases, and a strategy for identifying novel PAM sequences has been described by Shmakov 2015. It should also be noted that engineered RNA-guided nucleases can have PAM specificities that differ from the PAM specificities of reference molecules (for instance, in the case of an engineered RNA-guided nuclease, the reference molecule may be the naturally occurring variant from which the RNA-guided nuclease is derived, or the naturally occurring variant having the greatest amino acid sequence homology to the engineered RNA-guided nuclease). Examples of PAMs that may be used according to the embodiments herein include, without limitation, the nucleotide sequences set forth in SEQ ID NOs:l-7 (NGGNG (SEQ ID NO:1), NNAGAAW (SEQ ID NO:2), NAAR (SEQ ID NO:3), NNGRR (SEQ ID NO:4), NNGRRN (SEQ ID NO:5), NNGRRT (SEQ ID NO:6), and NNGRRV (SEQ ID NO:7)).
[0226] In addition to their PAM specificity, RNA-guided nucleases can be characterized by their DNA cleavage activity: naturally-occurring RNA-guided nucleases typically form DSBs in target nucleic acids, but engineered variants have been produced that generate only SSBs (discussed above and in Ran & Hsu 2013, incorporated by reference herein), or that do not cut at all.
Casl 2a
[0227] The crystal structure of Acidaminococcus sp. Casl 2a (also known as Cpfl) in complex with crRNA and a double-stranded (ds) DNA target including a TTTN PAM sequence has been solved by Yamano 2016 (incorporated by reference herein). Casl2a, like Cas9, has two lobes: a REC (recognition) lobe, and a NUC (nuclease) lobe. The REC lobe includes RECI and REC2 domains, which lack similarity to any known protein structures. The NUC lobe, meanwhile, includes three RuvC domains (RuvC-I, -II and -III) and a BH domain. However, in contrast to Cas9, the Casl2a REC lobe lacks an HNH domain, and includes other domains that also lack similarity to known protein structures: a structurally unique PI domain, three Wedge (WED) domains (WED-I, -II and -III), and a nuclease (Nuc) domain. [0228] While Cas9 and Casl2a share similarities in structure and function, it should be appreciated that certain Casl2a activities are mediated by structural domains that are not analogous to any Cas9 domains. For instance, cleavage of the complementary strand of the target DNA appears to be mediated by the Nuc domain, which differs sequentially and spatially from the HNH domain of Cas9. Additionally, the non-targeting portion of Casl2a gRNA (the handle) adopts a pseudoknot structure, rather than a stem loop structure formed by the repeat: antirepeat duplex in Cas9 gRNAs.
[0229] In certain embodiments, a Casl2a molecule may be a modified Casl2a molecule. In certain embodiments, a modified Casl2a molecule may include one or more modifications. In certain embodiments the modifications may be, without limitation, one or more mutations in a Casl2a nucleotide sequence or Casl2a polypeptide sequence, one or more additional sequences such as a His tag or a nuclear localization signal (NLS), or a combination thereof.
[0230] In certain embodiments, the Casl2a molecule may be derived from a Casl2a molecule selected from the group consisting of Acidaminococcus sp. strain BV3L6 Cpfl protein (AsCasl2a or AsCpfl), Lachnospiraceae bacterium ND2006 Cpfl protein (LbCpfl), and Lachnospiraceae bacterium MA2020 (Lb2 Cpfl). In certain embodiments, the Casl2a molecule may comprise a sequence selected from the group consisting of SEQ ID NOs: 18- 20, having the codon-optimized nucleic acid sequences of SEQ ID NOs:21-23, respectively.
[0231] In certain embodiments, the modified Casl2a molecule may comprise a nuclear localization signal (NLS). For example, but not by way of limitation, NLS sequences useful in connection with the methods and compositions disclosed herein will comprise an amino acid sequence capable of facilitating protein import into the cell nucleus. NLS sequences useful in connection with the methods and compositions disclosed herein are known in the art. Examples of such NLS sequences include the nucleoplasmin NLS having the amino acid sequence: KRPAATKKAGQAKKKK (SEQ ID NO:45) and the simian virus 40 “SV40” NLS having the amino acid sequence PKKKRKV (SEQ ID NO:46).
[0232] In certain embodiments, the NLS sequence of the modified Casl2a molecule is positioned at or near the C-terminus of the Casl2a protein sequence. For example, but not by way of limitation, the modified Casl2a molecule can be selected from the following: His- AsCpfl-nNLS (SEQ ID NO:8); His-AsCpfl-sNLS (SEQ ID NO:10) and His-AsCpfl-sNLS- sNLS (SEQ ID NO:9), where “His” refers to a six-histidine purification sequence, “AsCasl2a” refers to the Acidaminococcus sp. Casl2a protein sequence, “nNLS” refers to the nucleoplasmin NLS, and “sNLS” refers to the SV40 NLS. Additional permutations of the identity and C-terminal positions of NLS sequences, e.g., appending two or more nNLS sequences or combinations of nNLS and sNLS sequences (or other NLS sequences), as well as sequences with and without purification sequences, e.g., six -histidine sequences, are within the scope of the instantly disclosed subject matter.
[0233] In certain embodiments, the NLS sequence of the modified Casl2a molecule may be positioned at or near the N-terminus of the Casl2a protein sequence. For example, but not by way of limitation, the modified Casl2a protein may be selected from the following: His- sNLS-AsCpfl (SEQ ID NO: 11), His-sNLS-sNLS-AsCpfl (SEQ ID NO: 12), and sNLS- sNLS-AsCpfl (SEQ ID NO: 13). Additional permutations of the identity and N-terminal positions of NLS sequences, e.g., appending two or more nNLS sequences or combinations of nNLS and sNLS sequences (or other NLS sequences), as well as sequences with and without purification sequences, e.g., six-histidine sequences, are within the scope of the instantly disclosed subject matter.
[0234] In certain embodiments, the modified Casl2a molecule may comprise NLS sequences positioned at or near both the N-terminus and C-terminus of the Casl2a protein sequence.
For example, but not by way of limitation, the modified Casl2a protein may be selected from the following: His-sNLS-AsCpfl-sNLS (SEQ ID NO: 14) and His-sNLS-sNLS-AsCpfl- sNLS-sNLS (SEQ ID NO: 15). Additional permutations of the identity and N-terminal/C- terminal positions of NLS sequences, e.g., appending two or more nNLS sequences or combinations of nNLS and sNLS sequences (or other NLS sequences) to either the N- terminal/C-terminal positions, as well as sequences with and without purification sequences, e.g., six-histidine sequences, are within the scope of the instantly disclosed subject matter.
[0235] In certain embodiments, the modified Casl2a molecule may comprise an alteration (e.g., a deletion or substitution) at one or more cysteine residues of the Casl2a protein sequence. For example, but not by way of limitation, modified Casl2a molecule may comprise an alteration at a position selected from the group consisting of: C65, C205, C334, C379, C608, C674, C1025, and C1248. In certain embodiments, the modified Casl2a molecule may comprise a substitution of one or more cysteine residues for a serine or alanine. In certain embodiments, the modified Casl2a molecule may comprise an alteration selected from the group consisting of: C65S, C205S, C334S, C379S, C608S, C674S, C1025S, and C1248S. In certain embodiments, the modified Casl2a molecule may comprise an alteration selected from the group consisting of: C65A, C205A, C334A, C379A, C608A, C674A, C1025A, and C1248A. In certain embodiments, the modified Casl2a molecule may comprise alterations at positions C334 and C674 or C334, C379, and C674. In certain embodiments, the modified Casl2a molecule may comprise the following alterations: C334S and C674S, or C334S, C379S, and C674S. In certain embodiments, the modified Casl2a molecule may comprise the following alterations: C334A and C674A, or C334A, C379A, and C674A. In certain embodiments, the modified Casl2a molecule may comprise both one or more cysteine residue alteration as well as the introduction of one or more NLS sequences, e.g., His-AsCpfl-nNLS Cys-less (SEQ ID NO:16) or His-AsCpfl-nNLS Cys-low (SEQ ID NO: 17). In various embodiments, the Casl2a molecule comprises a deletion or substitution in one or more cysteine residues exhibiting reduced aggregation.
[0236] In certain embodiments, other modified Casl2a molecules known in the art may be used with the methods and systems described herein. For example, in certain embodiments, the modified Casl2a may be Casl2a containing the mutation S542R/K548V/N552R (“Cpfl RVR”). Casl2a RVR has been shown to cleave target sites with a TATV PAM. In certain embodiments, the modified Cas l2a may be Casl2a containing the mutation S542R/K607R (“Cpfl RR”). Cpfl RR has been shown to cleave target sites with a TYCV/CCCC PAM. AsCpfl RR and AsCpfl RVR are engineered AsCpfl variants which recognize TYCV/ACCC/CCCC and TATV/RATR PAMs, respectively (Gao 2017).
[0237] In some embodiments, a modified Casl2a molecule is used herein, wherein the modified Casl 2a molecule comprises mutations at one or more residues of AsCasl 2a (Acidaminococcus sp. BV3L6) selected from the group consisting of 11, 12, 13, 14, 15, 16, 17, 34, 36, 39, 40, 43, 46, 47, 50, 54, 57, 58, 111, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,
172, 173, 174, 175, 176, 177, 178, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542,
543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 565, 566, 567, 568,
569, 570, 571, 572, 573, 574, 575, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602,
603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620,
626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 642, 643, 644, 645, 646,
647, 648, 649, 651, 652, 653, 654, 655, 656, 676, 679, 680, 682, 683, 684, 685, 686, 687,
688, 689, 690, 691, 692, 693, 707, 711, 714, 715, 716, 717, 718, 719, 720, 721, 722, 739,
765, 768, 769, 773, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, or 1048 or the corresponding position of an AsCasl2a orthologue, homologue, or variant.
[0238] In certain embodiments, a modified Casl2a molecule as used herein may include any of the Casl2a (referred to also as Cpfl) proteins described in International Publication Number WO 2017/184768 Al by Zhang et al. (“’768 Publication”), which is incorporated by reference herein.
[0239] In certain embodiments, a modified Casl2a molecule used herein may comprise any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (exemplary Casl2a polypeptide sequences) or be encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (exemplary Casl2a polynucleotide sequences). Casl2a modified amino acid and nucleotide sequences that may be used herein are set forth in International Publication No. WO 2021/119040 (see, e.g., Table 14). Permutations of the identity and N-terminal/C- terminal positions of NLS sequences, e.g., appending two or more nNLS sequences or combinations of nNLS and sNLS sequences (or other NLS sequences) to either the N- terminal/C- terminal positions, as well as sequences with and without purification sequences, e.g., six-histidine sequences, are within the scope of the instantly disclosed subject matter.
[0240] In certain embodiments, any of the Casl2a molecules or modified Casl2a molecules disclosed herein may be complexed with one or more gRNA comprising the targeting domain set forth in Table 10 to alter the MYOC gene. In certain embodiments, any of the Casl2a molecules or modified Casl2a molecules disclosed herein may be complexed with one or more gRNA comprising a sequence set forth in Table 10. In certain embodiments, the modified Casl2a molecule may be His- AsCpfl -nNLS (SEQ ID NO:8) or His-AsCpfl-sNLS- sNLS (SEQ ID NO:9). In certain embodiments, a modified Casl2a molecule used herein may comprise any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (exemplary Casl2a polypeptide sequences) or be encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (exemplary Casl2a polynucleotide sequences).
[0241] In certain embodiments, the modified Casl2a molecule may include a modified Casl2a (i.e., Cpfl) molecule described in Kleinstiver 2019. For example, without limitation, in certain embodiments, the modified Casl2a molecule may be enAsCpfl. In certain embodiments, the modified Casl2a molecule may cleave target sites with a TTTV PAM. In certain embodiments, the modified Casl2a molecule may cleave target sites with a NWYN PAM. [0242] Provided below in Table 14 are exemplary Casl2a (Cpfl) polypeptide and nucleotide sequences.
[0243] In some embodiments, an RNA-guided nuclease has at least 80%, at least 85%, at least 86%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity relative to a wild-type RNA-guided nuclease and/or an RNA-guided nuclease disclosed herein (e.g., an RNA-guided nuclease comprising an amino acid sequence depicted in Table 14 or comprising any of SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48). In some embodiments, an RNA-guided nuclease has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations relative to a wild-type RNA-guided nuclease and/or an RNA- guided nuclease disclosed herein e.g., an RNA-guided nuclease comprising an amino acid sequence depicted in Table 14 or comprising any of SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30- 33, 34-36, 48). In some embodiments, an RNA-guided nuclease has less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations relative to a wild-type RNA- guided nuclease and/or an RNA-guided nuclease disclosed herein (e.g., an RNA-guided nuclease comprising an amino acid sequence depicted in Table 14 or comprising any of SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48).
Cas9
[0244] Crystal structures have been determined for S. pyogenes Cas9 (Jinek 2014), and for S. aureus Cas9 in complex with a unimolecular guide RNA and a target DNA (Nishimasu 2014; Anders 2014; and Nishimasu 2015).
[0245] A naturally occurring Cas9 protein comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which comprise particular structural and/or functional domains. The REC lobe comprises an arginine-rich bridge helix (BH) domain, and at least one REC domain (e.g., a RECI domain and, optionally, a REC2 domain). The REC lobe does not share structural similarity with other known proteins, indicating that it is a unique functional domain. While not wishing to be bound by any theory, mutational analyses suggest specific functional roles for the BH and REC domains: the BH domain appears to play a role in gRNA:DNA recognition, while the REC domain is thought to interact with the repeat: anti-repeat duplex of the gRNA and to mediate the formation of the Cas9/gRNA complex.
[0246] The NUC lobe comprises a RuvC domain, an HNH domain, and a PAM-interacting (PI) domain. The RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves the non-complementary (i.e., bottom) strand of the target nucleic acid. It may be formed from two or more split RuvC motifs (such as RuvC I, RuvCII, and RuvCIII in 5. pyogenes and S. aureus}. The HNH domain, meanwhile, is structurally similar to HNN endonuclease motifs, and cleaves the complementary (i.e., top) strand of the target nucleic acid. The PI domain, as its name suggests, contributes to PAM specificity.
[0247] While certain functions of Cas9 are linked to (but not necessarily fully determined by) the specific domains set forth above, these and other functions may be mediated or influenced by other Cas9 domains, or by multiple domains on either lobe. For instance, in S. pyogenes Cas9, as described in Nishimasu 2014, the repeat: antirepeat duplex of the gRNA falls into a groove between the REC and NUC lobes, and nucleotides in the duplex interact with amino acids in the BH, PI, and REC domains. Some nucleotides in the first stem loop structure also interact with amino acids in multiple domains (Pl, BH and RECI), as do some nucleotides in the second and third stem loops (RuvC and PI domains).
[0248] An exemplary S«Cas9 polypeptide sequence is: TGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKWKLSLKP YRFDVYLDNGV YKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKI SNQAEF IASFYNNDLIKINGELYRVIGV NNDLLNRIEVNMIDI TYREYLENMNDKRPPRI IKT IASKTQS IKKYSTD ILGNLYEVKSKKH PQI IKKGHEHHHH ( SEQ ID NO : 47 ) [0249] Examples of RNA-guided nucleases suitable for use in the context of the methods, strategies, and treatment modalities provided herein are listed in Table 3 below, and the methods, compositions, and treatment modalities disclosed herein can, in some embodiments, make use of any combination of RNA-guided nucleases disclosed herein, or known to those of ordinary skill in the art. Table 3. RNA-Guided Nucleases
Modifications of RNA-guided nucleases
[0250] The RNA-guided nucleases described above have activities and properties that can be useful in a variety of applications, but the skilled artisan will appreciate that RNA-guided nucleases can also be modified in certain instances, to alter cleavage activity, PAM specificity, or other structural or functional features.
[0251] Turning first to modifications that alter cleavage activity, mutations that reduce or eliminate the activity of domains within the NUC lobe have been described above.
Exemplary mutations that may be made in the RuvC domains, in the Cas9 HNH domain, or in the Casl2a Nuc domain are described in Ran & Hsu 2013 and Yamano 2016, as well as in Cotta-Ramusino. In general, mutations that reduce or eliminate activity in one of the two nuclease domains result in RNA-guided nucleases with nickase activity, but it should be noted that the type of nickase activity varies depending on which domain is inactivated. As one example, inactivation of a RuvC domain of a Cas9 will result in a nickase that cleaves the complementary or top strand as shown below (where C denotes the site of cleavage).
[0252] On the other hand, inactivation of a Cas9 HNH domain results in a nickase that cleaves the bottom or non-complementary strand.
[0253] Modifications of PAM specificity relative to naturally occurring Cas9 reference molecules has been described by Kleinstiver et al. for both 5. pyogenes (Kleinstiver 2015a) and 5. aureus (Kleinstiver 2015b). Kleinstiver et al. have also described modifications that improve the targeting fidelity of Cas9 (Kleinstiver 2016). Kleinstiver et al. have also described modifications of Casl2a (Cpfl) that provide increased activity and improved targeting ranges (Kleinstiver 2019). Each of these references is incorporated by reference herein.
