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WO2025186726A1 - Modulation de l'expression d'un gène agt (angiotensinogène) - Google Patents

Modulation de l'expression d'un gène agt (angiotensinogène)

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Publication number
WO2025186726A1
WO2025186726A1 PCT/IB2025/052358 IB2025052358W WO2025186726A1 WO 2025186726 A1 WO2025186726 A1 WO 2025186726A1 IB 2025052358 W IB2025052358 W IB 2025052358W WO 2025186726 A1 WO2025186726 A1 WO 2025186726A1
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WO
WIPO (PCT)
Prior art keywords
grna
agt
seq
subject
cas9
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Pending
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PCT/IB2025/052358
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WO2025186726A8 (fr
Inventor
Mary-Lee Dequeant
Viktoriia KYRYCHENKO
Sanjay D'SOUZA
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CRISPR Therapeutics AG
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CRISPR Therapeutics AG
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Publication of WO2025186726A1 publication Critical patent/WO2025186726A1/fr
Publication of WO2025186726A8 publication Critical patent/WO2025186726A8/fr
Pending legal-status Critical Current
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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
    • 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]
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification

Definitions

  • the present disclosure generally relates to the field of molecular biology and biotechnology, including gene editing.
  • Angiotensinogen is the precursor of the biologically active angiotensin II (Ang II).
  • CRISPR-Cas systems can be divided in two classes, with class 1 systems utilizing a complex of multiple Cas proteins (such as type I, III, and IV CRISPR-Cas systems) and class 2 systems utilizing a single Cas protein (such as type II, V, and VI CRISPR- Cas systems).
  • class 1 systems utilizing a complex of multiple Cas proteins (such as type I, III, and IV CRISPR-Cas systems) and class 2 systems utilizing a single Cas protein (such as type II, V, and VI CRISPR- Cas systems).
  • Type II CRISPR-Cas-based systems have been used for genome editing, and require a Cas polypeptide or variant thereof guided by a customizable guide RNA (gRNA) for programmable DNA targeting.
  • gRNA customizable guide RNA
  • gRNAs guide RNAs
  • AGT angiotensinogen genomic locus
  • the gRNA can, in some embodiments, comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, 127-129, and 155-162, or a portion thereof.
  • the spacer sequence comprises 16, 17, 18, or 19 nucleotides at the 3’ terminus of any one of the sequences of SEQ ID NOs: 64-71, 73, 75- 84, 127-129, and 155-162.
  • the spacer sequence comprises a sequence selected from SEQ ID Nos: 64-71, 73, 75-84, 127-129, and 155-162.
  • the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID Nos: 64, 67, 71, 73, 75, 78-82, and 84.
  • the gRNA comprises a spacer sequence comprising the sequence of SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, or SEQ ID NO: 162.
  • the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-67, or a portion thereof. In some embodiments, the spacer sequence comprises 16, 17, 18, or 19 nucleotides at the 3’ terminus of any one of the sequences of SEQ ID NOs: 64-67. In some embodiments, the gRNA comprises a spacer sequence comprising the sequence of SEQ ID NO: 65 or SEQ ID NO: 67, or a portion thereof. In some embodiments, the spacer sequence comprises 16, 17, 18, or 19 nucleotides at the 3’ terminus of SEQ ID NO: 65 or SEQ ID NO: 67.
  • the gRNAs disclosed herein can, for example, induce a cutting efficiency of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the gRNA induces a cutting efficiency of at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
  • the gRNA can be a single-guide RNA (sgRNA) and/or a chemically-modified gRNA.
  • the chemically-modified gRNA can comprise, for example, one or more phosphorothioate linkages and/or one or more 2’-O-methyl nucleotides at the 3’ end, the 5 ’ end, or both.
  • no more than 50% of the nucleotides of the gRNA comprise a 2’-O-methyl modification. In some embodiments, about 48% of the nucleotides of the gRNA comprise a 2’-O-methyl modification, wherein the 5’ end of the gRNA comprises three phosphorothioate linkages, and/or wherein the 3’ end of the gRNA comprises three phosphorothioate linkages.
  • compositions comprising: (a) a guide RNA (gRNA) that targets an angiotensinogen (AGT) genomic locus, comprising a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, 127-129, and 155-162, or a portion thereof, or a nucleic acid encoding the gRNA; and (b) a Cas9 endonuclease or a nucleic acid encoding a Cas9 endonuclease.
  • the spacer sequence can, for example, comprise a sequence selected from SEQ ID NOs: 64-71, 73, 75-84, 127-129, and 155-162.
  • the gRNA can be a singleguide RNA (sgRNA) and/or a chemically-modified gRNA.
  • the chemically- modified gRNA comprises one or more phosphorothioate linkages and/or one or more 2’-O- methyl nucleotides at the 3’ end, the 5’ end, or both.
  • no more than 50% of the nucleotides of the gRNA comprise a 2’-O-methyl modification.
  • the Cas9 endonuclease is selected from 5. pyogenes Cas9, . aureus Cas9, N meningitides Cas9, S. thermophilus CRISPR1 Cas9, S. thermophilus CRISPR 3 Cas9, and T. denticola Cas9.
  • the composition comprises (a) the AGT gRNA and (b) the Cas9 endonuclease, and the AGT gRNA and Cas 9 nuclease are formulated as a ribonucleoprotein particle (RNP).
  • the composition comprises (a) a nucleic acid encoding an AGT gRNA and (b) a nucleic acid encoding a Cas9 endonuclease, and wherein (a) and/or (b) is present on a viral vector.
  • the viral vector can be, for example, an adeno-associated viral vector.
  • the gRNA or the nucleic acid encoding a gRNA of (a), the Cas9 endonuclease or the nucleic acid encoding a Cas9 endonuclease of (b), or both are complexed with a liposome or lipid nanoparticle (LNP).
  • the lipid nanoparticle comprises one or more neutral lipids, charged lipids, ionizable lipids, steroids, and polymers conjugated lipids.
  • the lipid nanoparticle comprises cholesterol, a polyethylene glycol (PEG) lipid, or both.
  • Disclosed herein includes a method for treating an AGT-associated disease or disorder in a subject in need thereof.
  • the method comprises administering to the subject any one of the compositions disclosed herein, thereby treating the AGT-associated disease or disorder in the subject.
  • Disclosed herein includes a method for treating a subject that has or is suspected of having hypertension.
  • the method comprises administering to the subject any one of the compositions disclosed herein, thereby treating the hypertension.
  • Also disclosed herein includes a method for treating an AGT-associated disease or disorder in a subject in need thereof, comprising administering to the subject a composition.
  • the method comprises a plurality of nanoparticles complexed with: (a) a guide RNA (gRNA) that targets an AGT genomic locus or a nucleic acid encoding the gRNA; and (b) a Cas9 endonuclease or a nucleic acid encoding a Cas9 endonuclease, thereby treating the AGT- associated disease or disorder in the subject.
  • gRNA guide RNA
  • the gRNA that targets the AGT genomic locus comprises a spacer sequence that is 16, 17, 18, 19 or 20 nucleotides in length.
  • the gRNA that targets the AGT genomic locus comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, 127-129, and 155-162, or a portion thereof.
  • the method comprises administering to the subject a composition comprising a plurality of nanoparticles complexed with: (a) a guide RNA (gRNA) that targets an AGT genomic locus or a nucleic acid encoding the gRNA; and (b) a Cas9 endonuclease or a nucleic acid encoding a Cas9 endonuclease, thereby treating the hypertension.
  • gRNA guide RNA
  • the gRNA that targets the AGT genomic locus comprises a spacer sequence that is 16, 17, 18, 19 or 20 nucleotides in length.
  • the gRNA that targets the AGT genomic locus comprising a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, 127-129, and 155-162, or a portion thereof.
  • the Cas9 endonuclease is S. pyogenes Cas9, S. aureus Cas9, N. meningitides Cas9, S. thermophilus CRISPR1 Cas9, S. thermophilus CRISPR 3 Cas9, or T. denticola Cas9.
  • the plurality of nanoparticles are lipid nanoparticles.
  • the lipid nanoparticles comprise one or more neutral lipids, charged lipids, ionizable lipids, steroids, and polymers conjugated lipids.
  • the lipid nanoparticles comprise cholesterol, a polyethylene glycol (PEG) lipid, or both.
  • the methods can comprise administering to the subject the composition at a single dose of about 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, or 1.0 mg/kg of total nucleic acids of (a) and (b).
  • the method comprises a single administration of the composition to the subject.
  • the expression of AGT in the subject is reduced in the subject; optionally, wherein the expression of AGT is reduced in the liver of the subject; and wherein the reduction is relative to (a) the AGT expression of the subject prior to being administered the composition; (b) the AGT expression in one or more untreated subjects; and/or (3) a reference level of AGT expression of healthy subjects.
  • the expression of AGT in the subject is reduced by at least 20% after the administration. In some embodiments, the reduction is for at least two weeks, at least three weeks, at least four weeks, or at least a month.
  • the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent to the subject.
  • the additional therapeutic agent is an ACE inhibitor, an angiotensin-2 receptor blocker, a calcium channel blocker, a diuretic, a beta blocker, a renin inhibitor, a mineralocorticoid receptor antagonist, an AGT siRNA, or a combination thereof.
  • the additional therapeutic agent is enalapril, lisinopril, perindopril, ramipril, captopril, banezepril, quinapril, trandolapril, enalapril, fosinopril, candesartan, irbesartan, losartan, valsartan, olmesartan, azilsartan, telmisartan, amlodipine, felodipine, nifedipine, diltiazem, verapamil, indapamide, bendroflumethi azide, chlorothizaide, hydrochlorothiazide, chlorthalidone, metolazone, methyclothiazide, indapamide, furosemide, torsemide, bumetanide, acetazolamide, atenolol, bisoprolol, metoprolol, aliskiren, s
  • the subject has, or is suspected of having, hypertension, wherein the hypertension is resistant hypertension, refractory hypertension, or pregnancy-associated hypertension.
  • the subject has elevated blood pressure as compared to a reference value, optionally the elevated blood pressure is equal to or greater than 130/80 mmHg. In some embodiments, the blood pressure is reduced in the subject following administration of the composition.
  • the levels of angiotensin I and/or angiotensin II in the subject are reduced following administration of the composition; and wherein the reduction is relative to (a) the angiotensin I and/or angiotensin II levels of the subject prior to being administered the composition; (b) the angiotensin I and/or angiotensin n levels in one or more untreated subjects; and/or (3) a reference level of angiotensin I and/or angiotensin II of healthy subjects.
  • gRNAs guide RNAs
  • AGT angiotensinogen genomic locus
  • the gRNA can comprise a spacer sequence that is 16, 17, 18 or 19 nucleotides in length.
  • the spacer sequence comprises the 16, 17, 18, or 19 nucleotides at the 3’ terminus of any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, 127-129, and 155-162.
  • the gRNA induces a cutting efficiency of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
  • the gRNA induces a reduced off-target effect compared to a corresponding spacer sequence that is 20 nucleotides in length. In some embodiments, the gRNA induces fewer off-target events than a corresponding spacer sequence that is 20 nucleotides in length. In some embodiments, the gRNA does not edit any off-target site.
  • FIG. 1 illustrates the renin-angiotensin cascade.
  • FIG. 2A is a bar graph depicting on-target editing efficiencies of four selected gRNAs comprising the indicated spacer sequences in primary human and monkey hepatocytes. DNA editing and total AGT secreted in the media are shown as percentage of un-edited cells. The data is representative of 3-4 independent experiments for primary human hepatocyte (PHH) donors and 2 technical replicates for primary monkey hepatocyte (PMH) donor.
  • FIG. 2B is a bar graph depicting on-target editing efficiencies of selected gRNAs comprising the indicated spacer sequences in primary human hepatocytes. DNA editing and total AGT secreted in the media are shown as percentage of un-edited cells.
  • FIG. 3 is a bar graph depicting on-target editing efficiencies of selected gRNAs comprising the indicated spacer sequences in primary monkey hepatocytes. DNA editing and total AGT secreted in the media are shown as percentage of un-edited cells.