[0254] RNA-guided nucleases have been split into two or more parts, as described by Zetsche 2015 and Fine 2015 (both incorporated by reference herein).
[0255] RNA-guided nucleases can be, in certain embodiments, size-optimized or truncated, for instance via one or more deletions that reduce the size of the nuclease while still retaining gRNA association, target and PAM recognition, and cleavage activities. In certain embodiments, RNA guided nucleases are bound, covalently or non-covalently, to another polypeptide, nucleotide, or other structure, optionally by means of a linker. Exemplary bound nucleases and linkers are described by Guilinger 2014, incorporated by reference herein for all purposes.
[0256] RNA-guided nucleases also optionally include a tag, such as, but not limited to, a nuclear localization signal to facilitate movement of RNA-guided nuclease protein into the nucleus. In certain embodiments, the RNA-guided nuclease can incorporate C- and/or N- terminal nuclear localization signals. Nuclear localization sequences are known in the art and are described in Maeder and elsewhere.
[0257] The foregoing list of modifications is intended to be exemplary in nature, and the skilled artisan will appreciate, in view of the instant disclosure, that other modifications may be possible or desirable in certain applications. For brevity, therefore, exemplary systems, methods and compositions of the present disclosure are presented with reference to particular RNA-guided nucleases, but it should be understood that the RNA-guided nucleases used may be modified in ways that do not alter their operating principles. Such modifications are within the scope of the present disclosure.
Nucleic acids encoding RNA-guided nucleases
[0258] Nucleic acids encoding RNA-guided nucleases, e.g., Cas 12a or functional fragments thereof, are provided herein. Exemplary nucleic acids encoding RNA-guided nucleases have been described previously (see, e.g., Cong 2013; Wang 2013; Mali 2013; Jinek 2012).
[0259] In some cases, a nucleic acid encoding an RNA-guided nuclease can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified. In certain embodiments, an mRNA encoding an RNA-guided nuclease will have one or more (e.g., all) of the following properties: it can be capped; polyadenylated; and substituted with 5 -methylcytidine and/or pseudouridine.
[0260] Synthetic nucleic acid sequences can also be codon optimized, e.g., at least one noncommon codon or less-common codon has been replaced by a common codon. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., described herein. Examples of codon optimized Cas9 coding sequences are presented in Cotta-Ramusino.
[0261] In addition, or alternatively, a nucleic acid encoding an RNA-guided nuclease may comprise a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.
Functional analysis of candidate molecules
[0262] Candidate RNA-guided nucleases, gRNAs, and complexes thereof, can be evaluated by standard methods known in the art. See, e.g., Cotta-Ramusino. The stability of RNP complexes may be evaluated by differential scanning fluorimetry, as described below.
Differential Scanning Fluorimetry (DSF)
[0263] The thermostability of ribonucleoprotein (RNP) complexes comprising gRNAs and RNA-guided nucleases can be measured via DSF. The DSF technique measures the thermostability of a protein, which can increase under favorable conditions such as the addition of a binding RNA molecule, e.g., a gRNA.
[0264] A DSF assay can be performed according to any suitable protocol, and can be employed in any suitable setting, including without limitation (a) testing different conditions (e.g., different stoichiometric ratios of gRNA: RNA-guided nuclease protein, different buffer solutions, etc.) to identify optimal conditions for RNP formation; and (b) testing modifications (e.g., chemical modifications, alterations of sequence, etc.) of an RNA-guided nuclease and/or a gRNA to identify those modifications that improve RNP formation or stability. One readout of a DSF assay is a shift in melting temperature of the RNP complex; a relatively high shift suggests that the RNP complex is more stable (and may thus have greater activity or more favorable kinetics of formation, kinetics of degradation, or another functional characteristic) relative to a reference RNP complex characterized by a lower shift. When the DSF assay is deployed as a screening tool, a threshold melting temperature shift may be specified, so that the output is one or more RNPs having a melting temperature shift at or above the threshold. For instance, the threshold can be 5-10°C (e.g., 5°, 6°, 7°, 8°, 9°, 10°) or more, and the output may be one or more RNPs characterized by a melting temperature shift greater than or equal to the threshold.
[0265] Two non-limiting examples of DSF assay conditions are set forth below:
[0266] To determine the best solution to form RNP complexes, a fixed concentration (e.g., 2 pM) of Cas9 in water+10x SYPRO Orange® (Life Technologies cat#S-6650) is dispensed into a 384 well plate. An equimolar amount of gRNA diluted in solutions with varied pH and salt is then added. After incubating at room temperature for 10’ and brief centrifugation to remove any bubbles, a Bio- Rad CFX384™ Real-Time System C1000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20°C to 90°C with a 1°C increase in temperature every 10 seconds.
[0267] The second assay consists of mixing various concentrations of gRNA with fixed concentration (e.g., 2 pM) Cas9 in optimal buffer from assay 1 above and incubating (e.g., at RT for 10’) in a 384 well plate. An equal volume of optimal buffer + lOx SYPRO Orange® (Life Technologies cat#S-6650) is added and the plate sealed with Microseal® B adhesive (MSB- 1001). Following brief centrifugation to remove any bubbles, a Bio-Rad CFX384™ Real-Time System C1000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20°C to 90°C with a 1°C increase in temperature every 10 seconds.
Genome editing strategies
[0268] The genome editing systems described above are used, in various embodiments of the present disclosure, to generate edits in (i.e., to alter) targeted regions of DNA within or obtained from a cell. Various strategies are described herein to generate particular edits, and these strategies are generally described in terms of the desired repair outcome, the number and positioning of individual edits (e.g., SSBs or DSBs), and the target sites of such edits.
[0269] Genome editing strategies that involve the formation of SSBs or DSBs are characterized by repair outcomes including: (a) deletion of all or part of a targeted region; (b) insertion into or replacement of all or part of a targeted region; or (c) interruption of all or part of a targeted region. This grouping is not intended to be limiting, or to be binding to any particular theory or model, and is offered solely for economy of presentation. Skilled artisans will appreciate that the listed outcomes are not mutually exclusive and that some repairs may result in other outcomes. The description of a particular editing strategy or method should not be understood to require a particular repair outcome unless otherwise specified.
[0270] Replacement of a targeted region generally involves the replacement of all or part of the existing sequence within the targeted region with a homologous sequence, for instance through gene correction or gene conversion, two repair outcomes that are mediated by HDR pathways. HDR is promoted by the use of a donor template, which can be single-stranded or double stranded, as described in greater detail below. Single or double stranded templates can be exogenous, in which case they will promote gene correction, or they can be endogenous (e.g., a homologous sequence within the cellular genome), to promote gene conversion. Exogenous templates can have asymmetric overhangs (i.e., the portion of the template that is complementary to the site of the DSB may be offset in a 3’ or 5’ direction, rather than being centered within the donor template), for instance as described by Richardson 2016 (incorporated by reference herein). In instances where the template is single stranded, it can correspond to either the complementary (top) or non-complementary (bottom) strand of the targeted region.
[0271] Gene conversion and gene correction are facilitated, in some cases, by the formation of one or more nicks in or around the targeted region, as described in Ran & Hsu 2013 and Cotta-Ramusino. In some cases, a dual-nickase strategy is used to form two offset SSBs that, in turn, form a single DSB having an overhang (e.g., a 5’ overhang).
[0272] Interruption and/or deletion of all or part of a targeted sequence can be achieved by a variety of repair outcomes. As one example, a sequence can be deleted by simultaneously generating two or more DSBs that flank a targeted region, which is then excised when the DSBs are repaired, as is described in Maeder for the LCA10 mutation. As another example, a sequence can be interrupted by a deletion generated by formation of a double strand break with single-stranded overhangs, followed by exonucleolytic processing of the overhangs prior to repair.
[0273] One specific subset of target sequence interruptions is mediated by the formation of an indel within the targeted sequence, where the repair outcome is typically mediated by NHEJ pathways (including Alt-NHEJ). NHEJ is referred to as an “error prone” repair pathway because of its association with indel mutations. In some cases, however, a DSB is repaired by NHEJ without alteration of the sequence around it (a so-called “perfect” or “scarless” repair); this generally requires the two ends of the DSB to be perfectly ligated. Indels, meanwhile, are thought to arise from enzymatic processing of free DNA ends before they are ligated that adds and/or removes nucleotides from either or both strands of either or both free ends.
[0274] Because the enzymatic processing of free DSB ends may be stochastic in nature, indel mutations tend to be variable, occurring along a distribution, and can be influenced by a variety of factors, including the specific target site, the cell type used, the genome editing strategy used, etc. Even so, it is possible to draw limited generalizations about indel formation: deletions formed by repair of a single DSB are most commonly in the 1-50 bp range, but can reach greater than 100-200 bp. Insertions formed by repair of a single DSB tend to be shorter and often include short duplications of the sequence immediately surrounding the break site. However, it is possible to obtain large insertions, and in these cases, the inserted sequence has often been traced to other regions of the genome or to plasmid DNA present in the cells.
[0275] Indel mutations - and genome editing systems configured to produce indels - are useful for interrupting target sequences, for example, when the generation of a specific final sequence is not required and/or where a frameshift mutation would be tolerated. They can also be useful in settings where particular sequences are preferred, insofar as the certain sequences desired tend to occur preferentially from the repair of an SSB or DSB at a given site. Indel mutations are also a useful tool for evaluating or screening the activity of particular genome editing systems and their components. In these and other settings, indels can be characterized by (a) their relative and absolute frequencies in the genomes of cells contacted with genome editing systems and (b) the distribution of numerical differences relative to the unedited sequence, e.g., ±1, ±2, ±3, etc. As one example, in a lead-finding setting, multiple gRNAs can be screened to identify those gRNAs that most efficiently drive cutting at a target site based on an indel readout under controlled conditions. Guides that produce indels at or above a threshold frequency, or that produce a particular distribution of indels, can be selected for further study and development. Indel frequency and distribution can also be useful as a readout for evaluating different genome editing system implementations or formulations and delivery methods, for instance by keeping the gRNA constant and varying certain other reaction conditions or delivery methods. Multiplex Strategies
[0276] Genome editing systems according to this disclosure may also be employed for multiplex gene editing to generate two or more DSBs, either in the same locus or in different loci. Any of the RNA-guided nucleases and gRNAs disclosed herein may be used in genome editing systems for multiplex gene editing. Strategies for editing that involve the formation of multiple DSBs, or SSBs, are described in, for instance, Cotta-Ramusino. In certain embodiments, multiple gRNAs and an RNA-guided nuclease may be used in genome editing systems to introduce alterations (e.g., deletions, insertions) into the MYOC gene. In certain embodiments, the RNA-guided nuclease may be Casl2a.
Donor template design
[0277] Donor template design is described in detail in the literature, for instance in Cotta- Ramusino. DNA oligomer donor templates (oligodeoxynucleotides or ODNs), which can be single stranded (ssODNs) or double-stranded (dsODNs), can be used to facilitate HDR-based repair of DSBs or to boost overall editing rate, and are particularly useful for introducing alterations into a target DNA sequence, inserting a new sequence into the target sequence, or replacing the target sequence altogether.
[0278] Whether single-stranded or double stranded, donor templates generally include regions that are homologous to regions of DNA within or near (e.g., flanking or adjoining) a target sequence to be cleaved. These homologous regions are referred to here as “homology arms,” and are illustrated schematically below:
[5’ homology arm] — [replacement sequence] — [3’ homology arm].
[0279] The homology arms can have any suitable length (including 0 nucleotides if only one homology arm is used), and 3’ and 5’ homology arms can have the same length, or can differ in length. The selection of appropriate homology arm lengths can be influenced by a variety of factors, such as the desire to avoid homologies or microhomologies with certain sequences such as Alu repeats or other very common elements. For example, a 5’ homology arm can be shortened to avoid a sequence repeat element. In other embodiments, a 3’ homology arm can be shortened to avoid a sequence repeat element. In some embodiments, both the 5’ and the 3’ homology arms can be shortened to avoid including certain sequence repeat elements. In addition, some homology arm designs can improve the efficiency of editing or increase the frequency of a desired repair outcome. For example, Richardson 2016, which is incorporated by reference herein, found that the relative asymmetry of 3’ and 5’ homology arms of single stranded donor templates influenced repair rates and/or outcomes.
[0280] Replacement sequences in donor templates have been described elsewhere, including in Cotta-Ramusino. A replacement sequence can be any suitable length (including zero nucleotides, where the desired repair outcome is a deletion), and typically includes one, two, three or more sequence modifications relative to the naturally-occurring sequence within a cell in which editing is desired. One common sequence modification involves the alteration of the naturally-occurring sequence to repair a mutation that is related to a disease or condition of which treatment is desired. Another common sequence modification involves the alteration of one or more sequences that are complementary to, or then, the PAM sequence of the RNA-guided nuclease or the targeting domain of the gRNA(s) being used to generate an SSB or DSB, to reduce or eliminate repeated cleavage of the target site after the replacement sequence has been incorporated into the target site.
[0281] Where a linear ssODN is used, it can be configured to (i) anneal to the nicked strand of the target nucleic acid, (ii) anneal to the intact strand of the target nucleic acid, (iii) anneal to the plus strand of the target nucleic acid, and/or (iv) anneal to the minus strand of the target nucleic acid. An ssODN may have any suitable length, e.g., about, at least, or no more than 80-200 nucleotides (e.g., 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides).
[0282] It should be noted that a template nucleic acid can also be a nucleic acid vector, such as a viral genome or circular double stranded DNA, e.g., a plasmid. Nucleic acid vectors comprising donor templates can include other coding or non-coding elements. For example, a template nucleic acid can be delivered as part of a viral genome (e.g., in an AAV or lentiviral genome) that includes certain genomic backbone elements (e.g., inverted terminal repeats, in the case of an AAV genome) and optionally includes additional sequences coding for a gRNA and/or an RNA-guided nuclease. In certain embodiments, the donor template can be adjacent to, or flanked by, target sites recognized by one or more gRNAs, to facilitate the formation of free DSBs on one or both ends of the donor template that can participate in repair of corresponding SSBs or DSBs formed in cellular DNA using the same gRNAs. Exemplary nucleic acid vectors suitable for use as donor templates are described in Cotta- Ramusino, which is incorporated by reference. [0283] Whatever format is used, a template nucleic acid can be designed to avoid undesirable sequences. In certain embodiments, one or both homology arms can be shortened to avoid overlap with certain sequence repeat elements, e.g., Alu repeats, LINE elements, etc.
[0284] In certain embodiments, silent, non-pathogenic SNPs may be included in the ssODN donor template to allow for identification of a gene editing event.
Target cells
[0285] Genome editing systems according to this disclosure can be used to manipulate or alter a cell, e.g., to edit or alter a target nucleic acid. The manipulating can occur, in various embodiments, in vivo or ex vivo.
[0286] A variety of cell types can be manipulated or altered according to the embodiments of this disclosure, and in some cases, such as in vivo applications, a plurality of cell types are altered or manipulated, for example by delivering genome editing systems according to this disclosure to a plurality of cell types. In other cases, however, it may be desirable to limit manipulation or alteration to a particular cell type or types. For instance, it can be desirable in some instances to edit a cell with limited differentiation potential or a terminally differentiated cell, such as a photoreceptor cell in the case of Maeder, in which modification of a genotype is expected to result in a change in cell phenotype. In other cases, however, it may be desirable to edit a less differentiated, multipotent or pluripotent, stem or progenitor cell. By way of example, the cell may be an embryonic stem cell, induced pluripotent stem cell (iPSC), hematopoietic stem/progenitor cell (HSPC), or other stem or progenitor cell type that differentiates into a cell type of relevance to a given application or indication.
[0287] As a corollary, the cell being altered or manipulated is, variously, a dividing cell or a non-dividing cell, depending on the cell type(s) being targeted and/or the desired editing outcome.
[0288] When cells are manipulated or altered ex vivo, the cells can be used (e.g., administered to a subject) immediately, or they can be maintained or stored for later use. Those of skill in the art will appreciate that cells can be maintained in culture or stored (e.g., frozen in liquid nitrogen) using any suitable method known in the art. [0289] In an embodiment, a cell is manipulated by editing the MYOC target gene. In an embodiment, the expression of the MYOC target gene is modulated, e.g., in vivo. In another embodiment, the expression of the MYOC target gene is modulated, e.g., ex vivo.
[0290] The RNA-guided nuclease molecules (e.g., Casl2a) and gRNA molecules described herein can be delivered to a target cell. In an embodiment, the target cell is a cell from the eye, e.g., a trabecular mesh work cell, retinal pigment epithelial cell, a retinal cell, an iris cell, a ciliary body cell and/or the optic nerve. In an embodiment, the target cell is a trabecular meshwork cell. In an embodiment, the target cell is a retinal cell, e.g., a cell of the retinal pigment epithelium or a photoreceptor cell. In an embodiment, the target cell is a cone photoreceptor cell or cone cell, a rod photoreceptor cell or rod cell, or a macular cone photoreceptor cell. In an embodiment, cone photoreceptors in the macular are targeted, i.e., cone photoreceptors in the macular are the target cells.
[0291] In an embodiment, the target cell is removed from the subject, the MYOC gene is altered ex vivo, and the cell returned to the subject. In an embodiment, a photoreceptor cell is removed from the subject, the MYOC gene is altered ex vivo, and the photoreceptor cell is returned to the subject. In an embodiment, a cone photoreceptor cell is removed from the subject, the MYOC gene is altered ex vivo, and the cone photoreceptor cell is returned to the subject. In an embodiment, a trabecular meshwork cell is removed from the subject, the MYOC gene is altered ex vivo, and the trabecular meshwork cell is returned to the subject.
[0292] In an embodiment, the cells are induced pluripotent stem cells (iPS) cells or cells derived from iPS cells, e.g., iPS cells from the subject, modified to alter the gene and differentiated into trabecular meshwork cells, retinal progenitor cells or retinal cells, e.g., retinal photoreceptors, and injected into the eye of the subject, e.g., into the trabecular meshwork, or, e.g., subretinally, e.g., in the submacular region of the retina.
[0293] In an embodiment, the cells are targeted in vivo, e.g., by delivery of the components, e.g., a Casl2a molecule and gRNA molecules, to the target cells. In an embodiment, the target cells are trabecular meshwork cells, retinal pigment epithelium or photoreceptor cells. In an embodiment, AAV is used to transduce the target cells.
Delivery, formulations, and routes of administration
[0294] The genome editing systems of this disclosure can be implemented in any suitable manner, meaning that the components of such systems, including without limitation the RNA-guided nuclease, gRNA, and optional donor template nucleic acid, can be delivered, formulated, or administered in any suitable form or combination of forms that results in the transduction, expression or introduction of a genome editing system and/or causes a desired repair outcome in a cell, tissue or subject. Tables 4 and 5 set forth several, non-limiting examples of genome editing system implementations. Those of skill in the art will appreciate, however, that these listings are not comprehensive, and that other implementations are possible. With reference to Table 4 in particular, the table lists several exemplary implementations of a genome editing system comprising a single gRNA and an optional donor template. However, genome editing systems according to this disclosure can incorporate multiple gRNAs, multiple RNA-guided nucleases, and other components such as proteins, and a variety of implementations will be evident to the skilled artisan based on the principles illustrated in the table. In the table, [N/A] indicates that the genome editing system does not include the indicated component.
Table 4: Gene Editing System Components
[0295] Table 5 summarizes various delivery methods for the components of genome editing systems, as described herein. Again, the listing is intended to be exemplary rather than limiting.
Table 5: Delivery Methods
[0296] Table 6 describes exemplary promoter sequences that are capable of driving expression. Table 6: Promoter Sequences
[0297] In an embodiment, an RNA-guided nuclease molecule (e.g., Casl2a molecule) and a gRNA molecule or more (e.g., 1, 2, 3, 4, or more) gRNA molecules are delivered, e.g., by an AAV vector. In an embodiment, the sequence encoding the RNA-guided nuclease molecule (e.g., Casl2a molecule) and the sequence(s) encoding the one or more (e.g., 1, 2, 3, 4, or more) gRNA molecules are present on the same nucleic acid molecule, e.g., an AAV vector. When an RNA-guided nuclease molecule (e.g., Casl2a molecule) or gRNA molecule component is encoded as DNA for delivery, the DNA will typically, but not necessarily, include a control region, e.g., comprising a promoter, to effect expression. Useful promoters for an RNA-guided nuclease molecule (e.g., Casl 2a molecule) sequences include those set forth in Table 6 (EFS, EF-la, MSCV, PGK, CAG, DCN #1, DCN #2, ID3, MT2A, SERPINF1, SERPING1 #1, SERPING1 #2, TIMP1, CP, MT1X, OLFML3, PCOLCE, PDGFRA, PTN, RARRE SI, RBP4 #1, RBP4 #2, RBP4 #3, PDPN, APOD, B2M, PTGDS, EEF1A1, ANGPTL7, MGP, RPS24, PLA2G2A, CHI3L1, FTL, TAGLN, PCP4, MYL 9, MYOC, CMV, and mini-CMV promoters). In an embodiment, the promoter is a constitutive promoter. In another embodiment, the promoter is a tissue specific promoter. Useful promoters for gRNAs include Hl, 7SK, tRNA and U6 promoters. Promoters with similar or dissimilar strengths can be selected to tune the expression of components. Sequences encoding an RNA-guided nuclease molecule (e.g., Casl2a molecule) can comprise a nuclear localization signal (NLS), e.g., an SV40 NLS. In an embodiment, the sequence encoding an RNA-guided nuclease molecule (e.g., Casl2a molecule) comprises at least two nuclear localization signals. In an embodiment a promoter for an RNA-guided nuclease molecule (e.g., Casl2a molecule) or a gRNA molecule can be, independently, inducible, tissue specific, or cell specific.
Nucleic acid-based delivery of genome editing systems
[0298] Nucleic acids encoding the various elements of a genome editing system according to the present disclosure can be administered to subjects or delivered into cells by art-known methods or as described herein. For example, RNA-guided nuclease-encoding and/or gRNA- encoding DNA, as well as donor template nucleic acids can be delivered by, e.g., vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof.
[0299] Nucleic acids encoding genome editing systems or components thereof can be delivered directly to cells as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by the target cells (e.g., erythrocytes, HSCs). Nucleic acid vectors, such as the vectors summarized in Table 5, can also be used.
[0300] Nucleic acid vectors can comprise one or more sequences encoding genome editing system components, such as an RNA-guided nuclease, a gRNA and/or a donor template. A vector can also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), associated with (e.g., inserted into or fused to) a sequence coding for a protein. As one example, a nucleic acid vector can include a Cas9 coding sequence that includes one or more nuclear localization sequences (e.g., a nuclear localization sequence from S V40).
[0301] The nucleic acid vector can also include any suitable number of regulatory /control elements, e.g., promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRES). These elements are well known in the art, and are described in Cotta-Ramusino.
[0302] Nucleic acid vectors according to this disclosure include recombinant viral vectors. Exemplary viral vectors are set forth in Table 5, and additional suitable viral vectors and their use and production are described in Cotta-Ramusino. Other viral vectors known in the art can also be used. In addition, viral particles can be used to deliver genome editing system components in nucleic acid and/or peptide form. For example, “empty” viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to alter target tissue specificity.
[0303] In addition to viral vectors, non-viral vectors can be used to deliver nucleic acids encoding genome editing systems according to the present disclosure. One important category of non-viral nucleic acid vectors are nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art, and are summarized in Cotta-Ramusino. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. For instance, organic (e.g., lipid and/or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of this disclosure. In one embodiment, the vector is a lipid nanoparticle (LNP).
[0304] In certain embodiments, the LNP can have a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. In some embodiments, the LNP has a size ranging from about 1 nm to about 1000 nm. In some embodiments, the LNP has a size ranging from about 1 nm to about 500 nm. In some embodiments, the LNP has a size ranging from about 1 nm to about 250 nm. In some embodiments, the LNP has a size ranging from about 25 nm to about 200 nm. In some embodiments, the LNP has a size ranging from about 25 nm to about 100 nm. In some embodiments, the LNP has a size ranging from about 35 nm to about 75 nm. In some embodiments, the LNP has a size ranging from about 25 nm to about 60 nm.
[0305] In certain embodiments, but not by way of any limitation, LNPs can be made from ionizable lipids (e.g., cationic lipids), neutral lipids, structural lipids, helper lipids, and PEGylated lipids, or a combination of these. In some embodiments, fusogenic phospholipids (e.g. DOPE) and/or sterols (e.g. cholesterol), may be included in LNPs as ‘helper lipids’ to enhance transfection activity and/or LNP stability.
[0306] Exemplary lipids for use in nanoparticle formulations, and/or gene transfer are shown in Table 7, and Table 8 lists exemplary polymers for use in gene transfer and/or nanoparticle formulations.
Table 7: Lipids Used for LNPs and/or Gene Transfer
Table 8: Polymers Used for LNPs and/or Gene Transfer
[0307] In certain embodiments, the non-viral vectors may be lipid nanoparticles (LNPs). Lipid nanoparticles (LNPs) according to the present disclosure include, but are not limited to, LNPs formed with ionizable lipids. Ionizable lipids may include, but are not limited to, MC3 (also called Dlin-MC3-DMA), SM-102, ALC-0315, 5A2-SC8, and BAMEA-O16B. In certain embodiments, an ionizable lipid may include C12-200. In certain embodiments, LNPs may comprise about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% ionizable lipid. In certain embodiments, LNPs may further comprise cholesterol. In certain embodiments, LNPs may further comprise a phospholipid, e.g., distearoylphosphatidylcholine (DSPC). In certain embodiments, LNPs may further comprise a PEG lipid, e.g., dimethylglycine (DMG)-PEG2k.
[0308] In certain embodiments, the LNPs of the present disclosure comprise a cationic lipid, e.g., those cationic lipids disclosed in Table 7. In certain embodiments, the cationic lipids that find use in the LNPs, compositions, and methods of the present disclosure include, but are not limited to: l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA); 1,2- dilinolenyloxyN, N-dimethylaminopropane (DLenDMA); 2,2-dilinoleyl-4-(2-dimethyl- aminoethyl)-[l,3]dioxolane (DLin-K-C2-DMA.; '"XTC2"'); 2,2-dilinoleyl-4-(3-dimethyl- aminoopropyl)- [1,3] -dioxolane (DLin-K-C3-DMA); 2,2-dilinoieyl-4-(4-dimethylaminobutyl)- ll,3]-dioxolane (DLinK-C4-DMA); 2,2-dilinoleyl-5-dimethylaminomethyl-[J ,3]-dioxane (DLin-K6-DMA); 2,2-dilinoleyl-4-N-methylpepiazino-[l,3]-dioxolane (DLin-K-MPZ), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA); 1,2-dilinoleylcarbamoyl- oxy-3-dim.ethylaminopropane (DLin-C-DAP); 1 ,2-dilinoley oxy-3 -(dimethylamino) acetoxypropane (DLin-MAC); l,2-dilinoleyoxy-3-morpholinopropane (DLin-MA); L2-dilinoleoyl-3- dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3-dimethylaminopropane (DLin- SDMA); l-linoleoyi-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP); 1,2- dilinoleyloxy-3-trirmethylaminopropane chloride salt (DLin-TMA Cl), 1 ,2-dilinoleoyl-3- trimethylaminopropane chloride salt (DLin-TAP Cl); l,2-dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ); 3-(N,N-dilinoleylamino)-l,2-propanediol (DLinAP); 3-(N,N-dioleylamino)-l ,2-propanedio (DOAP); l,2-dilinoleyloxo-3-(2- N,Ndimethylamino) ethoxypropane (DLin-EG-DMA); N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); l,2-dioleyloxy-N,N-dimethylanrinopropane (DODMA); 1,2- distearyloxyN,N-dimethylamino-propane (DSDMA); N-(l-(2,3-dioley loxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(l-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3- (N - (N’,N'-dimethylamino-ethane)-carbanloyl)cholesterol (DC-Chol); N-(l,2- dirn.yristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE); 2,3- dioleyioxy-N-[2(spermine-carboxmnido)ethyl]-N,N-dimethyl-l- propanaminiumtrifluoroacetate (DOSPA); dioctadecyl- amidoglycyl spermine (DOGS); 3- dimethylamino-2-(cholest-5-en-3-betaoxybutan-4-oxy)-l-(cis,cis-9, 12- octadecadienoxy)propane (CLinDMA); 2-[ 5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3- dimethy 1- l-(cis,cis-9', l-2'-octadecadienoxy) propane (CpLinDMA); 1 ,2-Dilinoleoy Icarbamy 1-3-dimethyiaminopropane (DLinCDAP); N-dimethyi-3,4-dioleyloxybenzylamine (DMOBA); and l,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP).
[0309] In certain embodiments, the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: LP-01 or (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate or a pharmaceutically acceptable salt thereof. In certain embodiments, the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: (9Z,9'Z,12Z,12'Z)-2-(((3-(4-methylpiperazin-l- yl)propanoyl)oxy)methyl)propane-l,3-diyl bis(octadeca-9, 12-dienoate); (9Z,9'Z,12Z,12'Z)-2- (((4-(pyrrolidin-l-yl)butanoyl)oxy)methyl)propane-l,3-diylbis(octadeca-9, 12-dienoate); (9Z,9'Z, 12Z, 12'Z)-2-(((4-(piperidin- 1 -yl)butanoyl)oxy)methyl)propane- 1 , 3 -diyl bis(octadeca-
9.12-dienoate); (9Z,9Z,12Z,2'Z)-2-(((l,4-dimethylpiperidine-4- carbonyl)oxy)methyl)propane- 1,3 -diyl bis(octadeca-9,12-dienoate); (9Z,9'Z,12Z,12'Z)-2-(((l- (cyclopropylmethyl)piperidine-4-carbonyl)oxy)methyl)propane-l,3-diyl bis(octadeca-9,12- dienoate); (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-
(dimethylamino)propoxy)carbonyl)oxy)methyl) propyl octadeca-9, 12-dienoate; (9Z,12Z)-3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l-ethylpiperidin-3- yl)methoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; 2-((((2-
(diethylamino)ethoxy)carbonyl)oxy)methyl)propane-l,3-diylbis(2 -heptylundecanoate); (9Z,12Z)-3-(((2-(diethylamino)ethoxy)carbonyl)oxy)-2-(((2-heptylundecanoyl)oxy) methyl)propyl octadeca-9, 12-dienoate; 2-((((3-(dimethylamino)propoxy)carbonyl) oxy)methyl)propane-l,3-diyl bis(2-heptylundecanoate); (9Z,12Z)-3-(((3-
(diethylamino)propoxy)carbonyl)oxy)-2-(((2-heptylundecanoyl)oxy)methyl)propyl octadeca-
9.12-dienoate; (9Z,12Z)-3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)-2-(((3- octylundecanoyl)oxy)methyl)propyl octadeca-9, 12-dienoate; 2-((((3-
(diethylamino)propoxy)carbonyl)oxy)methyl)propane- 1 , 3 -diyl bis(3 -octylundecanoate); (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((3-octylundecanoyl)oxy)methyl )propyl octadeca-9, 12-dienoate; (9Z, 12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2- (((7-hexyltridecaonoyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z, 12Z)-3-(((3- (diethylamino)propoxy)carbonyl)oxy)-2-(((9-pentyltetradecanoyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z,12Z)-3-(((3-(diethylamino)propoxy)cabonyl)oxy)-2-(((5- heptyldodecanoyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9, 12Z)-3-(2,2- bis(heptyloxy)acetoxy)-2-((((2-(dimethylamino)ethoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z,12Z)-3-((6,6-bis(octyloxy)hexanoyl)oxy)-2-((((3-
(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; 2-(3-ethyl-l l- (((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)-8,14-dioxo-7,9,13-trioxa-3-azaheptadecan-17- yl)propane-l,3-diyl dioctanoate; (9Z,9'Z,12Z,12Z)-2-((((3-
(dimethylamino)propoxy)carbonyl)oxy)methyl)propane- 1 ,3 -diyl bis(octadeca-9, 12-dienoate); (9Z,9'Z,12Z,12'Z)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-l,3-diyl bis(octadeca-9, 12-dienoate); (9Z,9'Z,12Z,12'Z)-2-((((2-
(dimethylamino)ethoxy)carbonyl)oxy)methyl)propane- 1 , 3 -diyl bis(octadeca-9, 12-dienoate); 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 1- isopropylpiperidine-4-carboxylate; 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)- octadeca-9, 12-dienoyloxy)methyl)propyl l-(cyclopropylmethyl)piperidine-4-carboxylate; 3- ((4,4-bis(octyl oxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 1- methylpyrrolidine-3-carboxylate; (9Z,9'Z,12Z,12Z)-2-((((3-
(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((9Z,12Z)-octadeca-9,12- dienoyloxy)methyl)propane- 1,3 -diyl bis(octadeca-9,12-dienoate); (2S)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 1- methylpyrrolidine-2-carboxylate; (2R)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)- octadeca-9,12-dienoyloxy)methyl)propyl l-methylpyrrolidine-2-carboxylate; 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 4- methylmorpholine-2-carboxylate; 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca- 9,12-di enoyl oxy)methyl)propyl l,4-dimethylpiperidine-4-carboxylate; 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 1- methylpiperidine-4-carboxylate; (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l- methylpyrrolidin-3-yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z, 12Z)-3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((((( 1 -methylpiperidin-4- yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate; (9Z, 12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((((l-methylazetidin-3-yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate; (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l-ethylpiperidin-
4-yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((((l-methylpiperidin-4- yl)methoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((((l, 2,2,6, 6-pentamethylpiperidin-4- yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(dimethylamino)propyl)carbamoyl)oxy)methyl)propyl octadeca-9, 12-dienoate; 3-((4,4-bis((2-propylpentyl)oxy)butanoyl)oxy)-2-(((9Z,12Z)- octadeca-9, 12-dienoyloxy)methyl)propyl 1 ,4-dimethylpiperidine-4-carboxylate; 3-((6,6- bis((2-propylpentyl)oxy)hexanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12- dienoyloxy)methyl)propyl l,4-dimethylpiperidine-4-carboxylate; 2-(((l-methylpyrrolidine-3- carbonyl)oxy)methyl)propane- 1 ,3 -diyl bis(4,4-bis(octyloxy)butanoate) ; 2-((( 1 - methylpyrrolidine-3-carbonyl)oxy)methyl)propane-l,3-diyl bis(6,6-bis(octyloxy)hexanoate); 2-(((l-methylpyrrolidine-3-carbonyl)oxy)methyl)propane-l,3-diyl bis(6,6-bis((2- propylpentyl)oxy)hexanoate); 2-(5-(3-(dodecanoyloxy)-2-(((l-methylpyrrolidine-3- carbonyl)oxy)methyl)propoxy)-5-oxopentyl)propane-l,3-diyl dioctanoate; 2-(5-(3-((l- methylpyrrolidine-3-carbonyl)oxy)-2-((palmitoyloxy)methyl)propoxy)-5-oxopentyl)propane- 1 ,3-diyl dioctanoate; 2-(S-(3-((l-methylpyrrolidine-3-carbonyl)oxy)-2-
((tetradecanoyloxy)methyl)propoxy)-S-oxopentyl)propane- 1,3-diyl dioctanoate; 3-((4,4- bis(octyl oxy)butanoyl)oxy)-2-((dodecanoyloxy)methyl)propyl l-methylpyrrolidine-3- carboxylate; 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((tetradecanoyloxy)methyl)propyl 1- methylpyrrolidine-3-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((palmitoyloxy)methyl)propyl 1- methylpyrrolidine-3-carboxylate; l-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((l- methylpyrrolidine-3-carbonyl)oxy)methyl)propyl) 8-methyl octanedioate; l-(3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((l-methylpyrrolidine-3-carbonyl)oxy)methyl)propyl) 10- octyl decanedioate; l-(3-((6,6-bis((2-propylpentyl)oxy)hexanoyl)oxy)-2-(((l,4- dimethylpiperidine-4 carbonyl)oxy)methyl)propyl) 10-octyl decanedioate; l-(3-((6,6-bis((2- propylpentyl)oxy)hexanoyl)oxy)-2-(((l,4-dimethylpiperidine-4-carbonyl)oxy)methyl)propyl) 8-methyl octanedioate; and 8-dimethyl O'l,Ol-(2-(((l-methylpyrrolidine-3- carbonyl)oxy)methyl)propane- 1,3-diyl) dioctanedioate.