  • FIG. 4A displays bar graphs depicting editing efficiencies of gRNAs SpAgRl in SHR at a dose of 0, 0.5, 1.0, or 2.0 mg/kg.
  • FIG. 4B is a graph depicting serum AGT reduction.
  • FIG. 4C displays graphs depicting blood pressure change following the administration.
  • mpk stands for milligram per kilogram (mg/kg) of LNP/mouse weight.
  • FIGS. 5A-5B are bar graphs depicting editing efficiency and AGT protein reduction of gRNAs comprising truncated spacer sequences in comparison to their full-length counterparts in primary human hepatocytes.
  • FIGS. 6A-6C display graphs depicting editing efficiency and AGT protein reduction of exemplary AGT gRNAs in comparison to ANGPTL3 gRNA T6.
  • FIG. 7 displays comparison of on-target efficacy of two rat guides (e.g., SpAgRl and rAGT_E2G21) in vivo.
  • FIG. 8 displays bar graphs depicting editing efficiency and AGT protein reduction of batch 2 (B2) gRNAs in primary human hepatocytes (PHH) and primary monkey hepatocyte (PMH).
  • FIG. 9 displays bar graphs depicting editing efficiency and AGT protein reduction of batch 3 (B3) gRNAs in primary human hepatocytes (PHH) and primary monkey hepatocyte (PMH).
  • FIG. 10A displays non-limiting exemplary data depicting editing efficiency of gRNA rAGT_E2G21 in all SHRs at a dose of 0, 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg/kg.
  • FIG. 10B displays non-limiting exemplary data depicting editing efficiency of the same gRNAs in female and male SHRs at a dose of 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg/kg.
  • FIGs. 11A-D display non-limiting exemplary mean arterial pressure (MAP) of animals treated with LNPs at a dose ofO, 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg/kg, respectively.
  • MAP mean arterial pressure
  • FIGS. 12A-B display non-limiting exemplary data depicting mean arterial pressure (MAP) following the LNP administration in male rates (FIG. 12A) and female rates (FIG. 12B).
  • MAP mean arterial pressure
  • FIGS. 13A-B display non-limiting exemplary data depicting serum AGT (FIG. 13A) and renin levels (FIG. 13B) of treated animals in comparison to untreated animals.
  • UTD indicates “unable to determine”;
  • OOR indicates “out of range”, i.e., below 160 ngm/mL.
  • FIGS. 14A-B display non-limiting exemplary data depicting the percentage knockdown of AGT from baseline.
  • FIG. 14A excludes the out of range data
  • FIG. 14B includes the out of range data as 160 ng/mL.
  • FIG. 16A displays non-limiting exemplary data depicting editing efficiency and protein reduction percentage from baseline of four exemplary gRNAs xhAGT_E2_g30, xhAGT_E2_g48, xhAGT_E2_g83, and xhAGT_E2_glO3 in PHH donor 1 and PHH donor 2.
  • FIG. 16B displays non-limiting exemplary data depicting editing efficiency and protein reduction percentage from baseline of four exemplary gRNAs xhAGT_E2_g30, xhAGT_E2_g48, xhAGT_E2_g83, and xhAGT_E2_glO3 in PMH donor 1 and PMH donor 2.
  • FIG. 17 displays non-limiting exemplary data depicting liver gene editing efficiency and AGT protein deduction in serum from baseline of four exemplary gRNAs xhAGT_E2_g30, xhAGT_E2_g48, xhAGT_E2_g83, and xhAGT_E2_ lO3 in NHPs.
  • FIG. 18 displays non-limiting exemplary data depicting the pre-dose and postdose AGT concentration in NHP serum.
  • gRNAs guide RNAs
  • AGT angiotensinogen
  • the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, and 127-129, or a portion thereof.
  • compositions can comprise (a) a gRNA that targets an AGT genomic locus, comprising a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, and 127-129, or a nucleic acid encoding the gRNA; and (b) a Cas9 endonuclease or a nucleic acid encoding a Cas9 endonuclease.
  • One or more of the spacer sequences consisting of the sequence of SEQ ID NOs: 64-71, 73, 75-84, and 127- 129 can be part of a guide RNA used in prime editing, base editing, or both, to target and edit AGT gene locus.
  • Disclosed herein includes a method for treating an AGT-associated disease or disorder in a subject in need thereof. In some embodiments, the method comprises administering to the subject any one of the compositions disclosed herein, thereby treating the AGT-associated disease or disorder in the subject. Disclosed herein also includes a method for treating a subject that has or is suspected of having hypertension. In some embodiments, the method comprises administering to the subject any one of the compositions disclosed herein, thereby treating the hypertension.
  • RNA-guided endonuclease refers to a polypeptide capable of binding an RNA (e.g., a gRNA) to form a complex targeted to a specific DNA sequence (e.g. , in a target DNA).
  • RNA-guided endonuclease is a Cas polypeptide (e.g., a Cas endonuclease, such as a Cas9 endonuclease).
  • the RNA-guided endonuclease as described herein is targeted to a specific DNA sequence in a target DNA by an RNA molecule to which it is bound.
  • the RNA molecule can include a sequence that is complementary to and capable of hybridizing with a specific sequence within the target DNA, thus allowing for targeting of the bound polypeptide to a specific location within the target DNA.
  • guide RNA can refer to a site-specific targeting RNA that can bind an RNA-guided endonuclease to form a complex, and direct the activities of the bound RNA-guided endonuclease (such as a Cas endonuclease) to a specific sequence within a target nucleic acid (e.g., a specific gene or region within a gene).
  • the guide RNA can include one or more RNA molecules.
  • a “secondary structure” of a nucleic acid molecule refers to the base pairing interactions within the nucleic acid molecule.
  • Cas endonuclease or “Cas nuclease” refers to an RNA-guided DNA endonuclease associated with and/or derived from the CRISPR adaptive immunity system.
  • nuclease and “endonuclease” are used interchangeably herein to refer to an enzyme which possesses endonucleolytic catalytic activity for polynucleotide cleavage.
  • polynucleotide and “nucleic acid” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • a polynucleotide can be single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids/triple helices, or a polymer including purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • binding refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it means that the molecule X binds to molecule Y in a non-covalent manner).
  • Binding interactions can be characterized by a dissociation constant (Kd), for example a Kd of, or a Kd less than, 10' 6 M, 10' 7 M, 10' 8 M, 10' 9 M, 10' 10 M, 10’ 11 M, 10' 12 M, 10' 13 M, 10' 14 M, 10' 15 M, or a number or a range between any two of these values.
  • Kd can be dependent on environmental conditions, e.g., pH and temperature.
  • “Affinity” refers to the strength of binding, and increased binding affinity is correlated with a lower Kd.
  • hybridizing refers to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be achieved by any process in which a nucleic acid sequence joins with a substantially or fully complementary sequence through base pairing to form a hybridization complex. “Hybridizing” or “hybridize” can comprise denaturing the molecules to disrupt the intramolecular structure(s) (e.g., secondary structure(s)) in the molecule. In some embodiments, denaturing the molecules comprises heating a solution comprising the molecules to a temperature sufficient to disrupt the intramolecular structures of the molecules.
  • denaturing the molecules comprises adjusting the pH of a solution comprising the molecules to a pH sufficient to disrupt the intramolecular structures of the molecules.
  • two nucleic acid sequences or segments of sequences are “substantially complementary” if at least 80% of their individual bases are complementary to one another.
  • a splint oligonucleotide sequence is not more than about 50% identical to one of the two polynucleotides (e.g., RNA fragments) to which it is designed to be complementary.
  • the complementary portion of each sequence can be referred to herein as a “segment”, and the segments are substantially complementary if they have 80% or greater identity.
  • complementarity and “complementary” mean that a nucleic acid can form hydrogen bond(s) with another nucleic acid based on traditional Watson-Crick base paring rule, that is, adenine (A) pairs with thymine (T, or uracil (U) in RNA) and guanine (G) pairs with cytosine (C).
  • Complementarity can be perfect (e.g. complete complementarity) or imperfect (e.g. partial complementarity). Perfect or complete complementarity indicates that each and every nucleic acid base of one strand is capable of forming hydrogen bonds according to Watson-Crick canonical base pairing with a corresponding base in another, antiparallel nucleic acid sequence.
  • Partial complementarity indicates that only a percentage of the contiguous residues of a nucleic acid sequence can form Watson-Crick base pairing with the same number of contiguous residues in another, antiparallel nucleic acid sequence.
  • the complementarity can be at least 70%, 80%, 90%, 100% or a number or a range between any two of these values.
  • the complementarity is perfect, i.e., 100%.
  • the complementary candidate sequence segment is perfectly complementary to the candidate sequence segment, whose sequence can be deduced from the candidate sequence segment using the Watson-Crick base pairing rules.
  • nucleic acid and “polynucleotide” are interchangeable and refer to any nucleic acid, whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphorami date, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sultone linkages, and combinations of such linkages.
  • the terms “nucleic acid” and “polynucleotide” also specifically include nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).
  • DNA editing efficiency may be used interchangeably herein and can refer to the number or proportion of intended target sequences that are edited. For example, if a CRISPR-Cas9 system edits 10% of the intended target sequence (e.g, within a cell or within a population of cells), then the system can be described as being 10% efficient. In some embodiments, the efficiency can be reported as % indel, e.g., the proportion of insertions and/or deletions detected in the target sequence. Indels (e.g., insertion-deletions) can result from repair of double-stranded DNA breaks caused by Cas9 cleavage by processes including, but not limited to, non-homologous end joining (NHEJ) repair.
  • NHEJ non-homologous end joining
  • off-target editing frequency refers to the number or proportion of unintended DNA sequences that are edited.
  • On-target and off-target editing frequencies may be measured by the methods and assays described herein, further in view of techniques known in the art, including high-throughput sequencing reads.
  • high-throughput sequencing involves the hybridization of nucleic acid primers (e.g., DNA primers) with complementarity to nucleic acid (e.g., DNA) regions just upstream or downstream of the target sequence or off-target sequence of interest.
  • nucleic acid primers with sufficient complementarity to regions upstream or downstream of the Cas9-dependent off-target site may be designed using techniques and kits known in the art. These kits make use of polymerase chain reaction (PCR) amplification, which produces amplicons as intermediate products.
  • the target and off-target sequences may comprise genomic loci that further comprise protospacers and PAMs. Accordingly, the term “amplicons,” as used herein, may refer to nucleic acid molecules that constitute the aggregates of genomic loci, protospacers and PAMs.
  • High-throughput sequencing techniques used herein may further include Sanger sequencing and/or whole genome sequencing (WGS).
  • transfection refers to the introduction of a nucleic acid into a host cell, such as by contacting the cell with liposomes or nanoparticles (e.g., lipid nanoparticles) as described herein.
  • liposomes or nanoparticles e.g., lipid nanoparticles
  • treatment refers to a clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible.
  • the aim of treatment includes, but is not limited to, the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition.
  • Treatment refer to one or both of therapeutic treatment and prophylactic or preventative measures.
  • Subjects in need of treatment include those already affected by a disease or disorder or undesired physiological condition as well as those in which the disease or disorder or undesired physiological condition is to be prevented.
  • the terms “effective amount” or “pharmaceutically effective amount” or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
  • pharmaceutically acceptable excipient refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive or diluent for administration of a compound(s) of interest to a subject.
  • Pharmaceutically acceptable excipients can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.
  • a “subj ecf ’ refers to an animal for whom a diagnosis, treatment, or therapy is desired.
  • the subject is a mammal.
  • “Mammal,” as used herein, refers to an individual belonging to the class Mammalia and includes, but not limited to, humans, domestic and farm animals, zoo animals, sports and pet animals. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates, such as monkeys, chimpanzees and apes, and, in particular, humans.
  • the mammal is a primate.
  • the mammal is a human.
  • the mammal is not a human.
  • the subject has or is suspected of having an AGT- associated disease or disorder.
  • Angiotensinogen AGT
  • AGT angiotensinogen
  • the vectors, compositions, methods, and kits described herein can be particularly useful for treating AGT -associated diseases and conditions, such as hypertension (e.g., resistant hypertension, refractory hypertension, and pregnancy-associated hypertension).