[0310] In certain embodiments, the cationic lipids that find use in the LNPs, compositions, and methods of the present disclosure include, but are not limited to: ((4,4'-((((3- (Dimethylamino)propyl)thio)carbonyl) azanediyl)bis(butanoyl))bis(oxy))bis(propane-2, 1,3- triyl) tetranonanoate; ((4,4’-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl)bis (butanoyl))bis(oxy)) bis(propane-2,l,3-triyl) tetraoctanoate; bis(l,3-bis(Nonanoyloxy)propan- 2-yl) 5-((4-(dimethylamino)butanoyl)oxy)nonanedioate HCI salt; bis(l,3-bis(Octanoyloxy) propan-2-yl) 5-((4-(dimethylamino) butanoyl)thio)nonanedioate; bis(l,3-bis(N onanoyloxy) propan-2-yl) 4-((4-(dimethylamino) b utanoyl)oxy)heptanedioate; bis(l,3-bis(Octanoyloxy) propan-2-yl) 4-((4-(dimethylamino) butanoyl)thio)heptanedioate; ((2,2’-((((3-(Dimethyl- amino)propyl)thio) carbonyl)azanediyl)bis(acetyl))bis(oxy))bis(propane-2, 1 , 3 -triyl) tetranonanoate; bis(l,3-bis(N onanoyloxy )propan-2-yl) 4-((4-(dimethylamino) butanoyl)thio) heptanedioate ; ((4,4'-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl)bis (butanoyl)) bis(oxy))bis(propane-2,l,3-triyl) tetrakis(3-cyclohexylpropanoate); ((4,4’-((((3-(Dimethyl- amino)propyl)thio)carbonyl) azanediyl)bis(butanoyl))bis(oxy)) bis(propane-2, 1 , 3 -triyl) tetrakis(4-cyclohexyl butanoate); ((6,6'-((((3-(Dimethylamino)propyl)thio)carbonyl) azanedi- yl)bis(hexanoyl))bis(oxy))bis(propane-2,l,3-triyl)tetrakis(3-cyclohexylpropanoate);
Nonanoic acid 2-(3-{(3-dimethylaminopropylsulfanylcarbonyl)-[2-(2-nonanoyloxy-l- nonanoyloxymethyl-ethoxycarbonyl)-ethyl]amino}-propionyloxy)-3-octanoyloxy-propyl ester; ((4,4’-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl)bis(butanoyl))bis(oxy))bis (propane-2, 1,3-triyl) tetrakis(2-(4-methylcyclohexyl) acetate); ((4,4 '-((((3- (Dimethylamino)propyl)thio)carbonyl) azanediyl)bis(butanoyl))bis(oxy)) bis(propane-2, 1,3- triyl) tetrakis(4-ethylcyclo hexane- 1 -carboxylate); ((4,4’-((((3-(Dimethylamino) propyl)thio)carbonyl) azanediyl)bis(butanoyl)) bis(oxy))bis(propane-2, 1,3-triyl) tetrakis(3- cyclohexyl-2-methylpropanoate); ((4,4'-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl)bis(butanoyl)) bis(oxy))bis(propane-2, 1,3-triyl) tetrakis(2-methyloctanoate); ((4,4 '-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl) bis(butanoyl)) bis(oxy))bis(propane- 2,l,3-triyl)tetrakis(2,2-dimethylheptanoate); ((3,3'-((((3-(Dimethylamino) propyl)thio)carbonyl) azanediyl) bis(propanoyl)) bis(oxy))bis (methylene))bis(2- methylpropane-2, 1,3-triyl) tetrakis(3-(4-methyl cyclohexyl)propanoate); ((3,3'-((((3- (Dimethylamino)propyl)thio)carbonyl) azanediyl) bis(propanoyl))bis(oxy)) bis (methylene)) bis(2-methyl propane-2, 1,3-triyl) tetrakis(2-(4-ethylcyclohexyl) acetate); ((3,3'-((((3- (Dimethylamino)propyl)thio)carbonyl) azanediyl) bis(propanoyl))bis(oxy)) bis(methylene)) bis(2-methyl propane-2, 1,3-triyl) tetrakis(2-(4-ethylcyclohexyl)acetate); ((3,3'-((((3- (Dimethylamino)propyl)thio)carbonyl)azanediyl)bis(propanoyl))bis(oxy))bis(methylene))bis (2-methylpropane-2,l,3-triyl)tetrakis(3,3-dimethylheptanoate); ((3,3'-((((3-(Dimethylamino) propyl)thio)carbonyl)azanediyl)bis(propanoyl))bis(oxy))bis(methyl-ene))bis(2-methyl- propane-2, 1,3-triyl) tetrakis(octanoate); ((4,4'-((((3-(Dimethylamino) propyl)thio)carbonyl) azanediyl) bis(butanoyl))bis(oxy)) bis(methylene))bis(propane-2, 1,3-triyl) tetranonanoate; ((3,3'-((((2-(Dimethylamino)ethyl)thio)carbonyl)azanediyl)bis(propanoyl))bis(oxy)) bis(methylene))bis(propane-2,l,3-triyl)tetranonanoate; ((3,3'-((((3-(Dimethylamino)propyl) thio)carbonyl) azanediyl)bis(propanoyl))bis(oxy))bis(methylene))bis (propane-2, 1 ,3 - triyl) tetrakis(3-(4-methylcyclohexyl)propanoate); ((4,4'-((((3-(Dimethyl-amino)propyl)thio) carbonyl)azanediyl) bis(butanoyl))bis (oxy)bis(propane-2, 1,3-triyl) tetrakis(octahydro-lH- indene); ((4,4 '-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl) bis(butanoyl))bis(oxy) ) bis(propane-2, 1 ,3-triyl)tetrakis(octahydro-lH-indene-5-carboxylate); ((4,4'-(((3-(dimethyl- amino)propoxy)carbonyl) azanediyl)bis(butanoyl))bis(oxy)) bis(propane-2, 1 , 3 - triyl) tetranonanoate; ((4,4'-(((3-(dimethylamino)propyl)carbamoyl) azanediyl)bis (butanoyl)) bis(oxy))bis(propane-2, 1 ,3-triyl)tetranonanoate; ((4,4’-((((3-(dimethylamino) propyl)thio) carbonyl)azanediyl)bis(butanoyl)) bis(oxy)) bis(propane-2, 1,3-triyl) tetrakis(2-(p- tolyl)acetate); [2-[4-[3-(dimethylamino)propoxycarbonyl-[4-[2-(2-methyloctanoyloxy)-l-(2- methyloctanoyloxymethyl)ethoxy]-4-oxobutyl] amino]butanoyloxy]-3-(2-methyloctanoyl- oxy)propyl] 2-methyloctanoate; |2-|4-|3-(dimethylamino)propylcarbamoyl-|4-|2-(2- methyloctanoyl oxy)-l-(2-methyloctanoyloxymethyl)ethoxy ]-4-oxobutyl] amino Jbutanoyl- oxy]-3-(2-methyloctanoyloxy)propyl] 2-methyloctanoate; [2-[4-[[3-(dimethylamino)propyl- methyl-carbamoyl][4-[2-(2-methyloctanoyloxy)-l-(2-methyloctanoyloxymethyl)ethoxy]-4- oxobutyl] amino] butanoyloxy] -3 -(2- methyloctanoy loxy)propyl] 2-methyloctanoate; [2- [4- [5- (dimethylamino)pentanoyl- [4-[2-(2-methyloctanoyloxy)- 1 -(2-methyloctanoyloxym ethyl)ethoxy]-4-oxo-butyl] amino] butanoyloxy]-3-(2-methyloctanoyloxy)propyl] 2- methyloctanoate; [2-[4-[5-(dimethylamino)pentyl-[4-[2-(2-methyl octanoy loxy)-l-(2-methyl- octanoyloxym ethyl)ethoxy]-4-oxo-butyl] amino] butanoyloxy]-3-(2-methyloctanoyl- oxy)propyl] 2-methyloctanoate; bis [2-(2-methyloctanoyloxy)-l-(2-methyloctanoyl oxym ethyl)ethyl] 5-[ 4-(dimethylamino)butanoyloxy] nonanedioate; and ((4,4 '-((((3- (dimethylamino) propyl)thio)carbonyl)azanediyl)bis(butanoyl)) bis(oxy)) bis(propane-2, 1,3- triyl) tetrakis(2-methyl-4-(p-tolyl) butanoate).
[0311] In certain embodiments, the cationic lipids that find use in the LNPs, compositions, and methods of the present disclosure include, but are not limited to: 6,6'- (Methylazanediyl)Bis(N,N-Dioctylhexanamide); 6,6'-(Octylazanediyl)Bis(N,N- Dioctylhexan-amide); 6,6'-(Hexylazanediyl)Bis(N,N-Dioctylhexanamide); 10, 10'- (Methylazanediyl)Bis (N,N-Dioctyldecanamide); 8,8'-(Methylazanediyl)Bis(N,N- Didecyloctanamide); 6,6'-(Methylazanediyl)Bis(N,N-Didecylhexanamide); 6,6'- (Methylazanediyl)Bis(N,N-Didodecyl-hexanamide); 6,6'-((2- Hydroxyethyl)Azanediyl)Bis(N,N-Dioctylhexanamide); 6,6'-((6- Hydroxyhexyl)Azanediyl)Bis(N,N-Dioctylhexanamide); 6,6'-((2-Hydroxyethyl)Azanediyl) Bis(N,N-Didecylhexanamide); 8,8'-((6-Hydroxyhexy])Azanediyl)Bis(N,N-Dioctyloct- anamide); 10,10'-((6-Hydroxyhexyl)Azanediyl)Bis(N,N-Dioctyldecanamide); 10, 10'-((2- Hydroxyethyl)Azanediyl)Bis(N,N-Didecyldecanamide); 8,8'-((5-Hydroxypentyl)Azanediyl) Bis(N,N-Didecyloctanamide); 8,8’-((4-Hydroxybutyl)Azanediyl)Bis(N,N-Didecyloct- anamide); 8,8'-((6-Hydroxyhexyl)Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((2-Hydroxy- ethyl)Azanediyl)Bis(N,N-Didecyloctanamide); 10,10'-((4-Hydroxybutyl)Azanediyl)Bis(N,N- Didecyldecanamide); N,N’-( (Methylazanediyl )Bis(Hexane-6, 1-Diyl) )Bis(N,2- Dihexyldecanamide); N,N’-((Methylazanediyl)Bis(Hexane-6, l-Diyl))Bis(N-Hexyl Palmitamide); (9z,9'z,12z,12'z)-N,N'-((Methylazanediyl)Bis(Hexane-6,L-Diyl))Bis(N- Hexyloctadeca-9,L2-Dienamide); N,N-Didecyl-8-((8-(Hexadecylamino)-8-Oxooctyl) (Methyl)Amino)Octanamide; 8,8'-( (8-(Decylamino )-8-Oxooctyl )Azanediyl )Bis(N,N- Didecyloctanamide); 8,8'-((6-(Dihexylamino)-6-Oxohexyl)Azanediyl)Bis(N,N-Didecyloctan- amide); 8,8'-((5-(Decylamino)-5-Oxopentyl)Azanediyl)Bis(N,N-Didecyloctanamide); 6,6'-( (8-(Decylamino )-8-Oxooctyl )Azanediyl )Bis(N,N-Didecylhexanamide); 6,6'-((2- (Dihexylamino)Ethyl)Azanediyl)Bis(N,N-Didecylhexanamide); 10,10'-((2-(Dimethyl- amino)Ethyl)Azanediyl)Bis(N,N-Didecyldecanamide); 2-Butyloctyl 6-(Bis(6- (Dioctylamino)-6-Oxohexyl)Amino)Hexanamide; 6,6' -((4- Hydroxybutyl)Azanediyl)Bis(N,N-Bis(2-Ethylhexyl)Hexanamide); 8,8'-((2- Hydroxyethyl)Azanediyl)Bis(N,N-Didodecyl-octanamide); 6,6’-((6- Hydroxyhexyl)Azanediyl)Bis(N,N-Didodecylhexanamide); N,N-Didecyl-8-((8- (Hexadecyl(Methyl)Amino)-8-Oxooctyl)(Methyl )Amino)Octanamide; 8,8'- (Methylazanediyl)Bis(N,N-Didodecyloctanamide); 8,8'-((3-Hydroxypropyl)Azanediyl) Bis(N,N-Didecyloctanamide); 8,8’-( (2-(2-Hydroxyethoxy)Ethyl )Azanediyl )Bis(N,N- Didecyloctanamide); 8,8'-((5-Hydroxy-4,4-Dimethylpentyl)Azanediyl)Bis(N,N-Didecyl- octanamide); 8,8’-((3-(2-Methyl-Lh-Imidazol-L-Yl)Propyl)Azanediyl)Bis(N,N-Didecyloctan- amide); 8,8'-((7-Hydroxyheptyl)Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-( (2-(2- Methoxyethoxy)Ethyl)Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((8-Hydroxyoctyl) Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((3-(Lh-Imidazol-L-Yl)Propyl)Azanediyl) Bis(N,N-Didecyloctanamide); 8,8'-((2,2-Difluoro-3-Hydroxypropyl) Azanediyl)Bis(N,N- Didecyloctanamide); 8,8'-((3-((2-(Methylamino)-3,4-Dioxocyclobut-L-En-Lyl) Amino) Propyl)Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((2-Fluoro-3-Hydroxypropyl) Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((3,3,3-Trifluoro-2-(Hydroxymethyl)Propyl) Azanediyl) Bis(N,Ndidecyloctanamide); 8,8'-((5-Methoxypentyl)Azanediyl)Bis(N,N- Didecyloctanamide); N,N-Didecyl-8-((8-(Dioctylamino)-8-Oxooctyl)(Methyl)Amino) Octanamide; Tert-Butyl (3-(Bis(10-(Didecylamino)-10-oxodecyl)Amino)Propyl)Carbamate; 10,10'-((3-(Lh-Imidazol-L-Yl)Propyl)Azanediyl)Bis(N,N-Didecyldecanamide); 8,8'- (Methylazanediyl)Bis(N,N-Dinonyloctanamide); Tert-Butyl (3-(Bis( 10-(Didecylamino )- 10- oxodecyl )Amino )Propyl ) Carbamate; 10,10'-((3-((2-(Methylamino)-3,4-Dioxocyclobut-L- En-Lyl)Amino)Propyl) Azanediyl) Bis(N,N-Didecyldecanamide); N.N’-((methylazanediyl) bis(octane-8, l-diyl))bis(N-hexylhexanamide); N.N’-(((5-hydroxypentyl)azanediyl)bis (octane-8, l-diyl))bis(N-hexylhexanamide); N.N'-((methylazanediyl)bis(octane-8, 1- diyl))bis(N -octyl octanamide) ; N. N'-(((5-hydroxypentyl)azanediyl)bis(octane- 8, 1 - diyl))bis(N-octyl octanamide); N,N'-((methylazanediyl)bis(octane-8, l-diyl))bis(N- octyloctanamide); N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,l-diyl))bis(N- decyldecanamide); N,N'-((methylazanediyl)bis(octane-8, l-diyl))bis(N- dodecyldodecanamide); N,N'-(((5-hydroxypentyl)azanediyl)bis( octane-8, 1- diyl))bis(Ndodecyldodecanamide); N,N'-((methylazanediyl)bis(octane-8, l-diyl))bis(2- hexyldecanamide); N,N'-((methylazanediyl)bis (octane-8, l-diyl))bis(2-hexyl-N- methyldecanamide); N,N'-(((5-hydroxypentyl)azanediyl)bis (octane-8, l-diyl))bis(2- hexyldecanamide); N,N'-(((5-hydroxypentyl)azanediyl)bis( octane-8, l-diyl))bis(2-hexyl-N methyl decanamide); N-decyl-N-(8-((8-(didecylamino)-8 oxooctyl) (methyl) amino) octyl)decanamide; N-decyl-N-(8-((8-(didecylamino)-8-oxooctyl)(5- hydroxypentyl)amino)octyl)decanamide; 6,6'-((5-hydroxypentyl)azanediyl)bis(N,N-didecyl- hexanamide); 7, 7'-(methylazanediyl)bis(N,N-didecylheptanamide); 8,8'-((2-(dimethylamino) ethyl)azanediyl)bis(N,N-didecyloctanamide); 8,8'-((2-(pyrrolidin-l-yl)ethyl)azanediyl) bis(N,N-didecyloctanamide); N,N-didecyl-8-((8-(decyl(methyl)amino)-8-oxooctyl)(methyl) amino)octanamide; N,N-didecyl-8-((8-(decyl(methyl)amino)-8-oxooctyl)(2-hydroxyethyl) amino)octanamide; 8,8'-((5-Hydroxypentyl)Azanediyl)Bis(N,N-Dinonyloctanamide); 8,8'- ((5-Hydroxypentyl)Azanediyl)Bis(N,N-Didecyl-2-Fluorooctanamide); 8,8'-(Methyl- azanediyl)Bis(N,N-Didecyl-2-Fluorooctanamide); 2,2'-((5-Hydroxypentyl)Azanediyl) Bis(N,N-Didecylacetamide); and 4,4'-((5-Hydroxypentyl)Azanediyl)Bis(N,N-Didecylbutan- amide).
[0312] Moreover, each of the following references, which disclose ionizable cationic lipids that find use in connection with the LNPs, compositions, and methods disclosed herein, is hereby incorporated by reference in its entirety: PCT/US2014/066242, published as W02015074085A1; International Patent Application No. PCT/US2015/030218, published as W02016081029A1; U.S. Patent No. 10,227,302; U.S. Patent No. 10,383,952; U.S. Patent No. 10,526,284; International Patent Application No. PCT/US2016/069493, published as W02017117530A1; International Patent Application No. PCT/US2019/025246, published as W02019191780A; and U.S. Patent Application No. 16/823,212, published as US2020/0297634, and International Patent Application Publication Nos. WO2015/095340, W02022/011156, WO2022/133344, WO2023/086514, WO2023/081776, W02023/010128, WO2022/235972, WO2022/235935, WO2022/235923, WO2022/056413, W02022/036170, W02020/191103, WO2018/183901, WO2018/119163, W02020/097548, W02020/097540, W02020/097520, W02020/097493, WO2016/197133, WO2011/141705, WO2011/141704, WO2011/000107, WO2011/000106, W02010/144740, W02010/129709,W02010/088537, W02010/054406, WO2010/054405, WO2010/054401, W02010/054384, W02009/127060, W02008/042973, W02007/012191, W02006/074546, WO2005/121348, W02005/120461, W02005/120152, W02005/026372, W02005/007196, W02004/002453, W02002/087541, W02000/003683, WO2023/114944, WO2023/114943, WO2023/114937, W02022/016070, WO2021/030701, W02020/146805, W02020/081938, W02020/061426, WO2019/089828, WO2019/036030, WO2019/036028, W02019/036008, WO2019/036000, WO2018/200943, WO2018/191719, WO2018/191657, W02018/081480, WO2018/078053, WO2017/117528, WO2017/075531, W02017/004143, and WO2015/199952.
[0313] In certain embodiments, the LNPs of the present disclosure comprise a non-cationic lipid, e.g., those non-cationic lipids disclosed in Table 7. In certain embodiments, the noncationic lipids that find use in the LNPs, compositions, and methods of the present disclosure include, but are not limited to lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoyl- phosphatidylglycerol (DPPG), dioleoyl phosphatidyl ethanol amine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC) , palmitoy loleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-lcarboxylate (DOPE-mal), dipalmitoyl-phosphatidyl- ethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidy lethanolamine (DSPE), monomethyl-phosphatidyl ethanol amine, phosphatidylethanolamine (DEPE), dimethyl-phosphatidylethanolamine, dielaidoylstearoyloleoylphosphatidylethanolamine (SOPE).
[0314] In certain embodiments, the PEGylated lipids comprise a PEG molecule with a molecular weight from about 200Da to about 5000Da. In certain embodiments, the PEGylated lipids comprise a PEG molecule with a molecular weight of 2000Da (2kDa).
[0315] In certain embodiments, the PEG-lipids can include, but are not limited to, those identified in Table 8. For example, but not by way of limitation, the PEG-lipid can comprise PEG coupled to dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), methoxypolyethyleneglycol (PEG-DMG or PEG2000-DMG), PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides, PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof. In certain embodiments, the PEG-lipid comprises detergent-like PEG lipids (e.g., PEG-DSPE).
[0316] In certain embodiments, the PEG moiety is conjugated directly to the lipid. In certain embodiments, the PEG moiety is conjugated to the lipid via a linker moiety. Any linker moiety suitable for conjugating the PEG to a lipid can be used including, but not limited to, ester-containing linker moieties and/or non-ester-containing linker moieties. In certain embodiments, an ester-containing linker moiety is used to conjugate the PEG to the lipid. Exemplary ester-containing linker moieties include, e.g., carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof. Exemplary non-ester containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-0-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety).
[0317] In certain embodiments, phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form the PEG-lipid conjugate. In certain embodiments, phosphatidylethanolamines comprising saturated or unsaturated fatty acids with carbon chain lengths in the range of CIO to C20 are employed in connection with the LNPs, compositions, and methods disclosed herein. In certain embodiments, phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. In certain embodiments, the phosphatidylethanolamines that find use in connection with the LNPs, compositions, and methods disclosed herein include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).
[0318] In certain embodiments the PEG-DAA conjugate of the instant disclosure is a PEG- didecyloxypropyl (CIO) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG- dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16) conjugate, or a PEG- distearyloxypropyl (C18) conjugate. In certain of such embodiments, the PEG moiety has an average molecular weight of about 750 or about 2,000 daltons. In certain of such embodiments, the terminal hydroxyl group of the PEG moiety is substituted with a methyl group.
[0319] In addition to the foregoing, other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxy-propyl, methacrylamide, polymethacrylamide, and polydimethylacrylamide, polylactic acid, poly glycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
[0320] The lipids described herein can be combined in any number of molar ratios to produce an LNP. In certain embodiments, the LNP comprises a PEG-lipid where the PEG-lipid comprises at least 0. 1 mol% of the total lipid. For example, but not by way of limitation, the PEG-lipid component can comprise from about 0. 1 mol% to 5 mol% of the total lipid. In certain embodiments, the LNP comprises a cationic lipid where the cationic lipid component comprises at least 10 mol% of the total lipid. For example, but not by way of limitation, the cationic lipid component can comprise from about 10 mol% to 70 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 60 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 50 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 40 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 30 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 20 mol% of the total lipid. In certain embodiments, the LNP comprises cholesterol where cholesterol comprises at least 10 mol% of the total lipid. For example, but not by way of limitation, cholesterol can comprise from about 10 mol% to 70 mol% of the total lipid. In certain embodiments, the cholesterol can comprise from about 10 mol% to 60 mol% of the total lipid. In certain embodiments, the cholesterol can comprise from about 10 mol% to 50 mol% of the total lipid. In certain embodiments, the cholesterol can comprise from about 10 mol% to 40 mol% of the total lipid. In certain embodiments, the cholesterol can comprise from about 10 mol% to 30 mol% of the total lipid. In certain embodiments, the cholesterol can comprise from about 10 mol% to 20 mol% of the total lipid. In certain embodiments, the LNP comprises a non-cationic lipid where the non-cationic lipid comprises at least 10 mol% of the total lipid. For example, but not by way of limitation, the noncationic lipid can comprise from about 10 mol% to 70 mol% of the total lipid. In certain embodiments, the non-cationic lipid component can comprise from about 10 mol% to 60 mol% of the total lipid. In certain embodiments, the non-cationic lipid component can comprise from about 10 mol% to 50 mol% of the total lipid. In certain embodiments, the non-cationic lipid component can comprise from about 10 mol% to 40 mol% of the total lipid. In certain embodiments, the non-cationic lipid component can comprise from about 10 mol% to 30 mol% of the total lipid, the non-cationic lipid component can comprise from about 10 mol% to 20 mol% of the total lipid.
[0321] Non- viral vectors optionally include targeting modifications to improve uptake and/or selectively target certain cell types. These targeting modifications can include e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars (e.g., N-acetylgalactosamine (GalNAc)), and cell penetrating peptides. Such vectors also optionally use fusogenic and endosome-destabilizing peptides/polymers, undergo acid- triggered conformational changes (e.g., to accelerate endosomal escape of the cargo), and/or incorporate a stimuli-cleavable polymer, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.
[0322] In certain embodiments, one or more nucleic acid molecules (e.g., DNA molecules) other than the components of a genome editing system, e.g., the RNA-guided nuclease molecule component and/or the gRNA molecule component described herein, are delivered. In certain embodiments, the nucleic acid molecule is delivered at the same time as one or more of the components of the genome editing system. In certain embodiments, the nucleic acid molecule is delivered before or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the genome editing system are delivered. In certain embodiments, the nucleic acid molecule is delivered by a different means than one or more of the components of the genome editing system, e.g., the RNA-guided nuclease molecule component and/or the gRNA molecule component, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, e.g., an integration-deficient lentivirus, and the RNA-guided nuclease molecule component and/or the gRNA molecule component can be delivered by electroporation, e.g., such that the toxicity caused by nucleic acids (e.g., DNAs) can be reduced. In certain embodiments, the nucleic acid molecule encodes a therapeutic protein, e.g., a protein described herein. In certain embodiments, the nucleic acid molecule encodes an RNA molecule, e.g., an RNA molecule described herein.
Delivery ofRNPs and/or RNA encoding genome editing system components
[0323] RNPs (complexes of gRNAs and RNA-guided nucleases) and/or RNAs encoding RNA-guided nucleases and/or gRNAs, can be delivered into cells or administered to subjects by art-known methods, some of which are described in Cotta-Ramusino. In vitro, RNA- guided nuclease-encoding and/or gRNA-encoding RNA can be delivered, e.g., by microinjection, electroporation, transient cell compression or squeezing (see, e.g., Lee 2012). Lipid-mediated transfection, peptide-mediated delivery, GalNAc- or other conjugate- mediated delivery, and combinations thereof, can also be used for delivery in vitro and in vivo. A protective, interactive, non-condensing (PINC) system may be used for delivery.
[0324] In vitro delivery via electroporation comprises mixing the cells with the RNA encoding RNA-guided nucleases and/or gRNAs, with or without donor template nucleic acid molecules, in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. Systems and protocols for electroporation are known in the art, and any suitable electroporation tool and/or protocol can be used in connection with the various embodiments of this disclosure.
Route of administration
[0325] Genome editing systems, or cells altered or manipulated using such systems, can be administered to subjects by any suitable mode or route, whether local or systemic. Systemic modes of administration include oral and parenteral routes. Parenteral routes include, by way of example, intravenous, intramarrow, intrarterial, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. Components administered systemically may be modified or formulated to target the components to the eye.
[0326] Local modes of administration include, by way of example, intracameral, intraocular, intraorbital, subconjuctival, intravitreal, subretinal or transscleral routes, as well as delivery directly into the trabecular meshwork. In an embodiment, significantly smaller amounts of the components (compared with systemic approaches) may exert an effect when administered locally (for example, intravitreally) compared to when administered systemically (for example, intravenously). Local modes of administration can reduce or eliminate the incidence of potentially toxic side effects that may occur when therapeutically effective amounts of a component are administered systemically.
[0327] In an embodiment, components described herein are delivered subretinally, e.g., by subretinal injection. Subretinal injections may be made directly into the macular, e.g., submacular injection. [0328] In an embodiment, components described herein are delivered by intravitreal injection. Intravitreal injection has a relatively low risk of retinal detachment risk. In an embodiment, nanoparticle or viral, e.g., AAV vector, e.g., an AAV2 vector, e.g., a modified AAV2 vector, is delivered intravitreally.
[0329] Methods for administration of agents to the eye are known in the medical arts and can be used to administer components described herein. Exemplary methods include intraocular injection (e.g., retrobulbar, subretinal, submacular, intravitreal and intrachoridal), iontophoresis, eye drops, and intraocular implantation (e.g., intravitreal, sub-Tenons and subconjunctival).
[0330] Administration may be provided as a periodic bolus (for example, subretinally, intravenously or intravitreally) or as continuous infusion from an internal reservoir (for example, from an implant disposed at an intra- or extra-ocular location (see, U.S. Pat. Nos. 5,443,505 and 5,766,242)) or from an external reservoir (for example, from an intravenous bag). Components may be administered locally, for example, by continuous release from a sustained release drug delivery device immobilized to an inner wall of the eye or via targeted transscleral controlled release into the choroid (see, for example, PCT/US00/00207, PCT/US02/14279, Ambati 2000a, and Ambati 2000b). A variety of devices suitable for administering components locally to the inside of the eye are known in the art. See, for example, U.S. Patent Nos. 6,251,090, 6,299,895, 6,416,777, 6,413,540, and PCT/US00/28187.
[0331] In addition, components may be formulated to permit release over a prolonged period of time. A release system can include a matrix of a biodegradable material or a material which releases the incorporated components by diffusion. The components can be homogeneously or heterogeneously distributed within the release system. A variety of release systems may be useful. However, the choice of the appropriate system will depend upon rate of release required by a particular application. Both non-degradable and degradable release systems can be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents such as, but not limited to, calcium carbonate and sugar (for example, trehalose). Release systems may be natural or synthetic. However, synthetic release systems are preferred because generally they are more reliable, more reproducible and produce more defined release profiles. The release system material can be selected so that components having different molecular weights are released by diffusion through or degradation of the material. Administration can be provided as a periodic bolus (for example, intravenously) or as continuous infusion from an internal reservoir or from an external reservoir (for example, from an intravenous bag or implantable pump). Components can be administered locally, for example, by continuous release from a sustained release drug delivery device.
[0332] Representative synthetic, biodegradable polymers include, for example: polyamides such as poly(amino acids) and poly (peptides); polyesters such as poly(lactic acid), poly (glycolic acid), poly(lactic-co-glycolic acid), and poly (caprolactone); poly(anhydrides); poly orthoesters; polycarbonates; and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. Representative synthetic, non-degradable polymers include, for example: polyethers such as poly(ethylene oxide), poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymerspolyacrylates and polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and methacrylic acids, and others such as poly(vinyl alcohol), poly (vinyl pyrolidone), and poly (vinyl acetate); poly (urethanes); cellulose and its derivatives such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various cellulose acetates; poly siloxanes; and any chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof.
[0333] Poly(lactide-co-glycolide) microsphere can also be used. Typically, the microspheres are composed of a polymer of lactic acid and glycolic acid, which are structured to form hollow spheres. The spheres can be approximately 15-30 microns in diameter and can be loaded with components described herein. In some embodiments, genome editing systems, system components and/or nucleic acids encoding system components, are delivered with a block copolymer such as a poloxamer or a poloxamine.
Multi-modal or differential delivery of components
[0334] Skilled artisans will appreciate, in view of the instant disclosure, that different components of genome editing systems disclosed herein can be delivered together or separately and simultaneously or non-simultaneously. Separate and/or asynchronous delivery of genome editing system components can be particularly desirable to provide temporal or spatial control over the function of genome editing systems and to limit certain effects caused by their activity.
[0335] Different or differential modes as used herein refer to modes of delivery that confer different pharmacodynamic or pharmacokinetic properties on the subject component molecule, e.g., an RNA-guided nuclease molecule, gRNA, template nucleic acid, or payload. For example, the modes of delivery can result in different tissue distribution, different halflife, or different temporal distribution, e.g., in a selected compartment, tissue, or organ.