  • AGT is a member of the serpin family and is a component of the renin- angiotensin-aldosterone system (RAAS).
  • the RAAS pathway refers to a multi-component enzymatic pathway where the precursor component AGT is converted by various enzymes such as resin and angiotensin-converting enzymes (ACE) into downstream components such as angiotensin I and angiotensin II.
  • FIG. 1 illustrates the renin-angiotensin cascade.
  • AGT is primarily produced in the liver and is released into the circulation where renin converts it into angiotensin I, which is then converted by ACE to angiotensin II.
  • the RAAS pathway components including angiotensinogen and other downstream angiotensin peptides, have been demonstrated to be involved in maintaining blood pressure, body fluid and electrolyte homeostasis, and in the pathogenesis of essential hypertension and preeclampsia.
  • Mutations in AGT gene are associated with susceptibility to essential hypertension, and can cause renal tubular dysgenesis, a severe disorder of renal tubular development. Defects in AGT gene have also been associated with non- familial structural atrial fibrillation, and inflammatory bowel disease.
  • AGT is expressed in multiple tissues, plasma AGT levels are determined primarily by the rate of production by hepatocytes, which constitutively secrete AGT. Circulating levels of AGT are close to the Michaelis constant for renin, thus a small increase in AGT will increase angiotensin II synthesis.
  • Angiotensin II is a peptide hormone that can act on vascular smooth muscle as a potent vasoconstrictor which in turn can increase blood pressure.
  • RNAs, compositions, and methods for preventing and treating AGT-associated or RAAS-pathway associated diseases and disorders include guide RNAs, compositions, and methods for preventing and treating AGT-associated or RAAS-pathway associated diseases and disorders.
  • the gRNAs, compositions, and methods disclosed herein provide highly potent compounds to target liver, therefore limiting their distribution to other tissues.
  • the gRNAs disclosed herein target the AGT genomic locus, e.g., the chromosomal location of lq42.2.
  • the AGT gene sequence targeted by the gRNAs is annotated as NCBI reference sequence NC_000001.11.
  • the gRNAs disclosed herein target any one of the six exons of the AGT gene.
  • Gene editing is a type of genetic engineering in which nucleotide(s)/nucleic acid(s) is/are inserted, deleted, and/or substituted in a DNA sequence, such as in the genome of a targeted cell.
  • Targeted gene editing enables insertion, deletion, and/or substitution at pre-selected sites in the genome of a targeted cell (e.g. , in a targeted gene or targeted DNA sequence).
  • Targeted integration refers to a process involving insertion of one or more exogenous sequences, with or without deletion of an endogenous sequence at the insertion site. Targeted integration can result from targeted gene editing when a donor template containing an exogenous sequence is present.
  • Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease-dependent approach.
  • nuclease-independent targeted editing approach homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be introduced into an endogenous sequence through the enzymatic machinery of the host cell.
  • the exogenous polynucleotide can introduce deletions, insertions or replacement of nucleotides in the endogenous sequence.
  • the nuclease-dependent approach can achieve targeted editing with higher frequency through the specific introduction of double strand breaks (DSBs) by specific rare-cutting nucleases (e.g., endonucleases).
  • DSBs double strand breaks
  • nuclease-dependent targeted editing also utilizes DNA repair mechanisms, for example, non-homologous end joining (NHEI), which occurs in response to DSBs.
  • NHEI non-homologous end joining
  • DNA repair by NHEJ often leads to random insertions or deletions (indels) of a small number of endogenous nucleotides.
  • repair can also occur by a homology directed repair (HDR).
  • HDR homology directed repair
  • Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and RNA-guided CRISPR-Cas9 nuclease (CRISPR/Cas9; Clustered Regular Interspaced Short Palindromic Repeats Associated 9). Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxb 1 integrases may also be used for targeted integration.
  • ZFN zinc-finger nucleases
  • TALEN transcription activator-like effector nucleases
  • CRISPR/Cas9 Clustered Regular Interspaced Short Palindromic Repeats Associated 9
  • DICE dual integrase cassette exchange
  • ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain (ZFBD), which is a polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers.
  • ZFBD zinc finger DNA binding domain
  • a zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers.
  • a designed zinc finger domain is a domain not occurring in nature whose design/composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data.
  • a selected zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection.
  • ZFNs are described in greater detail in U.S. Patent Nos. 7,888,121 and 7,972,854. The most recognized example of a ZFN is a fusion of the FokI nuclease with a zinc finger DNA binding domain.
  • a TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain.
  • a “transcription activator-like effector DNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNA binding domain” is a polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains.
  • TAL effector DNA binding domain specificity depends on an effector-variable number of imperfect 34 amino acid repeats, which comprise polymorphisms at select repeat positions called repeat variable-diresidues (RVD).
  • RVD repeat variable-diresidues
  • TALENs are described in greater detail in US2011/0145940. The most recognized example of a TALEN in the art is a fusion polypeptide of the FokI nuclease to a TAL effector DNA binding domain.
  • Additional examples of targeted nucleases suitable for use as provided herein include, but are not limited to, Bxbl, phiC31, R4, PhiBTl, and W
  • targeted nucleases include naturally-occurring and recombinant nucleases, e.g., CRISPR/Cas9, restriction endonucleases, meganucleases homing endonucleases, and the like.
  • the vectors, compositions, methods, and kits described herein can be used in a gene editing system, such as in a CRISPR-Cas gene editing system, to genetically edit the AGT gene.
  • a gene editing system such as in a CRISPR-Cas gene editing system
  • the CRISPR-Cas9 system is a naturally-occurring defense mechanism in prokaryotes that has been repurposed as a RNA-guided DNA-targeting platform used for gene editing. It relies on the DNA nuclease Cas9, and two noncoding RNAs: crisprRNA (crRNA) and trans-activating RNA (tracrRNA) to target the cleavage of DNA.
  • crRNA crisprRNA
  • tracrRNA trans-activating RNA
  • crRNA drives sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing typically with a 20 nucleotide (nt) sequence in the target DNA.
  • the CRISPR-Cas9 complex only binds DNA sequences that contain a sequence match to the first 20 nt of the crRNA, single-guide RNA (sgRNA), if the target sequence is followed by a specific short DNA motif (with, for example, the sequence NGG) referred to as a protospacer adjacent motif (PAM).
  • sgRNA single-guide RNA
  • PAM protospacer adjacent motif
  • TracrRNA hybridizes with the 3’ end of crRNA to form an RNA-duplex structure that is bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.
  • CRISPR-Cas9 complex Once the CRISPR-Cas9 complex is bound to DNA at a target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, leaving a double-strand break (DSB) where both strands of the DNA terminate in a base pair (a blunt end).
  • DSB double-strand break
  • CRISPR-Cas9 gene editing system comprises an RNA-guided nuclease and one or more guide RNAs targeting one or more target genes.
  • RNA-guided endonuclease can be naturally-occurring or non-naturally occurring.
  • Non-limiting examples of RNA-guided endonucleases include a Cast, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, C
  • the RNA-guided endonuclease is a Cas9 endonuclease.
  • the Cas9 endonuclease can be from, e.g., Streptococcus pyogenes (SpCas9 or SpyCas9), Staphylococcus lugdunensis (SluCas9), or Staphylococcus aureus (SaCas9).
  • the RNA- guided endonuclease is a variant of Cas9, including but not limited to, a small Cas9, a dead Cas9 (dCas9), and a Cas9 nickase.
  • a Cas nuclease can comprise a RuvC or RuvC-like nuclease domain (e.g., Cpfl) and/or a HNH or HNH-like nuclease domain (e.g., Cas9).
  • the Cas9 endonuclease is S. pyogenes Cas9, 5. aureus Cas9, N. meningitides Cas9, S. thermophilus Cas9, S. thermophilus 3 Cas9, T. denticola Cas9, or a variant thereof.
  • the RNA-guided endonuclease can be a small RNA-guided endonuclease.
  • the small RNA-guided endonucleases can be engineered from portions of RNA-guided endonucleases derived from any of the RNA-guided endonucleases described herein and known in the art.
  • the small RNA-guided endonucleases can be, e.g., small Cas endonucleases.
  • a small RNA-guided nuclease is shorter than about 1,100 amino acids in length.
  • the RNA-guided endonuclease can be a mutant RNA-guided endonuclease.
  • the RNA-guided endonuclease can be a mutant of a naturally occurring RNA-guided endonuclease.
  • the mutant RNA-guided endonuclease can also be a mutant RNA-guided endonuclease with altered activity compared to a naturally occurring RNA-guided endonuclease, such as altered endonuclease activity (e.g., altered or abrogated DNA endonuclease activity without substantially diminished binding affinity to DNA).
  • Such modification can allow for the sequence-specific DNA targeting of the mutant RNA-guided endonuclease for the purpose of transcriptional modulation (e.g., activation or repression); epigenetic modification or chromatin modification by methylation, demethylation, acetylation or deacetylation, or any other modifications of DNA binding and/or DNA-modifying proteins known in the art.
  • the mutant RNA-guided endonuclease has no DNA endonuclease activity.
  • a nucleic acid encoding an RNA-guided endonuclease is administered to the subject.
  • the nucleic acid can be generated by an in vitro transcription reaction.
  • generating in vitro transcribed RNA comprises incubating a linear DNA template with an RNA polymerase and a nucleotide mixture under conditions to allow (run-off) RNA in vitro transcription.
  • the nucleotide mixture can be part of an in vitro transcription mix (IVT-mix).
  • the RNA polymerase is a T7 RNA polymerase.
  • the nucleotide mixture used in RNA in vitro transcription can additionally contain modified nucleotides as defined below.
  • the nucleotide mixture e. , the fraction of each nucleotide in the mixture
  • RNA in vitro transcription reactions can be optimized for the given RNA sequence (optimized NTP mix). Such methods are described, for example in WO2015/188933.
  • RNA obtained by a process using an optimized NTP mix is, in some embodiments, characterized by reduced immune stimulatory properties.
  • the nucleotide mixture is composed of (chemically) non-modified ribonucleoside triphosphates (NTPs) GTP, ATP, CTP and UTP.
  • NTPs non-modified ribonucleoside triphosphates
  • the in vitro transcription can include the presence of at least one cap analog, e.g., a capl trinucleotide cap analog, m7G(5’)ppp(5’)(2’OMeA)pG or m7G(5’)ppp(5’)(2’OMeG)pG, m7G(5’)ppp(5’)(2’OMeA)pG or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.
  • a capl trinucleotide cap analog e.g., a capl trinucleotide cap analog, m7G(5’)ppp(5’
  • the modified nucleoside triphosphate comprises pseudouridine ( ⁇ f), N1 -methylpseudouridine (ml ⁇
  • uracil nucleotides in the nucleotide mixture are replaced (either partially or completely) by pseudouridine ( ⁇
  • the chemically modified nucleotide is pseudouridine ( ⁇
  • the nucleotide mixture comprises at least one modified nucleotide and/or at least one nucleotide analogue or nucleotide derivative for incorporation into an RNA.
  • the modified nucleotide as defined herein can include nucleotide analogs/modifications, e.g., backbone modifications, sugar modifications or base modifications.
  • a backbone modification can comprise a modification, in which phosphates of the backbone of the nucleotides are chemically modified.
  • a sugar modification can comprise a chemical modification of the sugar of the nucleotides.
  • a base modification can comprise a chemical modification of the base moiety of the nucleotides.
  • nucleotide analogs or modifications can comprise nucleotide analogs which are applicable for transcription and/or translation.
  • the nucleotide mixture comprises least one modified nucleotide and/or at least one nucleotide analogues is selected from a backbone modified nucleotide, a sugar modified nucleotide and/or a base modified nucleotide, or any combination thereof.
  • modified nucleosides and nucleotides which may be included in the nucleotide mixture and incorporated into the RNA can be modified in the sugar moiety.
  • the 2’ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents.
  • “Deoxy” modifications include hydrogen, amino (e.g, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaiyl amino, diheteroaryl amino, or amino acid); or the amino group can be attached to the sugar through a linker, wherein the linker comprises one or more of the atoms C, N, and O.