[0336] Some modes of delivery, e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result in more persistent expression of and presence of a component. Examples include viral, e.g., AAV or lentivirus, delivery.
[0337] By way of example, the components of a genome editing system, e.g., an RNA- guided nuclease and a gRNA, can be delivered by modes that differ in terms of resulting halflife or persistent of the delivered component the body, or in a particular compartment, tissue or organ. In certain embodiments, a gRNA can be delivered by such modes. The RNA- guided nuclease molecule component can be delivered by a mode which results in less persistence or less exposure to the body or a particular compartment or tissue or organ.
[0338] More generally, in certain embodiments, a first mode of delivery is used to deliver a first component and a second mode of delivery is used to deliver a second component. The first mode of delivery confers a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ. The second mode of delivery confers a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.
[0339] In certain embodiments, the first pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property. [0340] In certain embodiments, the first mode of delivery is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.
[0341] In certain embodiments, the second mode of delivery is selected to optimize, e.g., maximize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.
[0342] In certain embodiments, the first mode of delivery comprises the use of a relatively persistent element, e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV or lentivirus. As such vectors are relatively persistent product transcribed from them would be relatively persistent.
[0343] In certain embodiments, the second mode of delivery comprises a relatively transient element, e.g., an RNA or protein.
[0344] In certain embodiments, the first component comprises gRNA, and the delivery mode is relatively persistent, e.g., the gRNA is transcribed from a plasmid or viral vector, e.g., an AAV or lentivirus. Transcription of these genes would be of little physiological consequence because the genes do not encode for a protein product, and the gRNAs are incapable of acting in isolation. The second component, an RNA-guided nuclease molecule, is delivered in a transient manner, for example as mRNA or as protein, ensuring that the full RNA-guided nuclease molecule/gRNA complex is only present and active for a short period of time.
[0345] Furthermore, the components can be delivered in different molecular forms or with different delivery vectors that complement one another to enhance safety and tissue specificity.
[0346] Use of differential delivery modes can enhance performance, safety, and/or efficacy, e.g., the likelihood of an eventual off-target modification can be reduced. Delivery of immunogenic components, e.g., Cas9 molecules, by less persistent modes can reduce immunogenicity, as peptides from the bacterially-derived Cas enzyme are displayed on the surface of the cell by MHC molecules. A two-part delivery system can alleviate these drawbacks.
[0347] Differential delivery modes can be used to deliver components to different but overlapping target regions. The formation active complex is minimized outside the overlap of the target regions. Thus, in certain embodiments, a first component, e.g., a gRNA is delivered by a first delivery mode that results in a first spatial, e.g., tissue, distribution. A second component, e.g., an RNA-guided nuclease molecule is delivered by a second delivery mode that results in a second spatial, e.g., tissue, distribution. In certain embodiments, the first mode comprises a first element selected from a liposome, nanoparticle, e.g., polymeric nanoparticle, and a nucleic acid, e.g., viral vector. The second mode comprises a second element selected from the group. In certain embodiments, the first mode of delivery comprises a first targeting element, e.g., a cell specific receptor or an antibody, and the second mode of delivery does not include that element. In certain embodiments, the second mode of delivery comprises a second targeting element, e.g., a second cell specific receptor or second antibody.
[0348] When the RNA-guided nuclease molecule is delivered in a virus delivery vector, a liposome, or polymeric nanoparticle, there is the potential for delivery to and therapeutic activity in multiple tissues, when it may be desirable to only target a single tissue. A two-part delivery system can resolve this challenge and enhance tissue specificity. If the gRNA molecule and the RNA-guided nuclease molecule are packaged in separated delivery vehicles with distinct but overlapping tissue tropism, the fully functional complex is only formed in the tissue that is targeted by both vectors.
EX MPLES
[0349] The following Examples are merely illustrative and are not intended to limit the scope or content of the disclosure in any way.
Example 1: Screening of . aureus Cas9 guide RNAs targeting the MYOC gene in T cells
[0350] 5. aureus Cas9 (5aCas9) guide RNAs (gRNAs) were designed to target the UTR and coding sequence of the MYOC gene using the PAM NNGRRT (see Table 9 for the sequences of the .S'aCas9 gRNA targeting domains) to identify ribonucleoproteins (RNPs) that introduce frameshift indels likely to disrupt expression of functional MYOC. RNPs comprising .S'«Cas9 gRNAs (Table 9) complexed with wildtype SaCas9 enzyme (S«Cas9 polypeptide sequence is set forth in SEQ ID NO:47) in a 2:1 ratio (guide to enzyme) were screened in a T cell assay to analyze the RNP gene editing.
[0351] Briefly, primary T cells were nucleofected with the RNPs at 8 pM. The genomic DNA was extracted four days post nucleofection and next-generation sequencing (NGS) was performed on the MYOC PCR products (see Table 15 for NGS primers used). The percentage of frameshift indels relative to the total percentage of indels that were introduced into the MYOC gene was analyzed (Fig. 1). The results showed that many of the tested .SV/CasQ RNPs introduced indels into the MYOC gene. In some cases, a large proportion of the indels were frameshift indels and therefore likely to disrupt the expression of a functional MYOC protein (Fig. 1).
[0352] Based on the results of the screening assay, seven .S'«Cas9 gRNAs that introduced greater than 40% indels and targeted the 5’UTR or exon 1 of the MYOC gene were selected for further assessment (Cas9-1, Cas9-5, Cas9-6, Cas9-24, Cas9-56, Cas9-57, Cas9-59 (Table 9)). Briefly, primary T cells were nucleofected with increasing concentrations (0.0891 pM,
0.178 pM, 0.356 pM, 0.7125 pM, 1.425 pM, 2.85 pM, 5.7 pM, and 11.4 pM) of RNP comprising an .ShCas9 gRNA complexed with wildtype 5aCas9 enzyme in a 2: 1 ratio. The genomic DNA was extracted four days post nucleofection and NGS was performed (see Table 15 for NGS primers used) on the MYOC PCR products to assess the total percentage of indels (Fig. 2A) and the percentage of frameshift indels (Fig. 2B).
Table 9: S'. aureus Cas9 Guide RNAs
Example 2: Screening of Acidaminococcus sp. Casl2a guide RNAs targeting the MYOC gene in T cells
[0353] Acidaminococcus sp. Casl2a (AsCasl2a) (also known as AvC I'l ) gRNAs were designed to target the MYOC gene (see Table 10 for the sequences of Acidaminococcus sp. Cast 2a gRNA targeting domains) to identify RNPs that introduce frameshift indels likely to disrupt expression of functional MYOC. RNPs comprising AvCas 12a gRNAs (Table 10) complexed with an AvCas 12a (AsCasl2a polypeptide sequence is set forth in SEQ ID NO:33) in a 2:1 ratio (guide RNA/Casl2a) were screened in a T cell assay to analyze the RNP gene editing.
[0354] Briefly, primary T cells were nucleofected with the RNPs at 8 pM. The genomic DNA was extracted four days post electroporation and NGS (see Table 16 for NGS primers used) was performed on the MYOC PCR products. The percentage of frameshift indels relative to the total percentage of indels that were introduced into the MYOC gene was analyzed (Fig. 3). Results indicated that RNPs comprising A.vCasl2a guide RNAs introduced indels in a high percentage of the cells (Fig. 3). In some cases, a large proportion of the indels were frameshift indels likely to disrupt the expression of functional MYOC protein.
[0355] Based on the results of the screening assay, six AvCas 12a gRNAs that introduced greater than 80% indels and targeted the 5’UTR or exon 1 of the MYOC gene were selected for further assessment (Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, Casl2a-8 (Table 10)). Briefly, primary T cells were nucleofected with increasing concentrations (0.010 pM, 0.030 pM, 0.080 pM, 0.25 pM, 0.80 pM, 2.53 pM, and 8.0 pM) of RNPs. The genomic DNA was extracted four days post nucleofection and NGS (see Table 16 for NGS primers used) was performed on the MYOC PCR products to assess the total percentage of indels (Fig. 4A) and percentage of frameshift indels (Fig. 4B).
Table 10: Acidaminococcus sp. Casl2a Guide RNAs
Example 3: Acidaminococcus sp. Casl2a guide RNAs targeting the MYOC gene provide an increase in gene editing in human trabecular meshwork cells
[0356] The rate of editing of .S'«Cas9 and A .vCas 12a RNPs targeting the MYOC gene was assessed in human trabecular meshwork cells (HTMCs). Briefly, HTMCs were electroporated with increasing concentrations of RNPs comprising an .S'aCas9 gRNA (Cas9-1 , Cas9-5, Cas9-6, Cas9-24 (Table 9)) complexed with S«Cas9 CS«Cas9 polypeptide sequence is set forth in SEQ ID NO:47) or an sCas l 2a gRNA (Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, or Casl2a-8 (Table 10)) complexed with AsCas 12a (A Casl2a polypeptide sequence is set forth in SEQ ID NO:33). The genomic DNA was extracted four days post nucleofection and NGS (see Tables 15 and 16 for NGS primers used) was performed on the MYOC PCR products to assess the percentage of total indels (Fig. 5A) and the percentage of frameshift indels (Fig. 5B). Results indicated that editing of the MYOC gene with AvCas 12a RNPs provided a significantly higher indel rate with a high percentage of frameshift indels relative to editing with S«Cas9 RNPs (compare Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8 (Table 10) with Cas9-1, Cas9-5, Cas9-6, Cas9- 24 (Table 9) in Figs. 5A and 5B).
Example 4: Promoters for driving expression in human trabecular meshwork cells
[0357] Various putative promoters were tested to identify promoters capable of driving expression in HTMCs. Based on single cell RNAseq data (Van Zyl 2020), genes that are strongly expressed in human trabecular meshwork cell populations were identified. The UCSC Browser and Eukaryotic Promoter Database were used to select the potential promoter and enhancer sequences that were then synthesized and cloned into plasmids. Briefly, HTMCs were nucleofected with 4.7E+10 copies (-200 ng/grl J of plasmid containing a putative promoter driving expression of enhanced green fluorescent protein (GFP or EGFP). The promoters tested were DCN #1, DCN #2, ID3, MT2A, SERPINF1, SERPING1 #1, SERPING1 #2, TIMP1, CP, MT IX, OLFML3, PCOLCE, PDGFRA, PTN, RARRE SI, RBP4 #1, RBP4 #2, RBP4 #3, PDPN, APOD, B2M, PTGDS, EEF1A1, ANGPTL7, MGP, RPS24, PLA2G2A, CHI3L1, FTL, TAGLN, PCP4, MYL 9, MYOC, CMV, and mini-CMV (see Table 6 for the sequences of the promoters). The following day cells were subjected to flow cytometry to determine the percentage of GFP+ cells and mean fluorescence intensity of GFP+ cells (Figs. 6A and 6B, respectively).
Example 5: Screening ionizable lipids for LNP delivery
[0358] To investigate the transfection efficiency of lipid nanoparticles (LNPs) formed with different ionizable lipids, LNPs were formulated with one of five different ionizable lipids, the structures of which are shown in Table 11 below: (1) MC3 (also called Dlin-MC3- DMA), (2) SM-102, (3) ALC-0315, (4) 5A2-SC8, and (5) BAMEA-O16B. LNPs (MC3, SM- 102, ALC-0315, BAMEA-O16B) were formed with 50% ionizable lipid, 38.5% cholesterol, 10% distearoylphosphatidylcholine (DSPC), and 1.5% dimethylglycine (DMG)-PEG2k and encapsulated GFP messenger RNA (mRNA) and irrelevant gRNA. 5A2-SC8 LNP was formed with 25% ionizable lipid, 48.5% cholesterol, 25% DSPC, and 1.5% DMG-PEG2k and encapsulated GFP mRNA and gRNA.
Table 11. Ionizable Lipids Used in LNPs
[0359] Briefly, LNPs were formulated with an amine-to-RNA-phosphate (N:P) ratio of 4-7. Unless otherwise specified, the N:P ratio = 6.88 was used. The lipid nanoparticle components (ionizable lipid, cholesterol, DSPC, and DMG-PEG2k) were dissolved in 100% ethanol and mixed in the indicated molar ratios. The RNA cargo, which included GFP mRNA and irrelevant gRNA (1:1 weight ratio (100% GFP mRNA:gRNA)) was dissolved in 50 mM citrate buffer (pH 4.5), resulting in a concentration of RNA cargo of approximately 0.12 mg/mL. LNPs were formed by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems Ignite or Spark Instrument, in accordance with the manufacturer’ s protocol.
[0360] After mixing, the LNPs were collected and ethanol was removed by one of the following two methods: 1) LNPs were diluted in PBS or TBS (1:40, vokvol) and loaded into Amicon™ Centrifugal Filter Units for ultrafiltration (MilliporeSigma, 30kD); or 2) LNPs were loaded into 10 kDa Slide-a-Lyzer G2 Dialysis Cassettes (ThermoFisher Scientific) for dialysis in TBS or PBS under gentle stirring (1 hour in room temperature and new buffer change, 3 hours in 4 °C and new buffer change, and overnight at 4 °C). The resultant mixture was concentrated to the target concentrations and then filtered using a 0.2-mm sterile filter. The filtrate was stored at 2 °C-8 °C for use within a week or stored at -80 °C for a longer time after addition of 10% sucrose.
[0361] To investigate LNP delivery to the eye, the various LNPs encapsulating GFP mRNA and gRNA were administered via intracameral injection to mice. Briefly, LNPs (50% ionizable lipid, 38.5% cholesterol, 10% DSPC, and 1.5% DMG-PEG2k) encapsulating GFP mRNA and gRNA were formulated with different ionizable lipids (i.e., MC3, SM-102, ALC- 0315, 5A2-SC8, BAMEA-O16B). Wild-type C57BL/6J mice (aged 6-8 weeks) were dosed with 500 pg/mL LNP-GFP mRNA (i.e., GFP mRNA encapsulated in LNPs) of each LNP formulation (5 mice per group for each LNP) via intracameral (i.e.) injections (1 p L in left eye only) and euthanized 24 hours later. Because the GFP mRNA and gRNA are at a 1 : 1 ratio by weight in the LNPs, if the mRNA concentration is 500 pg/mL, the gRNA concentration is also 500 pg/mL. Whole eyes (left only) were collected from the study animals and fixed in 4% paraformaldehyde (PF A) for 24 hours, then embedded in paraffin blocks. The paraffin blocks were sectioned (5 m thick) and 2 sections/eye were stained for GFP detection (primary antibody :rabbit monoclonal anti-GFP antibody (abl83734, Abeam); detection: BOND Polymer Refine Detection kit (Leica)). Sections were imaged on the P250 Scanner (3DHistech) and reviewed on the SlideViewer software (3DHistech). Images were qualitatively analyzed based on the strength and localization of GFP expression.
[0362] Histology data are shown in Figs. 7A (PBS (Vehicle)), 7B (LNPs formed with MC3), 7C (LNPs formed with SM-102), 7D (LNPs formed with ALC-0315), 7E (LNPs formed with 5A2-SC8), 7F (LNPs formed with BAMEA-O16B), 7G (LNPs formed with BAMEA- O16B), and 7H (LNPs formed with SM-102). Results showed that LNPs formed with MC3, SM-102, ALC-0315, and 5A2-SC8 ionizable lipids successfully mediated delivery of GFP mRNA to the trabecular meshwork (TM). MC3, ALC-0315, and 5A2-SC8 showed the highest specificity for the TM tissue, while SM-102 transfected multiple tissues within the anterior chamber.
Example 6: LNP ex vivo delivery to human and non-human primate corneal rims
[0363] To investigate LNP ex vivo delivery to the eye, various LNPs encapsulating GFP mRNA and irrelevant gRNA were administered to human and non-human primate (NHP) corneal rims. Briefly, LNPs were formulated as described in Example 5 with either 50% MC3 or 50% ALC-0315 ionizable lipids (Table 11), 38.5% cholesterol, 10% DSPC, and 1.5% DMG-PEG2k and encapsulated GFP mRNA and irrelevant gRNA using the Ignite instrument.
[0364] Human and NHP (cynomolgus monkey) comeal rims were dissected to create wedges and treated with 10 pL of LNPs (50% ionizable lipid, 38.5% cholesterol, 10% DSPC, and 1.5% DMG-PEG2k) encapsulating GFP mRNA and gRNA at a concentration of 0.5 mg/mL or 0.05 mg/mL LNP-GFP mRNA (i.e., GFP mRNA encapsulated in LNPs). Five days after treatment the wedges were imaged using confocal imaging (CQ1 confocal microscope) and the percent GFP expression in the TM region was calculated using Imaris software. Figs. 8A and 8B show that LNPs formed with ALC-0315 or MC3, respectively, efficiently transfected human and NHP trabecular mesh work region at 0.5 mg/mL and 0.05 mg/mL LNP-GFP mRNA. Example 7 : Investigation of varying concentrations of PEG on LNP delivery to murine eyes
[0365] Next, to test different percentages of DMG-PEG2k and DSPE-PEG2k used in LNPs, LNPs were formulated as described in Example 5 using the Ignite instrument with two different PEG lipids at different percentages: DMG-PEG2k (1%, 1.5%, 2%, 3%) or DSPE- PEG2k (1%, 1.5%, 2%, 3%), each encapsulating GFP mRNA and irrelevant gRNA (50% ALC-0315, 10% DSPC, 39%, 38.5%, 38%, or 37% cholesterol (adjusted to the respective PEG ratio)).