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a modified RNA molecule can include nucleotides containing, for instance, arabinose as the sugar.
  • the phosphate backbone can further be modified in the modified nucleosides and nucleotides, which can be included in the nucleotide mixture and incorporated into a modified in vitro transcribed RNA.
  • the phosphate groups of the backbone can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • the modified nucleosides and nucleotides can include the full replacement of an unmodified phosphate moiety with a modified phosphate as described herein.
  • modified phosphate groups include, but are not limited to, phosphor othioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene- phosphonates).
  • a nucleotide as described herein can be modified in the nucleobase moiety.
  • nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine and uracil.
  • nucleosides and nucleotides described herein can be chemically modified on the major groove face.
  • the major groove chemical modifications include an amino group, a thiol group, an alkyl group, or a halo group.
  • the nucleotide analogues/modifications comprise 2- amino-6-chloropurineriboside-5’ -triphosphate, 2-Aminopurine-riboside-5'-triphosphate; 2- aminoadenosine-5 ‘ -triphosphate, 2 ’ - Amino-2 ’ -deoxy cytidine-triphosphate, 2-thiocytidine-5 ’ - triphosphate, 2-thiouridine-5’ -triphosphate, 2’ -Fluorothymidine-5 ’-triphosphate, 2’-O-Methyl- inosine-5’ -triphosphate, 4-thiouridine-5 ’ -triphosphate, 5-aminoallylcytidine-5’-triphosphate, 5- aminoallyluridine-5’ -triphosphate, 5-bromocytidine-5’-triphosphate, 5-bromouridine-5’- triphosphate, 5 -Bromo-2 ’ -de
  • Base-modified nucleotides can comprise 5-methylcytidine-5’ -triphosphate, 7- deazaguanosine-5’ -triphosphate, 5-bromocytidine-5’ -triphosphate, and pseudouridine-5 ’- triphosphate, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4- thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl- uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-ps
  • At least one modified nucleotide and/or the at least one nucleotide analog can comprise 1 -methyladenosine, 2-methyladenosine, N6-methyladenosine, 2'-O-methyladenosine, 2-methylthio-N6-methyladenosine, N6-isopentenyladenosine, 2-methylthio-N6- isopentenyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6- hydroxynorvalylcarbamoyladenosine, 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine, inosine, 3 -methylcytidine, 2-O-methyl cytidine, 2-thiocytidine, N4-acet
  • chemical modifications comprise pseudouridine, Nl- methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4’-thiouridine, 5-methylcytosine, 5- methyluridine, 2-thio-l -methyl- 1-deaza-pseudouri dine, 2-thio-l-methyl-pseudouridine, 2-thio-5- aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4- methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4- thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2’-O- methyluridine.
  • 100% of the uracil in the coding sequence as defined herein can have a chemical modification.
  • a chemical modification is in the 5’-position of the uracil.
  • 100% of the uracil in the coding sequence (cds) of the RNA can have a chemical modification, e.g., a chemical modification that is in the 5’- position of the uracil.
  • at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the uracil nucleotides in the cds have a chemical modification, e.g., a chemical modification that is in the 5-position of said uracil nucleotides.
  • Such modifications may reduce the stimulation of the innate immune system (after in vivo administration of the RNA comprising such a modified nucleotide).
  • cds or “coding sequence” or “coding region” as used herein will be recognized and understood by the person of ordinary skill in the art, and are e.g., can refer to a sequence of several nucleotide triplets, which may be translated into a peptide or protein.
  • the cds of the RNA may comprise at least one modified nucleotide, wherein said at least one modified nucleotide may be selected from pseudouridine ( ⁇
  • the nucleotide mixture in an in vitro transcription reaction comprises a cap analog.
  • the cap analog is a capO, capl, cap2, a modified capO or a modified capl analog, or a capl analog as described below.
  • cap analog or “5 ’-cap structure” as used herein can refer to the 5’ structure of the RNA, particularly a guanine nucleotide, positioned at the 5 ’-end of an RNA, e.g., an mRNA.
  • the 5’-cap structure is connected via a 5 ’-5 ’-triphosphate linkage to the RNA.
  • a “5’-cap structure” or a “cap analogue” is not considered to be a “modified nucleotide” or “chemically modified nucleotides”.
  • 5’-cap structures which may be suitable include capO (methylation of the first nucleobase, e.g., m7GpppN), capl (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (anti-reverse cap analogue), modARCA (e.g., phosphothioate modARCA), inosine, Nl-methyl-guanosine, 2’- fluoro-guanosine, 7-deaza-guanosine, 8-ox
  • a 5 ’-cap (capO or capl) structure can be formed in chemical RNA synthesis, using capping enzymes, or in RNA in vitro transcription (co-transcriptional capping) using cap analogs.
  • cap analog as used herein can refer to a non-polymerizable di-nucleotide or tri-nucleotide that has cap functionality in that it facilitates translation or localization, and/or prevents degradation of the RNA when incorporated at the 5 ’-end of the RNA.
  • Non-polymerizable means that the cap analogue will be incorporated only at the 5 ’-terminus because it does not have a 5’ triphosphate and therefore cannot be extended in the 3 ’-direction by a template-dependent polymerase, (e.g., a DNA-dependent RNA polymerase).
  • a template-dependent polymerase e.g., a DNA-dependent RNA polymerase.
  • examples of cap analogues include m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogues (e.g., GpppG); dimethylated cap analogue (e.g., m2,7GpppG), trimethylated cap analogue (e.g. m2,2,7GpppG), dimethylated symmetrical cap analogues (e.g.
  • m7Gpppm7G anti reverse cap analogues
  • anti reverse cap analogues e.g., ARCA; m7,2’OmeGpppG, m7,2’dGpppG, m7,3’OmeGpppG, m7,3’dGpppG and their tetraphosphate derivatives.
  • Further cap analogues have been described previously, e.g., W02008/016473, WO2008/157688, WO2009/149253, WO2011/015347, and WO2013/059475.
  • cap analogues in that context are described in, e.g., WO2017/066793, WO2017/066781, WO20 17/066791, WO2017/066789, WO2017/053297, WO2017/066782, WO2018/075827 and WO2017/066797 wherein the disclosures relating to cap analogues are incorporated herewith by reference.
  • a capl structure is generated using tri-nucleotide cap analogue as disclosed in WO2017/053297, WO2017/066793, WO2017/066781, WO20 17/066791, WO2017/066789, WO2017/066782, WO2018/075827 and WO2017/066797.
  • any cap analog derivable from the structure disclosed in claim 1-5 of WO2017/053297 may be suitably used to co-transcriptionally generate a capl structure.
  • any cap analog derivable from the structure described in WO2018/075827 can be suitably used to co-transcriptionally generate a capl structure.
  • the capl analog is a capl trinucleotide cap analog.
  • the capl structure of the in vitro transcribed RNA is formed using co-transcriptional capping using tri -nucleotide cap analog m7G(5’)ppp(5')(2’OMeA)pG or m7G(5’)ppp(5’)(2’OMeG)pG.
  • the capl analog is m7G(5’)ppp(5’)(2’OMeA)pG.
  • the RNA (e.g., mRNA) comprises a 5 ’-cap structure, e.g., a capl structure.
  • the 5’ cap structure can improve stability and/or expression of the mRNA.
  • a capl structure comprising mRNA (produced by, e.g., in vitro transcription) has several advantageous features including an increased translation efficiency and a reduced stimulation of the innate immune system.
  • the in vitro transcribed RNA comprises at least one coding sequence encoding at least one peptide or protein.
  • the protein is an RNA-guided endonuclease.
  • the RNA- guided endonuclease is Cas9 or a derivative thereof.
  • the site-directed endonuclease is a Cas nuclease.
  • the Cas nuclease is a Cas9 polypeptide.
  • the Cas9 polypeptide is a Streptococcus pyogenes-&QVNQ& Cas9 (SpCas9) polypeptide.
  • the ORF further comprises one or more nucleotide sequences encoding a nuclear localization signal, such as one described herein.
  • the ORF comprises a nucleotide sequence encoding a site-directed endonuclease, such as a SpCas9 polypeptide and at least one NLS that is a nucleoplasmin and/or SV40 NLS.
  • the ORF comprises a nucleotide sequence encoding an N-terminal and/or C-terminal NLS operably-linked to a site-directed endonuclease, such as a SpCas9 polypeptide.
  • the disclosure provides an mRNA comprising a nucleotide sequence that is at least 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleotide sequence of SEQ ID NO: 122. In some embodiments, the disclosure provides an mRNA comprising a nucleotide sequence that is 100% identical to the nucleotide sequence of SEQ ID NO: 122.
  • the mRNA comprises a codon-optimized sequence comprising a nucleotide sequence that is at least 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleotide sequence of SEQ ID NO: 122.
  • the disclosure provides an mRNA comprising a nucleotide sequence that is at least 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the disclosure provides an mRNA comprising a nucleotide sequence that is 100% identical to the nucleotide sequence of SEQ ID NO: 121.
  • the mRNA can comprise at least one chemically modified nucleoside and/or nucleotide.
  • the chemically modified nucleoside and/or nucleotide is selected from pseudouridine, N1 -methylpseudouridine, and 5- methoxyuridine.
  • the chemically modified nucleoside is Nl- methylpseudouridine (e.g., 1 -methylpseudouridine).
  • At least about 80% or more (e.g, about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) of uridines in the mRNA are modified or replaced with N1 -methylpseudouridine.
  • 100% of the uridines (e. ., uracils) in the mRNA are modified or replaced with N1 -methylpseudouridine.
  • the disclosure provides an mRNA comprising a nucleotide sequence that is at least 85% ormore e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identical to the nucleotide sequence of SEQ ID NO: 121, wherein 100% of the uridines or uracils of the mRNA are modified or replaced with N1 -methylpseudouridine.
  • two or more e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
  • uridine or uracil residues are Nl- methylpseudouridine.
  • Some embodiments provide an mRNA comprising a nucleotide sequence that is at least 85% ormore (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleotide sequence of SEQ ID NO: 123.
  • the disclosure provides an mRNA comprising a nucleotide sequence that has one, two, three, four, or five mismatches to the nucleotide sequence of SEQ ID NO: 123.
  • the disclosure provides an mRNA comprising a nucleotide sequence that is 100% identical to the nucleotide sequence of SEQ ID NO: 123.
  • Some embodiments provide an mRNA comprising a nucleotide sequence that is 100% identical to the nucleotide sequence of SEQ ID NO: 121, wherein 100% of the uridines (e.g., uracils) of the mRNA are modified or replaced with N1 -methylpseudouridine.
  • the disclosure provides an mRNA comprising or consisting of the nucleotide sequence of SEQ ID NO: 123.
  • a mRNA can further comprise a 5’ cap, such as one described herein.
  • the 5’ cap is a cap-0, a cap-1, or a cap-2 structure.
  • SEQ ID NO: 122 is the sequence of a non-limiting exemplary parent Cas9 mRNA.
  • SEQ ID NO: 52 is codon-optimized sequence derived from the parent Cas9 mRNA, and some u are N1 -methylpseudouridines in SEQ ID NO: 123.
  • the CRISPR/Cas-mediated gene editing system used to genetically edit an AGT gene comprises a genome-targeting nucleic acid (e.g., a guide RNA) that can direct the activities of an RNA-guided endonuclease to a specific target sequence within the AGT gene.
  • a guide RNA comprises at least a spacer sequence that hybridizes to a specific nucleic acid sequence of interest, and a CRISPR repeat sequence.
  • the gRNA can be a single-molecule guide RNA (sgRNA) or a double-molecule guide RNA.
  • the RNA-guided endonuclease can be, for example a Cas endonuclease, including Cas9 endonuclease.
  • the Cas9 endonuclease can be, for example, a SpCas9, a SaCas9, or a SluCas9 endonuclease.
  • the RNA- endonuclease is a Cas9 variant.
  • the RNA-guided endonuclease is a small RNA-guided endonuclease.
  • the RNA-guided endonuclease is a small Cas endonuclease.
  • the gRNA comprises 5 ’ to 3 ’ : a crRNA and a tracrRNA, wherein the crRNA and tracrRNA hybridize to form a duplex.
  • the crRNA comprises a spacer sequence capable of targeting a target sequence in a target nucleic acid (e.g., genomic DNA molecule) and a crRNA repeat sequence.
  • the tracrRNA comprises a tracrRNA anti-repeat sequence and a 3’ tracrRNA sequence.
  • the 3’ end of the crRNA repeat sequence is linked to the 5’ end of the tracrRNA anti-repeat sequence, e.g., by a tetraloop, wherein the crRNA repeat sequence and the tracrRNA anti -repeat sequence hybridize to form the sgRNA.
  • the sgRNA comprises 5’ to 3’ : a spacer sequence, a crRNA repeat sequence, a tetraloop, a tracrRNA anti-repeat sequence, and a 3’ tracrRNA sequence.
  • the sgRNA comprise a 5’ spacer extension sequence.
  • the sgRNA comprise a 3’ tracrRNA extension sequence.
  • the 3’ tracrRNA can comprise, or consist of, one or more stem loops, for example, one, two, three, or more stem loops.
  • a spacer sequence in a gRNA is a sequence (e.g., a 20-nucleotide sequence) that defines the target sequence (e.g., a DNA target sequences, such as a genomic target sequence) of a target gene of interest (e.g., AGT gene).
  • the spacer sequence ranges from 15 to 30 nucleotides.
  • the spacer sequence can be, can be about, can be at least, or can be at most 10, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, or a number or a range between any of these values, of nucleotides in length.
  • a spacer sequence contains 20 nucleotides.
  • the gRNA is capable of hybridizing to the forward strand of the target dsDNA.
  • the gRNA is capable of hybridizing to the reverse strand of the target dsDNA.
  • the gRNA is capable of hybridizing to a DNA strand that is complementary to a target PAM-strand in a dsDNA.
  • a spacer sequence is a shortened or truncated modified oligonucleotide.
  • the shortened or truncated spacer sequence can have one or more nucleotides (e.g., 1, 2, 3, or 4) deleted from the 5’ end (i.e., 5’ truncation), from the 3’ end (i.e., 3’ truncation) or both (i.e., 5’ and 3’ truncations) of a spacer sequence.
  • a spacer sequence used herein can be a shortened or truncated modified oligonucleotide of a corresponding spacer sequence that is 20 nucleotides in length.
  • the spacer sequence can be 16, 17, 18, or 19 nucleotides in length.
  • the truncated spacer sequence has one, two, three or four nucleotides deleted from the 5’ terminus of the 20-nucloetide (nt) spacer sequence.
  • a truncated spacer sequence can comprise 16, 17, 18, or 19 nucleotides at the 3’ terminus of a 20-nt spacer sequence.
  • a truncated spacer sequence can have one, two, three or four nucleotides deleted from the 3’ terminus of the 20-nt spacer sequence.
  • a truncated spacer sequence can have one, two, three, four or more nucleotides deleted from both the 5’ terminus and the 3’ terminus of a spacer sequence (e.g., a 20-nt spacer).
  • a gRNA comprising a shortened or truncated spacer sequence as described herein can lead to a reduced off-target effect compared to a gRNA comprising a corresponding spacer sequence that is not truncated (e.g., a 20-nt spacer sequence), while still retaining high on-target cutting efficiency despite the truncation.
  • a gRNA comprising a truncated spacer sequence can induce a cutting efficiency of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
  • a truncated spacer sequence (e.g., 16, 17, 18, or 19-nt spacer sequence) can induce fewer off-target events than a corresponding spacer sequence that is not truncated (e.g., a 20-nt spacer sequence).
  • a truncated spacer sequence described herein does not edit any off-target site.
  • bioinformatics tools known and publicly available that can be used to evaluate off-target sites.
  • Exemplary bioinformatics tools for evaluating off-target sites include, for example, COSMID (CRISPR Off-target Sites with Mismatches, Insertions and Deletions available on the web at crispr.bme.gatech.edu) and CCTop (available on the web at cctop.cos.uni-heidelberg.de).
  • gRNAs can also be tested for off-target activity using cell-free methods including Digenome-seq (digested genome sequencing), DIG-seq (Digenome-seq using cell-free chromatin DNA), Extru-seq, SITE-seq (selective enrichment and identification of tagged genomic DNA ends by sequencing) and CIRCLE-seq (circularization for in vitro reporting of cleavage effects by sequencing) and cell culture-based methods including whole genome sequencing (WGS), Cas9 ChlP-seq (chromatin immunoprecipitation followed by high- throughput sequencing), IDLVs (integrase defective lentiviral vectors), GUIDE-seq (genomewide, unbiased identification of DSBs enabled by sequencing), LAM-HTGTS (linear amplification-mediated high-throughput genome-wide sequencing), BLESS (breaks labeling, enrichment on streptavidin, and next-generation sequencing), and BLISS (breaks labeling in situ and sequencing) in addition to
  • target nucleic acid may be used interchangeably throughout and can refer to any nucleic acid sequence that may be targeted by a gRNA sequence described herein.
  • the “target sequence” is in a target gene that is adjacent to a PAM sequence and is the sequence to be modified by an RNA-guided nuclease e.g., Cas9).
  • the “target sequence” can be on the so-called PAM-strand in a “target nucleic acid,” which is a double-stranded molecule containing the PAM-strand and a complementary non-PAM strand.
  • the gRNA spacer sequence hybridizes to the complementary sequence located in the non-PAM strand of the target nucleic acid of interest.
  • the gRNA spacer sequence is the RNA equivalent of the target sequence.
  • the spacer of a gRNA interacts with a target nucleic acid of interest in a sequence-specific manner via hybridization (i.e., base pairing).
  • the nucleotide sequence of the spacer thus varies depending on the target sequence of the target nucleic acid of interest.
  • the target sequence of the AGT gene is within exon 2, 3, 4, or 5 of the AGT gene.
  • the spacer sequence is designed to hybridize to a region of the target nucleic acid that is located 5' of a PAM recognizable by a Cas9 enzyme used in the system.
  • the spacer can perfectly match the target sequence or can have mismatches.
  • Each Cas9 enzyme has a particular PAM sequence that it recognizes in a target DNA.
  • S. pyogenes recognizes in a target nucleic acid a PAM that comprises the sequence 5'-NRG-3', where R comprises either A or G, where N is any nucleotide and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.
  • the target nucleic acid sequence has 20 nucleotides in length. In some embodiments, the target nucleic acid has less than 20 nucleotides in length. In some embodiments, the target nucleic acid has more than 20 nucleotides in length. In some embodiments, the target nucleic acid has at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid has at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid sequence has 20 bases immediately 5' of the first nucleotide of the PAM.
  • the target nucleic acid in a sequence comprising 5'-NNNNNNNNNNNNNNNNN ⁇ can be the sequence that corresponds to the Ns, wherein N can be any nucleotide, and the underlined NRG sequence (R is G or A) is the S. pyogenes PAM.
  • the PAM sequence used in the compositions and methods of the present disclosure as a sequence recognized by SpCas9 is NGG, wherein N can be A, T, C or G.
  • the percent complementarity between the spacer sequence and the target nucleic acid is about, at least, at least about, at most or at most about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.
  • the spacer sequence of the guide RNA and the target nucleic acid in the target gene is 100% complementary.
  • the percent complementarity between the spacer sequence and the target nucleic acid is 100% over the six contiguous 5'-most nucleotides of the target sequence of the complementary strand of the target nucleic acid.
  • the percent complementarity between the spacer sequence and the target nucleic acid is at least 60% over about 20 contiguous nucleotides.
  • the spacer sequence of the guide RNA and the target sequence in the target gene can contain up to 10 mismatches, e.g., up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 mismatch.
  • the gRNA is a chemically modified gRNA.
  • RNA modifications can be introduced to the gRNAs to enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes as described in the art.
  • the gRNAs described herein can comprise one or more modifications including intemucleoside linkages, purine or pyrimidine bases, or sugar.
  • a modification is introduced at the terminal of a gRNA with chemical synthesis or with a polymerase enzyme. Examples of modified nucleic acids and their synthesis are disclosed in WO2013/052523. Synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).
  • the chemically-modified gRNA comprises phosphorothioated 2'-O-methyl nucleotides at the 3' end and the 5' end of the gRNA. In some embodiments, the chemically-modified gRNA comprises phosphorothioated 2'-O-methyl nucleotides at the 3' end of the gRNA. In some embodiments, the chemically-modified gRNA comprises phosphorothioated 2'-O-methyl nucleotides at the 5'end of the gRNA.
  • the chemically-modified gRNA comprises three or four phosphorothioated 2'-O- methyl nucleotides at the 3' end and/or three or four at the 5' end of the gRNA.
  • any one of a gRNA comprising any of SEQ ID NOs: 64-71, 73, 75-84, and 127- 129, or a portion thereof can be chemically modified to have four or more phosphorothioated 2'- O-methyl nucleotides at the 3' end and/or three at the 5' end of the gRNA.
  • the number and position of the phosphorothioate linkages can vary.
  • the linkage can be between the first and second, the second and third, the third and fourth position, fourth and fifth, fifth and sixth, sixth and seventh, seventh and eighth, eighth and ninth, ninth or tenth, or further, position from the 5’ end of the gRNA.
  • the linkage can be between the first and second, the second and third, the third and fourth position, fourth and fifth, fifth and sixth, sixth and seventh, seventh and eighth, eighth and ninth, ninth or tenth, or further, position from the 3’ end of the gRNA.
  • the nucleotide analogues/modifications can comprise 2- amino-6-chloropurineriboside-5’ -triphosphate, 2-Aminopurine-riboside-5'-triphosphate; 2- aminoadenosine-5 ‘ -triphosphate, 2 ’ - Amino-2 ’ -deoxy cytidine-triphosphate, 2-thiocytidine-5 ’ - triphosphate, 2-thiouridine-5 ’ -triphosphate, 2’ -Fluorothymidine-5 ’-triphosphate, 2’-O-Methyl- inosine-5’ -triphosphate, 4-thiouridine-5 ’ -triphosphate, 5-aminoallylcytidine-5’-triphosphate, 5- aminoallyluridine-5’ -triphosphate, 5-bromocytidine-5’-triphosphate, 5-bromouridine-5’- triphosphate, 5 -Bromo-2 ’
  • Base-modified nucleotides can comprise 5-methylcytidine-5’ -triphosphate, 7- deazaguanosine-5’ -triphosphate, 5-bromocytidine-5’ -triphosphate, and pseudouridine-5 ’- triphosphate, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4- thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl- uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2 -thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-p
  • At least one modified nucleotide and/or the at least one nucleotide analog can comprise 1 -methyladenosine, 2-methyladenosine, N6-methyladenosine, 2'-O-methyladenosine, 2-methylthio-N6-methyladenosine, N6-isopentenyladenosine, 2-methylthio-N6- isopentenyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6- hydroxynorvalylcarbamoyladenosine, 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine, inosine, 3 -methylcytidine, 2-O-methyl cytidine, 2-thiocytidine, N4-acet
  • Chemical modifications can comprise pseudouridine, Nl- methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4’-thiouridine, 5-methylcytosine, 5- methyluridine, 2-thio-l -methyl- 1-deaza-pseudouri dine, 2-thio-l-methyl-pseudouridine, 2-thio-5- aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4- methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4- thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine or 2’-O- methyluridine.
  • the modification comprises a 2’-O-methyluridine (2'OMe- rU), a 2-O-methylcytidine (2'OMe-rC), 2'-O-methyladenosine (2'OMe-rA), or 2'-O- methylguanosine (2'OMe-rG).
  • the gRNA can comprise any number of modified nucleic acids.
  • the percentage of nucleic acids in a gRNA molecule that are modified can be, can be at least, can be about, or can be at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
  • the nucleotides of the gRNA comprise a 2’-O- methyl modification.
  • more than one guide RNA can be used with a CRISPR/Cas nuclease system.