[0366] To investigate LNP delivery to the eye, the various LNPs encapsulating GFP mRNA and gRNA were administered via intracameral injection to mice. Briefly, wild-type C57BL/6J mice (aged 6-8 weeks) were dosed with 500 pg/mL of LNP-GFP mRNA (5 mice per group for each LNP) via intracameral (i.c.) injections (1 L in left eye only) and euthanized 24 hours later. Whole eyes (left only) were collected from the study animals and fixed in 4% PFA for 24 hours, then embedded in paraffin blocks. The paraffin blocks were sectioned (5 pm thick) and 2 sections/eye were stained for GFP detection (primary antibody: rabbit monoclonal anti-GFP antibody (abl 83734, Abeam); detection: BOND Polymer Refine Detection kit (Leica)). Sections were imaged on the P250 Scanner (3DHistech) and reviewed on the SlideViewer software (3DHistech). Images were qualitatively analyzed and scored based on the strength and localization of GFP expression.
[0367] Fig. 9A shows the GFP expression using LNPs formed with different PEG lipids and ratios (DMG-PEG2k (1%, 1.5%, 2%, 3%) and DSPE-PEG2k (1%, 1.5%, 2%, 3%)). Figs. 9B and 9C show histology data from transfection of the mouse TM in vivo with Vehicle (PBS) or 1.5% DMG-PEG, respectively.
Example 8: LNP delivery to primary human trabecular mesh work cells and HEK293 T cells
[0368] Next, to test the effect of different percentages of ALC-0315, cholesterol, and DSPC on LNP delivery, LNPs encapsulating GFP mRNA and irrelevant gRNA formed with different percentages of ALC-0315, cholesterol, and DSPC with a fixed amount of 1.5% DMG-PEG2k were screened for transfection of primary human TM cells and HEK293T cells. Table 12 shows the percent transfection of primary human TM cells and HEK293T cells, along with the biophysical properties of LNPs formulated with different ratios of ALC- 0315, cholesterol, and DSPC with a fixed 1.5% DMG-PEG2K (i.e., percentage of encapsulation of GFP-mRNA (Encap. (%)), particle size in nanometers (nm) (Z Ave. (nm)), and polydispersity index). The LNPs listed in Table 12 were formulated as described in Example 5. LNPs were formed by microfluidic mixing of the lipid and RNA solutions using the Spark Instrument from Precision NanoSystems, in accordance with the manufacturer’s protocol. Encapsulation efficiencies were determined by RiboGreen assay. Particle size and polydispersity were measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument. Cells were treated with LNPs with either 0.05 pg/ml LNP-GFP mRNA (i.e., GFP mRNA encapsulated in LNPs) for TM cells or 0.005 pg/ml LNP-GFP mRNA for HEK293T cells. 24 hours later the percent GFP positive cells were determined by flow cytometry. Table 12. Biophysical Properties of LNPs and Transfection and Encapsulation
[0369] Based on the initial lipid ratio screening (Table 12), seven LNPs were selected (LNP1, LNP2, LNP5, LNP8, LNP14, LNP17, LNP19) for in vitro confirmation and comparison to the LNP lipid ratio (LNP15) tested in Example 6. TM cells and HEK293T cells were treated with LNPs of increasing concentrations (TM cells: 4.6 x 1 O’8 mg/mL, 1.4 x 10‘7 mg/mL, 4.1 x 10'7 mg/mL, 1.2 x 10‘6 mg/mL, 3.7 x 10’6 mg/mL, 1.1 x 10 s mg/mL, 3.3 x 10"5 mg/mL, 1.0 x 10"4 mg/mL; HEK293T cells: 5.0 x 10"9 mg/mL, 1.5 x 10"8 mg/mL, 4.5 x 10"8 mg/mL, 1.4 x 10"7 mg/mL, 4.1 x 10'7 mg/mL, 1.2 x 10"6 mg/mL, 3.7 x 10'6 mg/mL, 1.0 x 10"5 mg/mL) of LNP-GFP mRNA for 24 hours followed by flow cytometry to determine the percent GFP positive cells. Figs. 10A and 10B show concentration dependent transfection of primary human TM cells and HEK293T cells, respectively, with LNPs (LNP1, LNP2, LNP5, LNP8, LNP14, LNP15, LNP17, LNP19 (Table 12)) with varying lipid ratios (ALC-0315, cholesterol, DSPC, and DMG-PEG2k).
Example 9: In vivo screening of LNPs for delivery to murine eyes
[0370] Next, in vivo screening of LNP1, LNP8, LNP14, LNP15, and LNP19 (Table 12) for transfection of the mouse trabecular meshwork was performed. LNPs encapsulating GFP mRNA and irrelevant gRNA were generated as described in Example 5 and Example 8. Wild-type C57BL/6J mice (aged 6-8 weeks) were dosed LNPs with 500 pg/mL of LNP-GFP mRNA (i.e., GFP mRNA encapsulated in LNPs) (5 mice per group for each LNP) via intracameral (i.c.) injections (1 pL in left eye only) and euthanized 24 hours later. Whole eyes (left only) were collected from the study animals and fixed in 4% PFA for 24 hours, then embedded in paraffin blocks. The paraffin blocks were sectioned (5 pm thick) and 2 sections/eye were stained for GFP detection (primary antibody: rabbit monoclonal anti-GFP antibody (abl83734, Abeam); detection: BOND Polymer Refine Detection kit (Leica)). Sections were imaged on the P250 Scanner (3DHistech) and reviewed on the SlideViewer software (3DHistech). Images were qualitatively analyzed based on the strength and localization of GFP expression. Figs. 11A (Vehicle, PBS), 11B (LNP1), 11C (LNP8), 11D (LNP14), HE (LNP15), and HF (LNP19) show that LNP1, LNP8, LNP14, LNP15, and LNP19 successfully mediated delivery of GFP mRNA to the TM. Example 10: LNP delivered AsCasl2a mRNA and gRNA mediated ex-vivo editing of MYOC gene in human corneal rims
[0371] To test ex vivo editing of the TM in a human model, LNPs (50% MC3, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k) were formulated as described in Example 5 using the Ignite Instrument to encapsulate mRNA expressing an AsCasl2a enzyme (SEQ ID NO:33) plus Casl2a-8 gRNA (Table 10). The RNA cargo concentration of AsCasl2a mRNA and gRNA are at a 1:1 ratio by weight in the LNPs. Human donor corneal rims were dissected to create wedges and were treated with LNPs with 0.5 mg/ml or 0.05 mg/ml LNP- AsCasl2a mRNA (i.e., AsCasl2a mRNA encapsulated in LNPs). Six days after treatment the trabecular meshwork was dissected and the percentage of indels in the MYOC gene was determined using IlLseq (see Table 16 for NGS primers used). Fig. 12 shows results of editing using LNP encapsulating AsCasl2a mRNA and Casl2a-8 gRNA.
Example 11: LNP delivered AsCasl2a mRNA and gRNA mediated in vivo editing of MYOC gene in non-human primate trabecular meshwork tissue
[0372] To test in vivo editing efficiency in Cynomolgus monkeys (Macacafascicularis), LNPs (50% ALC-0315, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k) encapsulating mRNA encoding AsCasl2a (SEQ ID NO:33) plus Casl2a-1 gRNA or Casl2a-2 gRNA (Table 10) were formulated as described in Example 5 using the Ignite instrument. The RNA cargo concentration of AsCasl2a mRNA and gRNA are at a 1:1 ratio by weight in the LNPs. Cynomolgus monkeys received intracameral injections of LNPs in both eyes (7.5 pg AsCasl2a mRNA/eye) and were euthanized one week later (Table 13).
Table 13. Ocular Dosing of Non-Human Primates
[0373] The eyes were collected and trabecular meshwork collected. The percentage of indels in the MYOC gene was determined using IlLSeq (see Table 16 for NGS primers used). Fig. 13 shows the percent genomic DNA editing observed in the non-human primate (NHP) TM tissue.
Example 12: LNP delivered AsCasl2a mRNA and gRNA mediated editing of MYOC gene in human primary trabecular meshwork cells
[0374] To test editing efficiency in primary human TM cells, LNPs (40% ALC-0315, 46% cholesterol, 12.5% DSPC, and 1.5% DMG-PEG2K) encapsulating mRNA encoding AsCasl2a (SEQ ID NO:33) plus Casl2a-1 gRNA (Table 10) were formulated as described in Example 5 using the Ignite instrument. The RNA cargo concentration of AsCasl2a mRNA and gRNA are at a 1 : 1 ratio by weight in the LNPs. Fig. 14 shows data from primary human TM cells treated with LNPs with increasing concentrations (1.4 x 10'6 mg/mL, 4.1 x 10"6 mg/mL, 1.2 x 105 mg/mL, 3.7 x 10 5 mg/mL, 1.1 x 10^ mg/mL, 3.3 x 104 mg/mL, 1.0 x 10 3 mg/mL) of LNP-AsCasl2a mRNA. Three days after treatment, gDNA was isolated and the resulting percentage of indels introduced into the MYOC gene determined by IlLSeq (see Table 16 for NGS primers used).
Example 13: LNP delivered AsCasl2a mRNA and gRNA mediated in vivo editing of MYOC gene in murine trabecular meshwork cells from a humanized mouse model for myocilin associated glaucoma
[0375] To test in vivo editing efficiency in mouse TM tissue from a humanized mouse model for myocilin associated glaucoma, LNPs (40% ALC-0315, 46% cholesterol, 12.5% DSPC, and 1.5% DMG-PEG2K) encapsulating mRNA encoding AsCasl2a (SEQ ID NO:33) plus Casl2a-1 gRNA (Table 10) were formulated as described in Example 5 using the Ignite instrument. A humanized mouse model homozygous for the human MYOC gene with the Y437H mutation at the MYOC locus (MYOCY437H'V437H ), called the MY01-H0M mouse model, was developed. Briefly, the mouse Myoc gene (jnMyoc) was replaced with the human MYOC gene carrying the pathogenic Y437H mutation (hMYOCY437H) (Fig. 15A). The knock- in gene includes the full-length human mutant MYOCY437H gene in addition to 1.5 kb upstream, which contains a portion of the human promoter (Fig. 15A). Intraocular pressure (IOP) was measured monthly on unanesthetized mice using a tonometer. The MYO 1 -HOM mouse model (MYOCY437H7Y437H') presents with elevated IOP levels compared to wild-type control mice (Fig. 15B). The mouse TM tissue is too small to dissect, therefore anterior chambers were collected followed by extraction of mRNA and gDNA. The anterior chamber has a mixed cell population that includes the TM tissue, cornea, ciliary body, and other ancillary tissues. Fig. 15B shows the percentage of myocilin mRNA remaining as determined by RT-ddPCR. Fig. 15C shows the percentage of indels introduced into the MYOC gene determined by Ill-Seq (see Table 16 for NGS primers used).
Example 14: Non-human primate study to determine the dose dependency and ocular tolerability of editing using LNP delivery
[0376] Dose dependency and ocular tolerability using LNP delivery was assessed in a non- human primate (NHP) model. LNPs (40% ALC0315, 46% cholesterol, 12.5% DSPC and 1.5% DMG-PEG2K) encapsulating mRNA encoding AsCasl2a (SEQ ID NO:33) plus Casl2a-1 gRNA (Table 10) were formulated as described in Example 5 in TBS and 10% sucrose. The RNA cargo concentration of AsCasl2a mRNA and gRNA are at a 1:1 ratio by weight in the LNPs. Cynomolgus monkeys received intracameral injections of LNPs at increasing concentrations of AsCasl2a mRNA (100 pg/mL, 300 pg/mL, or 500 pg/mL mRNA). NHP were given 40 mg methylprednisolone intramuscularly (IM) at day -1 (i.e., 1 day prior to dosing) and were euthanized after 4 weeks and editing was assessed in the dissected TM tissue. An assessment of inflammation was performed including an ophthalmologic examination on Day 2 post-treatment, which showed that most of the animals had dose-dependent mild to moderate inflammation that resolved with daily topical Durezol treatment for a week. In addition, an endpoint histopathology showed dose-dependent minimal to mild mononuclear infiltrates that were not considered adverse. Fig. 16A shows the percentage of indels generated by editing at the DNA level. Fig. 16B shows the percentage of myocilin mRNA remaining. Editing showed dose-dependent trends in the NHP TM tissues.
Example 15: Concentration dependent editing of myocilin and reduction of endoplasmic reticulum stress in in vitro phenotypic assay
[0377] Concentration dependent editing of myocilin and reduction of endoplasmic reticulum stress (ER) stress were assessed using an in vitro assay in HEK293T cells stably expressing wildtype myocilin and Y437H mutant myocilin. Normally myocilin is secreted from the cell but mutations in myocilin prevent secretion resulting in a build-up of the mutant protein inside the cell and subsequent increase in ER stress (i.e., expression of GRP78 protein). As shown in Fig. 17A, HEK293T cells stably expressed either wildtype or Y437H mutant myocilin. The Y437H mutation is one of the more aggressive mutations and is associated with early onset of primary open angle glaucoma. LNPs (40% ALC0315, 46% cholesterol, 12.5% DSPC and 1.5% DMG-PEG2K) encapsulating mRNA encoding AsCasl2a (SEQ ID NO:33) plus Casl2a-1 gRNA (Table 10) were formulated as described in Example 14 in TBS and 10% sucrose. The RNA cargo concentration of AsCasl2a mRNA and gRNA are at a 1:1 ratio by weight in the LNPs. HEK293T cells were treated with LNPs with increasing concentrations (2.3 x IO’6, 6.9 x IO’6, 2.1 x 105, 6.2 x 10 5, 1.9 x 104, 5.6 x IO 4, 1.7 x IO’3, 5.0 x 10'3 mg/mL) of LNP-AsCasl2a mRNA. Three days after treatment, gDNA, mRNA, and protein were extracted for downstream analysis. Results show the percentage of indels generated by editing MYOC at the DNA level by Ill-Seq (Fig. 17B), myocilin mRNA expression normalized to the housekeeping gene GAPDH as determined by RT-ddPCR (Fig. 17C), the expression of myocilin protein (intracellular) normalized to GAPDH as determined by Jess Automated Western Blot (Fig. 17D), and the expression of GRP78 protein normalized to GAPDH as determined by Jess Automated Western blot (Fig. 17E).
Example 16: LNP delivery of AsCasl2a mRNA and gRNA demonstrates editing of human MYOC and reduction of IQP in the MYO 1 -HOM mouse model
[0378] LNP delivery of AsCasl2a mRNA and gRNA and editing was assessed in a MY01- HOM mouse model. LNPs (40% ALC0315, 46% cholesterol, 12.5% DSPC and 1.5% DMG- PEG2K) encapsulating mRNA encoding AsCasl2a plus Casl2a-1 gRNA (Table 10) were formulated using a Knauer IJM Single-Core system. The RNA cargo concentration of AsCasl2a mRNA and gRNA are at a 1: 1 ratio by weight in the LNPs. The mRNA sequence encoded the AsCasl2a set forth in SEQ ID NO:48 with the addition of two C-terminal nuclear localization signals (NLS). Similar to the method described in Example 5, lipid mixture of 40% ALC0315, 46% cholesterol, 12.5% DSPC and 1.5% DMG-PEG2K in ethanol and the AsCasl2a mRNA and gRNA solution in 50 mM citrate buffer (pH 4.5) were prepared separately before formulation. Lipid mixture, AsCasl2a mRNA and gRNA solution, and inline dilution buffer (1 x TBS, pH 7.4) were mixed in a 1:3:2 ratio (v/v/v) through Knauer’s Impingement Jets Mixing (UM-2) mixer with a total flow speed of 15 mL/min. The formulation coming out of the Knauer system was subjected to the ethanol removal and purification process as described in Example 5. The final LNP product was aliquoted and stored in TBS with 10% sucrose at -80 °C before using. MY01-H0M mice were dosed with 1 pL LNPs at various concentrations of LNP-AsCasl2a mRNA (25 pg/mL, 100 pg/mL, or 500 pg/mL). IOP was measured prior to dosing and at weeks 4 and 6. Mice were euthanized at week 6 and editing was assessed. Fig. 18A shows the IOP pre-dosing and 4 and 6 weeks after dosing. Fig. 18B shows the percentage of indels generated by editing at the DNA level.
Fig. 18C shows the percentage of myocilin mRNA remaining.