  • Each guide RNA can contain a different targeting sequence, such that the CRISPR/Cas system cleaves more than one target nucleic acid.
  • one or more guide RNAs can have the same or differing properties such as activity or stability within the Cas9 RNP complex. Where more than one guide RNA is used, each guide RNA can be encoded on the same or on different vectors.
  • the gRNAs described herein can be produced by in vitro transcription (IVT), synthetic and/or chemical synthesis methods, or a combination thereof.
  • IVT in vitro transcription
  • One or more of enzymatic IVT, solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods can be utilized.
  • the gRNAs are made using IVT enzymatic synthesis methods. Methods of making polynucleotides by IVT are known in the art and are described in WO2013/151666. Polynucleotides constructs and vectors can be used to in vitro transcribe a gRNA described herein.
  • gRNAs guide RNAs
  • AGT angiotensinogen
  • the gRNA for targeting an AGT gene locus is 20 nucleotides in length.
  • the gRNA is shortened or truncated, and is 16, 17, 18, or 19 nucleotides in length.
  • the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, 127-129, and 155-162, or a portion thereof.
  • the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, 127-129, and 155-162, or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 64- 71, 73, 75-84, 127-129, and 155-162.
  • the spacer sequence can comprise or consist of a sequence selected from SEQ ID NOs: 64-71, 73, and 75-84.
  • the spacer sequence is shortened or truncated, and comprises 16, 17, 18, or 19 nucleotides at the 3’ terminus of any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, 127-129, and 155-162.
  • the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64, 67, 71, 73, 75, 78-82, 84, 127-129, and 155-162, or a portion thereof.
  • the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64, 67, 71, 73, 75, 78-82, 84, 127-129, and 155-162, or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 64, 67, 71, 73, 75, 78-82, 84, 127-129, and 155-162.
  • the spacer sequence can comprise or consist of a sequence selected from SEQ ID Nos: 64, 67, 71, 73, 75, 78-82, and 84. In some embodiments, the spacer sequence can comprise or consist of a sequence selected from SEQ ID Nos: 64-67, 70, 73, 75, 78, 83, 127-129, and 155-162.
  • the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-67, or a portion thereof. In some embodiments, the spacer sequence comprises 16, 17, 18, or 19 nucleotides at the 3’ terminus of any one of the sequences of SEQ ID Nos: 64-67. In some embodiments, the gRNA comprises a spacer sequence comprising the sequence of SEQ ID NO: 65 or SEQ ID NO: 67, or a portion thereof. In some embodiments, the spacer sequence comprises 16, 17, 18, or 19 nucleotides at the 3’ terminus of SEQ ID NO: 65 or SEQ ID NO: 67.
  • the gRNA induces a cutting efficiency of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% (e.g, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
  • the gRNA induces a cutting efficiency of at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% (e.g, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values).
  • the gRNA can be a single-guide RNA (sgRNA).
  • the gRNA can be a chemically-modified gRNA.
  • the chemically-modified gRNA can comprise one or more phosphorothioate linkages.
  • the chemically-modified gRNA can comprise one or more 2’-O- methyl nucleotides at the 3’ end, the 5’ end, or both. In some embodiments, 50% or less of the nucleotides of the gRNA comprise a 2’-O-methyl modification.
  • gRNA e.g., an sgRNA
  • 50 or less of the nucleotides can be a 2’-O-methyl nucleotide (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 of the nucleotides can be or can comprise 2’-O-methyl nucleotides).
  • 2’-O-methyl nucleotide e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 of the nucleotides can be or can comprise 2’-O-methyl nucleotides).
  • the 2’-O-methyl nucleotides can be at any position within the gRNA.
  • the 3 nucleotides at the 5’ end of the gRNA comprise or are 2’-O-methyl nucleotides.
  • approximately the last 35 or less of the nucleotides at the 3’ end of the gRNA comprise or are 2’-O-methyl nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 of the last nucleotides at the 3’ end of the sgRNA).
  • the nucleotides at positions 25-41 of the sgRNA can be or can comprise 2’-O-methyl nucleotides.
  • About 48% of the nucleotides of the gRNA can comprise a 2’-O-methyl modification.
  • the 5’ end of the gRNA can comprise three phosphorothioate linkages and the 3’ end of the gRNA can comprise three phosphorothioate linkages.
  • the linkage can be between the first and second, the second and third, and/or the third and fourth position from the 5’ end of the gRNA. In some embodiments, the linkage can be between the first and second, the second and third, and/or the third and fourth position from the 3 ’ end of the gRNA.
  • a gene can be edited using base editing.
  • Base editing is a genome editing method that directly generates point mutations within a specific region of the genomic DNA without causing double-stranded breaks (DSB).
  • DNA base editors (BEs) comprise fusions between a catalytically impaired Cas nuclease and a base-modification enzyme.
  • Nucleobase editors typically include a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., adenosine deaminase, cytidine deaminase).
  • a polynucleotide programmable nucleotide binding domain when in conjunction with a bound guide polynucleotide (e g., gRNA), can specifically bind to a target polynucleotide sequence and thereby localize the base editor to the target nucleic acid sequence desired to be edited.
  • base editing can be used to introduce a loss-of-function mutation (e.g., premature stop codons, destabilizing mutations, altering splicing, etc.).
  • base editing can be used to correct, a mutation (e.g., a disease-causing mutation).
  • base editors comprising a polynucleotide programmable nucleotide binding domain comprise all or a portion (e.g., a functional portion) of a CRISPR protein.
  • the polynucleotide programmable nucleotide binding domain comprises a nickase domain.
  • nickase shall be given its ordinary meaning, and shall also refer to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that is capable of cleaving only one strand of the two strands in a double- stranded nucleic acid molecule (e.g., DNA).
  • a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9
  • the Cas9-derived nickase domain can include a D10A mutation and a histidine at position 840.
  • a Cas9-derived nickase domain comprises an H840A mutation, while the amino acid residue at position 10 remains a D.
  • a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain (e g., the Cas9 is a nickase, referred to as an “nCas9” protein).
  • base editors comprise a polynucleotide programmable nucleotide binding domain which is catalytically dead (e.g., incapable of cleaving a target polynucleotide sequence).
  • the Cas9 can comprise both a D10A mutation and an H840A mutation.
  • a catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A orH840A) as well as a deletion of all or a portion (e.g., a functional portion) of a nuclease domain.
  • a base editor comprises an adenosine deaminase domain. Such an adenosine deaminase domain of a base editor can facilitate the editing of an adenine (A) nucleobase to a guanine (G) nucleobase by deaminating the A to form inosine (I), which exhibits base pairing properties of G.
  • an A-to-G base editor further comprises an inhibitor of inosine base excision repair, for example, a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine specific nuclease.
  • a uracil glycosylase inhibitor UGI domain or catalytically inactive inosine specific nuclease
  • the UGI domain or catalytically inactive inosine specific nuclease can inhibit or prevent base excision repair of a deaminated adenosine residue (e.g., inosine), which can improve the activity or efficiency of the base editor.
  • the adenosine deaminase can be derived from any suitable organism (e.g., E.
  • the adenine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations. Details of A to G nucleobase editing proteins are described W02018/027078 and Gaudelli, N.M., et al., “Programmable base editing of A»T to G»C in genomic DNA without DNA cleavage” Nature, 551, 464-471 (2017), the entire contents of which are hereby incorporated by reference.
  • a base editor comprises a fusion protein or complex comprising cytidine deaminase capable of deaminating a target cytidine (C) base of a polynucleotide to produce uridine (U), which has the base pairing properties of thymine.
  • C target cytidine
  • U uridine
  • the uridine base can then be substituted with a thymidine base (e.g., by cellular repair machinery) to give rise to a C:G to a T:A transition.
  • deamination of a C to U in a nucleic acid by a base editor cannot be accompanied by substitution of the U to a T.
  • the deamination of a target C in a polynucleotide to give rise to a U is a non-limiting example of a type of base editing that can be executed by a base editor described herein.
  • a base editor comprising a cytidine deaminase domain can mediate conversion of a cytosine (C) base to a guanine (G) base.
  • a U of a polynucleotide produced by deamination of a cytidine by a cytidine deaminase domain of a base editor can be excised from the polynucleotide by a base excision repair mechanism (e.g., by a uracil DNA glycosylase (UDG) domain), producing an abasic site.
  • the nucleobase opposite the abasic site can then be substituted (e.g., by base repair machinery) with another base, such as a C, by for example a translesion polymerase.
  • base repair machinery e.g., by base repair machinery
  • substitutions e.g., A, G or T
  • substitutions e.g., A, G or T
  • a base editor described herein comprises a deamination domain (e.g., cytidine deaminase domain) capable of deaminating a target C to a U in a polynucleotide.
  • the base editor can comprise additional domains which facilitate conversion of the U resulting from deamination to, in some embodiments, a T or a G.
  • a base editor comprising a cytidine deaminase domain can further comprise a uracil glycosylase inhibitor (UGI) domain to mediate substitution of a U by a T, completing a C- to-T base editing event.
  • UMI uracil glycosylase inhibitor
  • the base editor can comprise a uracil stabilizing protein as described herein.
  • a base editor can incorporate a translesion polymerase to improve the efficiency of C-to-G base editing, since a translesion polymerase can facilitate incorporation of a C opposite an abasic site (i.e., resulting in incorporation of a G at the abasic site, completing the C-to-G base editing event).
  • a base editor comprising a cytidine deaminase as a domain can deaminate a target C in any polynucleotide, including DNA, RNA and DNA-RNA hybrids.
  • a cytidine deaminase of a base editor comprises all or a portion (e.g., a functional portion) of an apolipoprotein B mRNA editing complex (APOBEC) family deaminase.
  • APOBEC apolipoprotein B mRNA editing complex
  • APOBEC is a family of evolutionarily conserved cytidine deaminases. Members of this family are C-to-U editing enzymes.
  • the N-terminal domain of APOBEC like proteins is the catalytic domain, while the C-terminal domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc dependent cytidine deaminase domain and is important for cytidine deamination.
  • APOBEC family members include APOBEC 1, AP0BEC2, AP0BEC3A, AP0BEC3B, APOBEC3C, AP0BEC3D (“AP0BEC3E” now refers to this), APOBEC3F, AP0BEC3G, AP0BEC3H, AP0BEC4, and Activation-induced (cytidine) deaminase.
  • the deaminases are activation-induced deaminases (AID).
  • an APOBEC deaminase incorporated into a base editor can comprise one or more mutations selected from the group consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBECl; D316R, D317R, R320A, R320E, R313A, W285A, W285Y, and R326E of hAPOBEC3G; and any alternative mutation at the corresponding position, or one or more corresponding mutations in another APOBEC deaminase.
  • a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of an APOBEC 1 deaminase.
  • a polynucleotide programmable nucleotide binding domain when in conjunction with a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence (i.e., via complementary base pairing between bases of the bound guide nucleic acid and bases of the target polynucleotide sequence) and thereby localize the base editor to the target nucleic acid sequence desired to be edited (e.g., a double- stranded DNA target).
  • the guide polynucleotide is a gRNA.
  • the guide polynucleotide is at least one single guide RNA (“sgRNA” or “gRNA”).
  • the methods described herein can utilize an engineered Cas protein.
  • a guide RNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user- defined ⁇ 20 nucleotide spacerthat defines the genomic target to be modified.
  • the specificity of the Cas protein for the genomic target of the Cas protein is partially determined by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome.
  • the spacer is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length.
  • the spacer of a gRNA can be or can be about 19, 20, or 21 nucleotides in length.
  • Provided herein includes a method of using genome editing to edit AGT thereby functionally reducing the expression of the AGT gene.
  • the method can be used to treat a subject, e.g., a patient with an AGT-associated diseases or condition.
  • the method comprises administering to the subject a plurality of nanoparticles complexed with (a) a guide RNA (gRNA) or a nucleic acid encoding a gRNA that targets AGT gene, and (b) a nucleic acid encoding an RNA-guided endonuclease, thereby relieving the AGT-related disease or disorder in the subject.
  • gRNA guide RNA
  • the subject can be administered with the plurality of nanoparticles one time.
  • the subject can be administered with the plurality of nanoparticles two or more times, for example twice, for the treatment.
  • Two administrations of the nanoparticles to the subject can be separated by a suitable time period.
  • the suitable time period is, or is about, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, three months, four months, five months, six months, a year, two years, three years, or more.
  • two of the two or more administrations are about two weeks to about two months apart, for example about three weeks
  • each two of the two or more administrations are about two weeks to about two months apart, for example about three weeks.
  • the suitable time period between two administrations can be the same as or different from the suitable time period between another two administrations.
  • the plurality of nanoparticles can be administered to the subject, for example, at a dose of about 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg
  • the plurality of nanoparticles is administered to the subject at a dose of about 0.05-10 mg/kg, for example 0.05-5 mg/kg, 0.05-2 mg/kg, 0.5-3 mg/kg or 0.1-1 mg/kg, per administration.
  • the AGT gRNA or the nucleic acid encoding the AGT gRNA is administered to the subject at a dose of, or a dose of about, 0.05-10 mg/kg, for example 0.1-1 mg/kg gRNA per administration.
  • the nucleic acid encoding the RNA-guided endonuclease is administered to the subject at a dose of, or a dose of about, 0.1-5 mg/kg, for example 0.5-3 mg/kg or 0.3-2 mg/kg per administration.
  • the dose can be the same or different for each of the administration to the subject.
  • the gRNA targets within or near a coding sequence in the AGT gene. In some embodiments, the gRNA targets a sequence within one of the 6 exons of the AGT gene. In some embodiments, the gRNA targets a sequence within exon 2, 3, 4, or 5 of the AGT gene.
  • the gRNA can comprise a spacer sequence complementary to a target sequence within exon 2, 3, 4, or 5 of the AGT gene.
  • the spacer(s) are complementary to a sequence within or near (for example, within any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bases from) exon 2, 3, 4, or 5 of the AGT gene.
  • the complementarity between the spacer of the gRNA and the target sequence in the AGT gene can be perfect or imperfect. In some embodiments, the complementarity can be at least 70%, 80%, 90%, 100% or a number or a range between any two of these values. In some embodiments, the complementarity is perfect, i.e., 100%.
  • the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, and 127-129, or a portion thereof.
  • the gRNA comprises a spacer sequence selected from SEQ ID NOs: 64-71, 73, 75- 84 and 127-129 or variants thereof having about, at least, at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology to any spacer of SEQ ID NOs: 64-71, 73, 75-84, and 127- 129.
  • the gRNA comprises a spacer sequence selected from SEQ ID NOs: 64-71, 73, 75-84, and 127-129 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 64-71, 73, 75-84, and 127-129.
  • the gRNA comprises or consists of a spacer sequence selected from SEQ ID NOs: 64-71, 73, 75-84, and 127- 129.
  • the gRNAs used in the methods herein can comprise two or more gRNAs, each comprising a spacer complementary to a sequence at the AGT gene locus (e.g., any one of SEQ ID NOs: 64-71, 73, 75-84, and 127-129 or variants thereof having at least 85% homology to any one of SEQ ID Nos: 64-71, 73, 75-84, and 127-129 or variants having no more than 3 mismatches compared to any one of SEQ ID NOs: 64-71, 73, 75-84, and 127- 129).
  • a spacer complementary to a sequence at the AGT gene locus e.g., any one of SEQ ID NOs: 64-71, 73, 75-84, and 127-129 or variants thereof having at least 85% homology to any one of SEQ ID Nos: 64-71, 73, 75-84, and 127-129 or variants having no more than 3 mismatches compared to any one of
  • the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64, 67, 71, 73, 75, 78-82, 84, and 127-129, or a portion thereof.
  • the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64, 67, 71, 73, 75, 78-82, 84, and 127-129 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 64, 67, 71, 73, 75, 78-82, 84, and 127-129.
  • the spacer sequence can comprise or consist of a sequence selected from SEQ ID NOs: 64, 67, 71, 73, 75, 78-82, 84, and 127-129.
  • the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-67, 70, 73, 75, 78, 83, and 127-129, or a portion thereof. In some embodiments, the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-67, 70, 73, 75, 78, 83, and 127-129 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 64-67, 70, 73, 75, 78, 83, and 127- 129.
  • the spacer sequence can comprise or consist of a sequence selected from SEQ ID NOs: 64- 67, 70, 73, 75, 78, 83, and 127-129.
  • the gRNAs used herein can enhance on-target activity while significantly reducing potential off-target effects (z.e., cleaving genomic DNA at undesired locations other than AGT gene).
  • the off-target binding is reduced by about, at least or at least about 80%, 85%, 90%, 95%, 98%, 99% or 100%.
  • the gRNA induces a cutting efficiency of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% (e.g., at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,
  • the gRNA induces a cutting efficiency of at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% (e.g., at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values).
  • the DNA endonuclease is a Cas endonuclease described herein or known in the art.
  • the Cas endonuclease can be naturally-occurring or non-naturally- occurring (e.g, recombinant or with mutations).
  • the DNA endonuclease is selected from a Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, or Cpfl endonuclease, or a functional derivative thereof.
  • the DNA endonuclease is a Cas9 endonuclease or a variant thereof.
  • the Cas9 endonuclease is from Streptococcus pyogenes (SpyCas9 or SpCas9).
  • the Cas9 endonuclease is from Staphylococcus lugdunensis (SluCas9).
  • the CRISPR/Cas nuclease system disclosed herein comprising a guide RNA (gRNA) or a nucleic acid sequence encoding the gRNA and an RNA-guided nuclease or a nucleic acid sequence encoding the RNA-guided endonuclease, can be delivered to a target cell via a conventional method.
  • components of a CRISPR/Cas nuclease system as disclosed herein is delivered to a target cell separately, either simultaneously or sequentially.
  • the components of the CRISPR/Cas nuclease system is delivered into a target together, for example, as a complex.
  • a gRNA and an RNA-guided nuclease are pre-complexed together to form a ribonucleoprotein (RNP), which can be delivered into a target cell.
  • RNP ribonucleoprotein
  • RNPs are useful for gene editing, at least because they minimize the risk of promiscuous interactions in a nucleic acid-rich cellular environment and protect the RNA from degradation.
  • Methods for forming RNPs are known in the art.
  • an RNP containing an RNA-guided nuclease e.g, a Cas nuclease, such as a Cas9 nuclease
  • a guide RNA targeting the AGT gene can be delivered to a target cell.
  • an RNP can be delivered to the target cell by electroporation.
  • an RNA-guided nuclease can be delivered to a cell in a DNA vector that expresses the RNA-guided nuclease in the cell.
  • an RNA- guided nuclease can be delivered to a cell in an RNA that encodes the RNA-guided nuclease and expresses the nuclease in the cell.
  • a gRNA targeting a gene can be delivered to a cell as a RNA, or a DNA vector that expresses the gRNA in the cell.
  • Delivery of an RNA-guided nuclease, gRNA, and/or an RNP can be through direct injection or cell transfection using known methods, for example, electroporation or chemical transfection. Other cell transfection methods may be used.
  • one or more of the nucleic acid sequences and/or polypeptides can be delivered to cells, either in vitro or in vivo, via viral based or non-viral based delivery systems, including adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, lentiviral vectors, herpes virus vectors, liposomes, lipid nanoparticles, poxviruses, naked DNA administration, plasmids, cosmids, phages, encapsulated cell technology, and the like.
  • AAV adeno-associated virus
  • retrovirus vectors retrovirus vectors
  • lentiviral vectors lentiviral vectors
  • herpes virus vectors herpes virus vectors
  • liposomes liposomes
  • lipid nanoparticles poxviruses
  • naked DNA administration plasmids, cosmids, phages, encapsulated cell technology, and the like.
  • LNP Lipid nanoparticle
  • the compounds of the compositions disclosed herein can be formulated in a liposome or lipid nanoparticle.
  • the compounds of the composition are formulated in a lipid nanoparticle (LNP).
  • LNP is a non-viral delivery system that can safely and effectively deliver nucleic acids to target organs (e.g., liver).
  • lipid nanoparticle refers to a nanoscopic particle composed of lipids having a size measured in nanometers (e.g., 1-5,000 nm).
  • the LNPs have a mean diameter of about, at least, at least about, at most or at most about 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm or a number or a range between any of these values.
  • the lipid nanoparticle particle size is about 50 to about 200 nm in diameter, or about 70 to about 180 nm in diameter, or about 80 to about 150 nm in diameter.
  • the particle size (e.g., mean diameter) of the LNP is in the 85-95 nm range.
  • the particle size (e.g., mean diameter) of the LNP is about 190 nm, 195 nm, 200 nm, 205 nm, or a range between any two of these values. Without being bound by any particular theory, it is believed that it can be advantageous to use small size LNP to deliver payload to the trabecular meshwork.
  • LNP with the size of 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, or a number or a range between any two of these values to deliver a CRISPR/Cas-mediated gene editing system to the trabecular meshwork cells of a subject.
  • the LNPs can comprise one or more ionizable cationic lipid described herein.
  • the LNP can comprise one or more ionizable cationic lipids selected from the group consisting of: C 12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA, or DOTAP.
  • the ionizable cationic lipid can be from about 30 mol % to about 70 mol % (e.g 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70 mol %) of the total lipid present in the LNP.
  • mol percent refers to a component’s molar percentage relative to total mols of all lipid components in the LNP (i.e., total mols of cationic lipids, neutral lipids, sterol and polymer conjugated lipids).
  • the LNP include from about 40% to about 60% ionizable cationic lipid of the total lipid in the LNP.
  • the lipid nanoparticles can include about 40%, 45%, 50% or 60% ionizable cationic lipid of the total lipid on a molar basis (based upon 100% total moles of lipids in the LNP).
  • the LNP comprises about 50 mol percent ionizable cationic lipids described herein.
  • the LNPs described herein can further comprise one or more non-cationic lipids (helper lipids).
  • the LNP can further comprise one or more neutral lipids, charged lipids, sterols, and polymers conjugated lipids.
  • the lipid nanoparticle comprises one or more neutral or zwitterionic lipids.
  • neutral lipid refers to any of a number of lipid species which exist either in an uncharged or neutral zwitterionic form at physiological pH. The selection of neutral lipids and other non-cationic lipids for use in the particles described herein is generally guided by consideration of, for example, lipid particle size and stability of the lipid particle in the bloodstream.
  • the non-cationic lipids contain saturated fatty acids with carbon chain lengths in the range of Cio to C20. In some embodiments, non-cationic lipids with mono- or di-unsaturated fatty acids with carbon chain lengths in the range of Cio to C20 are used. Additionally, non-cationic lipids having mixtures of saturated and unsaturated fatty acid chains can be used.
  • Suitable neutral lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dimyristoyl phosphatidylcholine (DMPC
  • the helper lipid is, or comprises, a PC class lipid (e.g., DLPC (12:0), DMPC (14:0), DPPC (16:0), DSPC (18:0), DOPC (18:1), DUPC (18:2), POPC (16:0, 18: 1), SOPC (18:0, 18:1)); a PE class like lipid (e.g, DOPE (18:1), DSPE (18:0), DPPE (16:0), DMPE (14:0) SOPE (18:0, 18: 1), POPE (16:0, 18:1)); a PG class like lipid e.g., DOPG (18: 1), DPPG (16:0)), or a mixture thereof.
  • a PC class lipid e.g., DLPC (12:0), DMPC (14:0), DPPC (16:0), DSPC (18:0), DOPC (18:1), DUPC (18:2), POPC (16:0, 18:1)
  • a PE class like lipid e
  • the helper lipid is, or comprise, l,2-dilauroyl-sn-glycero-3 -phosphocholine (DLPC), DMPC, DPPC, DSPC, DOPC, diundecanoylphosphatidylcholine (DUPC), POPC, 1- Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), DOPE, DSPE, DPPE, DMPE, 1- stearoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (SOPE, 18:0-18:1 PE), POPE, Dioleoyl phosphatidylglycerol (DOPG), Dipalmitoyl-sn-glycero-3-PG (DPPG), or a mixture thereof.