[0379] LNPs used in the in vivo study were also assessed for editing in primary TM cells in vitro to confirm activity. Briefly, TM cells were treated with LNPs with increasing concentrations (1.4 x 10'6 mg/mL, 4.1 x 10"6 mg/mL, 1.2 x 10’5 mg/mL, 3.7 x 10'5 mg/mL, 1.1 x 10’4 mg/mL, 3.3 x 10’4 mg/mL, 1.0 x 10'3 mg/mL) of LNP-AsCasl2a mRNA that had been diluted for injection into the MYO 1 -HOM mice. Three days after treatment, gDNA was isolated and the resulting percentage of indels introduced into the MYOC gene determined by IlLSeq. Fig. 18D shows the percentage of indels in the MYOC gene in vitro in primary TM cells with LNPs with various concentrations of LNP-AsCasl2a mRNA.
SEQUENCES
Additional Sequences
[0380] Primers used for next-generation sequencing (NGS) to analyze editing by Cas9 and Casl2a RNP are set forth in Table 15 and Table 16, respectively. Table 14: Casl2a Polypeptide, Nucleotide, and mRNA Sequences
Table 15: S. aureus Cas9 Next- Generation Sequencing Primers
Table 16: Casl2a RNP Next-Generation Sequencing Primers
INCORPORATION BY REFERENCE
[0381] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
EQUIVALENTS
[0382] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.
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Claims

Other embodiments are within the following claims. What is claimed is:
1. A guide RNA (gRNA) molecule comprising a targeting domain that binds a target domain of a MYOC gene.
2. The gRNA molecule of claim 1, wherein the targeting domain is complementary to the target domain of the MYOC gene.
3. The gRNA molecule of claim 1 or 2, wherein the targeting domain is configured to, in combination with an RNA-guided nuclease, provide a cleavage event selected from a double strand break and a single strand break, within 500, 400, 300, 200, 100, 50, 25, or 10 nucleotides of a MYOC target position.
4. The gRNA molecule of any one of claims 1-3, wherein the MYOC target position is in a region selected from the group consisting of a 5’ untranslated region (“UTR”), exon 1, an exon 1/intron 1 border, intron 1, an intron 1/exon 2 border, exon 2, an exon 2/intron 2 border, intron 2, an intron 2/exon 3 border, exon 3, an exon 3/intron 3 border, and 3’ UTR of the MYOC gene.
5. The gRNA molecule of any one of claims 1-4, wherein the targeting domain comprises a sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence from Table 10.
6. The gRNA molecule of any one of claims 1-5, wherein the targeting domain comprises a sequence that is the same as a targeting domain sequence from Table 10.
7. The gRNA molecule of any one of claims 1-5, wherein the targeting domain comprises a sequence selected from the group consisting of a targeting domain sequence in Table 10.
8. The gRNA molecule of any one of claims 1-7, wherein the targeting domain comprises a sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain selected from the group consisting of a Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8 targeting domain (Table 10).
9. The gRNA molecule of any one of claims 1-8, wherein the targeting domain comprises a sequence selected from the group consisting of a Casl2a-1, Casl2a-2, Casl2a-3, Casl2a-4, Casl2a-5, and Casl2a-8 targeting domain (Table 10).
10. The gRNA molecule of any one of claims 1-9, wherein the targeting domain is a length selected from the group consisting of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26 nucleotides or more in length.
11. The gRNA molecule of any one of claims 1-10, comprising a targeting domain and a crRNA direct repeat extension.
12. The gRNA molecule of claim 11, wherein the crRNA direct repeat extension comprises a sequence that is the same as, or differs by no more than 3, 4, 5, or 6 nucleotides from, a crRNA direct repeat extension sequence of UAAUUUCUACUCUUGUAGAU (SEQ ID NO:49).
13. The gRNA molecule of claim 10 or claim 11, wherein the crRNA direct repeat extension sequence is UAAUUUCUACUCUUGUAGAU (SEQ ID NO:49).
14. The gRNA molecule of any one of claims 1-13, wherein the gRNA molecule is a modular gRNA molecule or a chimeric gRNA molecule.
15. The gRNA molecule of claim 11, wherein the gRNA molecule comprises a sequence that is the same as, or differs by no more than 3 nucleotides from, a crRNA direct repeat extension sequence of UAAUUUCUACUCUUGUAGAU (SEQ ID NO:49).
16. A nucleic acid that comprises: (a) a sequence that encodes the gRNA molecule of any one of claims 1-15.
17. The nucleic acid of claim 16, further comprising: (b) a sequence that encodes an RNA-guided nuclease molecule.
18. The nucleic acid of claim 16 or 17, wherein the RNA-guided nuclease molecule is a Casl2a molecule.
19. The nucleic acid of claim 18, wherein the Casl2a molecule comprises any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Casl2a polypeptide sequences).
20. The nucleic acid of claim 18, wherein the Casl2a molecule is encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Casl2a polynucleotide sequences).
21. The nucleic acid of any one of claims 18-20, wherein the nucleic acid comprises a promoter operably linked to the sequence that encodes the Casl2a molecule of (b).
22. The nucleic acid of claim 21, wherein the promoter operably linked to the sequence that encodes the Casl2a molecule of (b) comprises a sequence selected from the group consisting of the sequences set forth in Table 6 (DCN #1, DCN #2, ID3, MT2A, SERPINF1, SERPING1 #1, SERPING1 #2, TIMP1, CP, MT1X, OLFML3, PCOLCE, PDGFRA, PTN, RARRE SI, RBP4 #1, RBP4 #2, RBP4 #3, PDPN, APOD, B2M, PTGDS, EEF1A1, ANGPTL7, MGP, RPS24, PLA2G2A, CHI3L1, FTL, TAGLN, PCP4, MYL 9, MYOC, CMV, and mini-CMV promoter).
23. The nucleic acid of any one of claims 16-22, further comprising: (c) a sequence that encodes a second gRNA molecule having a targeting domain that is complementary to a second target domain of the MYOC gene.
24. The nucleic acid of claim 23, wherein the second gRNA molecule is a gRNA molecule of any one of claims 1-15.
25. The nucleic acid of claim 23 or 24, further comprising a third gRNA molecule.
26. The nucleic acid of claim 25, further comprising a fourth gRNA molecule.
27. The nucleic acid of any one of claims 17-26, wherein each of (a) and (b) is present on the same nucleic acid molecule.
28. The nucleic acid any one of claims 16-27, wherein the nucleic acid molecule is an AAV vector.
29. The nucleic acid of any one of claims 17-26, wherein: (a) is present on a first nucleic acid molecule; and (b) is present on a second nucleic acid molecule.
30. The nucleic acid of claim 29, wherein the first and second nucleic acid molecules are AAV vectors.
31. The nucleic acid of claim 29, wherein the first nucleic acid molecule is other than an AAV vector and the second nucleic acid molecule is an AAV vector.
32. The nucleic acid of any one of claims 16-31, wherein the nucleic acid comprises a promoter operably linked to the sequence that encodes the gRNA molecule of (a).
33. A lipid nanoparticle (LNP) comprising one or more ionizable lipids and encapsulating the gRNA molecule of any one of claims 1-15.
34. The LNP of claim 33, wherein the one or more ionizable lipids is selected from the group consisting of MC3, SM-102, ALC-0315, 5A2-SC8, and BAMEA-O16B.
35. The LNP of claims 33 or 34, further comprising cholesterol.
36. The LNP of any one of claims 33-35, further comprising distearoylphosphatidylcholine (DSPC).
37. The LNP of any one of claims 33-36, further comprising a PEG-lipid.
38. The LNP of claim 37, wherein the PEG lipid is dimethylglycine (DMG)-PEG2k.
39. The LNP of any one of claims 33-38, wherein the LNP encapsulates a nucleic acid encoding an RNA-guided nuclease.
40. The LNP of claim 39, wherein the nucleic acid encoding the RNA-guided nuclease is messenger RNA (mRNA).
41. The LNP of claim 39 or 40, wherein the RNA-guided nuclease comprises any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Casl2a polypeptide sequences) or is encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Cast 2a polynucleotide sequences).
42. A composition comprising the (a) gRNA molecule of any one of claims 1-15.
43. The composition of claim 42, further comprising (b) an RNA-guided nuclease molecule or a nucleic acid encoding the RNA-guided nuclease molecule.
44. The composition of claim 43, wherein the (b) RNA-guided nuclease molecule is a Casl2a molecule.
45. The composition of claim 44, wherein the Casl2a molecule comprises any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Casl2a polypeptide sequences).
46. The composition of claim 44, wherein the Casl2a molecule is encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Casl2a polynucleotide sequences).
47. The composition of any one of claims 42-46, further comprising (c) a second gRNA molecule of any one of claims 1-15.
48. The composition of claim 48, further comprising a third gRNA molecule.
49. The composition of claim 49, further comprising a fourth gRNA molecule.
50. The composition of any one of claims 42-49, wherein the gRNA molecule is encapsulated in an LNP of any one of claims 33-41.
51. The composition of any one of claims 43-50, wherein the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease is encapsulated in the LNP of any one of claims 33-41.
52. The composition of claim 51, wherein the nucleic acid encoding the RNA-guided nuclease is messenger RNA (mRNA).
53. A method of altering a cell comprising contacting the cell with:
(a) a gRNA molecule of any one of claims 1-15;
(b) an RNA-guided nuclease molecule or a nucleic acid encoding the RNA-guided nuclease molecule; and optionally, (c) a second gRNA molecule of any one of claims 1-15.
54. The method of claim 53, wherein the RNA-guided nuclease molecule is a Casl2a molecule.
55. The method of claim 54, wherein the Casl2a molecule comprises any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Casl2a polypeptide sequences).
56. The method of claim 54, wherein the Casl2a molecule is encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Casl2a polynucleotide sequences).
57. The method of any one of claims 53-56, further comprising contacting the cell with a third gRNA molecule.
58. The method of claim 57, further comprising contacting the cell with a fourth gRNA molecule.
59. The method of any one of claims 53-58, comprising contacting the cell with (a), (b), and (c).
60. The method of any one of claims 53-59, wherein the cell is from a subject suffering from POAG.
61. The method of any one of claims 53-60, wherein the cell is an ocular cell.
62. The method of any one of claims 53-60, wherein the cell is a trabecular meshwork cell.
63. The method of any one of claims 53-62, wherein the contacting step is performed in vivo.
64. The method of any one of claims 53-63, wherein the contacting step comprises contacting the cell with a nucleic acid that encodes at least one of (a), (b), and optionally (c).
65. The method of any one of claims 53-64, wherein the contacting step comprises contacting the cell with a nucleic acid of any one of claims 16-32.
66. The method of any one of claims 53-64, wherein the contacting step comprises delivering to the cell the RNA-guided nuclease molecule of (b) a nucleic acid which encodes the gRNA molecule of (a), and optionally a nucleic acid which encodes the second gRNA molecule of (c).
67. The method of any one of claims 53-64, wherein the contacting step comprises delivering to the cell the RNA-guided nuclease molecule of (b), the gRNA molecule of (a) and optionally the second gRNA molecule of (c).
68. The method of any one of claims 53-64, wherein the contacting step comprises delivering to the cell a nucleic acid that encodes the RNA-guided nuclease molecule of (b), the gRNA molecule of (a), and optionally the second gRNA molecule of (c).
69. The method of any one of claims 53-68, wherein the gRNA molecule is encapsulated in an LNP of any one of claims 33-41.
70. The method of any one of claims 53-69, wherein the RNA-guided nuclease molecule or the nucleic acid encoding the RNA-guided nuclease molecule is encapsulated in the LNP of any one of claims 33-41.
71. The method of claim 70, wherein the nucleic acid encoding the RNA-guided nuclease molecule is messenger RNA (mRNA).
72. The method of claim 71, wherein the mRNA encodes any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Casl2a polypeptide sequences).
73. A method of treating a subject, comprising contacting a subject (or a cell from the subject) with:
(a) a gRNA molecule of any one of claims 1-15;
(b) an RNA-guided nuclease molecule or a nucleic acid encoding the RNA- guided nuclease molecule; and optionally, (c) a second gRNA molecule of any one of claims 1-15.
74. The method of claim 73, further comprising contacting the subject with a third gRNA molecule.
75. The method of claim 74, further comprising contacting the subject with a fourth gRNA molecule.
76. The method of any one of claims 73-75, comprising contacting the subject with (a), (b), and (c).
77. The method of any one of claims 73-76, wherein the subject is suffering from POAG.
78. The method of any one of claims 73-77, wherein the subject has a mutation in the MYOC gene.
79. The method of any one of claims 73-78, wherein the contacting step is performed in vivo.
80. The method of any one of claims 73-79, wherein the contacting step comprises subretinal delivery.
81. The method of claim 80, wherein the contacting step comprises subretinal injection.
82. The method of any one of claims 73-79, wherein the contacting step comprises intravitreal delivery.
83. The method of claim 82, wherein the contacting step comprises intravitreal injection.
84. The method of any one of claims 73-83, wherein the contacting step comprises contacting the subject with a nucleic acid that encodes at least one of (a), (b), and optionally (c).
85. The method of any one of claims 73-83, wherein the contacting step comprises contacting the subject with a nucleic acid of any one of claims 16-32.
86. The method of any one of claims 73-83, wherein the contacting step comprises delivering to the subject the RNA-guided nuclease molecule of (b), a nucleic acid that encodes the gRNA molecule of (a), and optionally a nucleic acid that encodes the second gRNA molecule of (c).
87. The method of any one of claims 73-83, wherein the contacting step comprises delivering to the subject the RNA-guided nuclease molecule of (b), the gRNA molecule of (a) and optionally the second gRNA molecule of (c).
88. The method of any one of claims 73-83, wherein the contacting step comprises delivering to the subject a nucleic acid that encodes the RNA-guided nuclease molecule of (b), the gRNA molecule of (a), and optionally the second gRNA molecule of (c).
89. The method of any one of claims 73-83, wherein the gRNA molecule is encapsulated in an LNP of any one of claims 33-41.
90. The method of any one of claims 73-89, wherein the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease is encapsulated in the LNP of any one of claims 33-41.
91. The method of claim 90, wherein the nucleic acid encoding the RNA-guided nuclease is messenger RNA (mRNA).
92. A reaction mixture comprising a gRNA molecule, a nucleic acid, or a composition described herein, and a cell from a subject having POAG, or a subject having a mutation in the MYOC gene.
93. A kit comprising, (a) gRNA molecule of any one of claims 1-15, or a nucleic acid that encodes the gRNA molecule, and one or more of the following:
(b) an RNA-guided nuclease molecule or a nucleic acid encoding the RNA-guided nuclease molecule; and optionally
(c) a second gRNA molecule of any one of claims 1-15; and/or
(e) a nucleic acid that encodes one or more of (b) and optionally (c).
94. The kit of claim 93, wherein the (b) RNA-guided nuclease molecule is a Casl2a molecule.
95. The kit of claim 94, wherein the Casl2a molecule comprises any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Cas 12a polypeptide sequences).
96. The kit of claim 94, wherein the Cast 2a molecule is encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Casl2a polynucleotide sequences).
97. The kit of any one of claims 93-96, comprising a nucleic acid that encodes one or more of (a), (b), and optionally (c).
98. The kit of any one of claims 93-97, further comprising a third gRNA molecule targeting a MYOC target position.
99. The kit of claim 98, further comprising a fourth gRNA molecule targeting a MYOC target position.
100. A gRNA molecule of any one of claims 1-15 for use in treating POAG in a subject.
101. The gRNA molecule of claim 100, wherein the gRNA molecule is used in combination with (b) an RNA-guided nuclease molecule or a nucleic acid molecule encoding the RNA-guided nuclease molecule.
102. The gRNA molecule of claim 101, wherein the (b) RNA-guided nuclease molecule is a Casl2a molecule.
103. The gRNA molecule of claim 102, wherein the Casl2a molecule comprises any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Casl2a polypeptide sequences).
104. The gRNA molecule of claim 102, wherein the Casl2a molecule is encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Casl2a polynucleotide sequences).
105. The gRNA molecule of any one of claims 100-104, wherein the gRNA molecule is used in combination with (c) a second gRNA molecule of any of claims 1-15.
106. Use of a gRNA molecule of any one of claims 1-15 in the manufacture of a medicament for treating POAG in a subject.
107. The use of claim 106, wherein the medicament further comprises (b) an RNA- guided nuclease molecule.
108. The use of claim 107, wherein the (b) RNA-guided nuclease molecule is a Casl2a molecule.
109. The use of claim 108, wherein the Casl2a molecule comprises any of the sequences set forth in SEQ ID NOs:8, 9, 10-20, 24, 25-29, 30-33, 34-36, 48 (Casl2a polypeptide sequences).
110. The use of claim 108, wherein the Casl2a molecule is encoded by any of the sequences set forth in SEQ ID NOs:21-23, 37-44 (Casl2a polynucleotide sequences).
111. The use of any one of claims 106- 110, wherein the medicament further comprises (c) a second gRNA molecule of any one of claims 1-15.
112. A nucleic acid of any one of claims 16-32 for use in treating POAG in a subject.
113. An LNP of any one of claims 33-41 for use in treating POAG in a subject.
114. A composition of any one of claims 42-52 for use in treating POAG in a subject.
PCT/US2025/023321 2024-04-05 2025-04-04 Crispr/rna-guided nuclease related methods and compositions for treating primary open angle glaucoma Pending WO2025213138A1 (en)

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