  • DLPC l,2-dilauroyl-sn-glycero-3 -phosphocholine
  • DMPC DPPC
  • DOPC diundecanoylphosphat
  • the neutral lipids can be from about 5 mol % to about 20 mol % (e.g., about 5 mol %, 10 mol %, 15 mol %, 20 mol %) of the total lipid present in the LNP.
  • the LNP include from about 10% neutral lipid of the total lipid in the LNP on a molar basis (based upon 100% total moles of lipids in the LNP).
  • the LNP can further comprise a sterol, such as cholesterol.
  • the sterol can be about 10 mol % to about 60 mol %, optionally about 20 mol % to about 50 mol %, more optionally about 30% to about 40% of the total lipid present in the LNP. In some embodiments, the sterol is about 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, or 50 mol % of the total lipid present in the LNP.
  • the sterol can be one or more of cholesterol, sitosterol, campesterol, plant sterols (also called phytosterols, e.g., stigmasterol, p-sitosterol ), sterols from algae (e.g., fucosterol), sterols from animals (also called “zoosterols”), and sterols from fungi and protozoa (e.g., ergosterol).
  • the LNPs disclosed herein can comprise tocopherols and hopanoids (Diploptene and Diplopterol) classes of compounds.
  • tocopherols and hopanoids (Diploptene and Diplopterol) classes of compounds are for replacing the sterols in the LNPs. In some embodiments, tocopherols and hopanoids (Diploptene and Diplopterol) classes of compounds are present in the LNP in addition to the sterol.
  • the LNP can further comprise polymer conjugated lipids such as polyethylene glycol (PEG)-modified lipids.
  • PEG-conjugated lipid include, for example, a PEG- diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), a PEG-dimyristoyl glycerol (DMG), or a mixture thereof.
  • the PEG conjugated lipid can be about 0 mol % to about 10 mol % of the total lipid in the LNP.
  • the PEG conjugated lipid is about 0 mol %, 0.5 mol %, 1 mol %, 1.5 mol %, 2 mol %, 3 mol %, 4 mol %, 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol % or 10 mol % (or a number or a range between any two of these values) of the total lipid present in the LNP.
  • the polymer conjugated lipid e.g., PEG conjugated lipid
  • the PEG-modified lipid can be or can comprise, for example, DMG-PEG, DSG-PEG, a PEG-ceramide, a PEG- phospholipid, or a combination thereof.
  • the ionizable cationic lipid may be C12-200, cKK-E12, DL1N-MC3, DLIN-MC4, DLIN-MC5, DODMA, or DOTAP;
  • the helper lipid may be l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-Dioleoyl-sn- glycero-3 -phosphocholine (DOPC);
  • the sterol may be cholesterol or sitosterol; and the PEG-lipid may be DMG-PEG.
  • the LNP comprises about 50 mol% of C12-200, DLIN- MC3, DLIN-MC4, DLIN-MC5, DODMA and/or DOTAP, about 10 mol% of DSPC, about 37.0- 39.5 mol% of cholesterol or sitosterol, and about 0.5-3 0% of DMG-PEG.
  • the LNP comprises about 50 mol% of C12-200, about 10 mol% of DSPC, about 37.0- 39.5 mol% of sitosterol, and about 0.5-1.5% of DMG-PEG.
  • the lipid nanoparticles can comprise varying concentration of constituent lipids.
  • the molar percent of an ionizable lipid in the total lipid of a lipid nanoparticle is about, at least, at least about, at most or at most about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or a number or range between any two of these values.
  • the molar percent of an ionizable lipid in a lipid nanoparticle is in a range between about 40-70% (e.g., about 60%).
  • the lipid nanoparticle can further comprise a helper lipid (e.g., DSPC), a sterol lipid (e.g., cholesterol), and PEG lipid or a phospholipid PEG conjugate.
  • a helper lipid e.g., DSPC
  • a sterol lipid e.g., cholesterol
  • PEG lipid or a phospholipid PEG conjugate e.g., PEG lipid or a phospholipid PEG conjugate.
  • the molar percent of a helper lipid in a lipid nanoparticle is about 5%-20% (e.g., about 10.5%)
  • the molar percent of a sterol lipid is about 10%-40% (e.g, about 21%)
  • the molar percent of a PEG lipid is about 0.5%- 10% (e.g., about 8.5%).
  • the LNP uptake into hepatocytes can be mediated by the Apolipoprotein E- low density lipoprotein receptor (ApoE-LDLR) or the /V-Acetyl-/J- galactosamine/asialoglycoprotein receptor pathway (GalNAc-ASGPR) (Sato et al., 2020, Journal of Controlled Release, 322, 217-226.).
  • Apolipoprotein E- low density lipoprotein receptor Apolipoprotein E- low density lipoprotein receptor (ApoE-LDLR) or the /V-Acetyl-/J- galactosamine/asialoglycoprotein receptor pathway (GalNAc-ASGPR) (Sato et al., 2020, Journal of Controlled Release, 322, 217-226.).
  • the LNP herein described for delivery of gRNA and Cas endonuclease to the cells can be formulated to follow the ApoE-LDLR uptake pathway.
  • the LNP herein described for delivery of gRNA and Cas endonuclease to the cells can be formulated to follow the GalNAc-ASGPR uptake pathway.
  • the LNP formulations herein described can be used to treat a subject with a disease or disorder that presents as heterozygous (HeFH) or homozygous (HoFH) for the loss of low density lipoprotein receptor (LDLR).
  • HeFH heterozygous
  • HoFH homozygous
  • LDLR low density lipoprotein receptor
  • the lipid nanoparticles comprise N- Acetylgalactosamine (GalNAc), an amino sugar derivative of galactose.
  • GalNAc is present in the LNP in a molar percentage of about, at least, at least about, at most, or at most about 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, or 6.0%.
  • GalNAc is present in the LNP in a molar percentage of about 2.5%.
  • the lipid nanoparticles comprise GalNAc in a molar percentage of no more than about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, or less.
  • the LNP can be administered by any appropriate route.
  • an effective amount of the LNP may be administered to the patient via injection, e.g., intravenous injection.
  • Cas9 mRNA, sgRNA targeting one or two loci in AGT gene, and donor DNA can each be separately formulated into lipid nanoparticles, or are all coformulated into one lipid nanoparticle.
  • Cas9 mRNA is formulated in a lipid nanoparticle, while sgRNA and donor DNA are delivered in an AAV vector.
  • Options are available to deliver the Cas9 nuclease as a DNA plasmid, as mRNA or as a protein.
  • the guide RNA can be expressed from the same DNA, or can also be delivered as an RNA.
  • the RNA can be chemically modified to alter or improve its half-life, or decrease the likelihood or degree of immune response.
  • the endonuclease protein can be complexed with the gRNA prior to delivery.
  • Viral vectors allow efficient delivery; split versions of Cas9 and smaller orthologs of Cas9 can be packaged in AAV, as can donors for HDR.
  • a range of non- viral delivery methods also exist that can deliver each of these components, or non- viral and viral methods can be employed in tandem.
  • nanoparticles can be used to deliver the protein and guide RNA, while AAV can be used to deliver a donor DNA.
  • a composition for carrying out the methods disclosed herein.
  • a composition can include one or more gRNA(s), an RNA-guided endonuclease or a nucleotide sequence encoding the RNA-guided endonuclease described herein.
  • the composition can further comprise a polynucleotide to be inserted (e.g., a donor template) in the AGT gene to affect the desired genetic modification of the methods disclosed herein.
  • compositions comprising (a) any of the gRNAs disclosed herein or a polynucleotide encoding the gRNA and (b) a Cas9 endonuclease or a nucleic acid encoding a Cas9 endonuclease.
  • the composition comprises: (a) a guide RNA (gRNA) that targets an angiotensinogen (AST) genomic locus, comprising a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, and 127-129 or a portion thereof, or a nucleic acid encoding the gRNA; and (b) a Cas9 endonuclease or a nucleic acid encoding a Cas9 endonuclease.
  • gRNA guide RNA
  • AST angiotensinogen
  • compositions can comprise any of the spacers and/or gRNAs disclosed herein.
  • the spacer sequence comprises a sequence selected from SEQ ID NOs: 64-71, 73, 75-84, and 127-129 or a portion thereof.
  • the gRNA can be a single-guide RNA (sgRNA).
  • the gRNA can be a chemically-modified gRNA.
  • the chemically-modified gRNA can comprise one or more phosphorothioate linkages.
  • the chemically-modified gRNA can comprise one or more 2’-O-methyl nucleotides at the 3’ end, the 5’ end, or both.
  • 50% or less of the nucleotides of the gRNA comprise a 2’-O-methyl modification (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or a number or a range between any two of these values).
  • a 2’-O-methyl modification e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,
  • about 48% of the nucleotides of the gRNA comprise a 2’-O-methyl modification (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or a number or a range between any two of these values).
  • a 2’-O-methyl modification e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,
  • the 5’ end of the gRNA comprises three phosphorothioate linkages and the 3’ end of the gRNA comprises three phosphorothioate linkages.
  • the Cas9 endonuclease is selected from S. pyogenes Cas9, S. aureus Cas9, N. meningitides Cas9, S. thermophilus CRISPR1 Cas9, S. thermophilus CRISPR 3 Cas9, and T. denticola Cas9.
  • the composition can comprise (a) the AGT gRNA and (b) the Cas9 endonuclease, and the AGT gRNA and Cas 9 nuclease can be formulated as a ribonucleoprotein particle (RNP).
  • the composition can comprise (a) a nucleic acid encoding an AGT gRNA and (b) a nucleic acid encoding a Cas9 endonuclease.
  • (a) and/or (b) is present on a viral vector.
  • the viral vector can be an adeno-associated viral vector.
  • the gRNA or the nucleic acid encoding a gRNA of (a), the Cas9 endonuclease or the nucleic acid encoding a Cas9 endonuclease of (b), or both can be complexed with a liposome or lipid nanoparticle (LNP).
  • the lipid nanoparticle can comprise one or more neutral lipids, charged lipids, ionizable lipids, steroids, and polymers conjugated lipids.
  • the lipid nanoparticle can comprise cholesterol, a polyethylene glycol (PEG) lipid, or both.
  • the one or more gRNA(s) each comprises a spacer complementary to a genomic sequence within or near (for example, within any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bases from) any exon of the AGE gene.
  • the gRNA targets a sequence within any one of exons 2-5 of the AGE gene.
  • the gRNA can comprise a spacer sequence complementary or identical to a target sequence within any one of exons 2-5 of the AGE gene.
  • a gRNA comprises a spacer sequence of any one of SEQ ID NOs: 64-71, 73, 75-84, and 127-129 or a variant thereof having at least 85% homology to the spacer sequence of any one of SEQ ID NOs: 64-71, 73, 75-84, and 127-129.
  • the RNA-guided endonuclease is a Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cpfl endonuclease, or a functional derivative thereof.
  • the DNA endonuclease is Cas9.
  • the Cas9 endonuclease is from Streptococcus pyogenes (SpyCas9).
  • the Cas9 endonuclease is from Staphylococcus lugdunensis (SluCas9).
  • a DNA sequence that is transcribed to the nucleic acid encoding the DNA endonuclease is codon optimized.
  • the nucleic acid encoding the DNA endonuclease (e.g, an mRNA) comprises a 5’ CAP structure and 3’ polyA tail.
  • the nucleic acid encoding the DNA endonuclease is linked to the gRNA via a covalent bond.
  • one or more of the nucleic acid sequences and/or polypeptides can be delivered to cells, either in vitro or in vivo, via viral based or non-viral based delivery systems, including adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, lentiviral vectors, herpes virus vectors, liposomes, lipid nanoparticles, poxviruses, naked DNA administration, plasmids, cosmids, phages, encapsulated cell technology, and the like.
  • AAV adeno-associated virus
  • the compounds of the compositions disclosed herein can be formulated in a liposome or lipid nanoparticle.
  • the compounds of the composition are formulated in a lipid nanoparticle (LNP).
  • LNP is a non-viral delivery system that can safely and effectively deliver nucleic acids to target organs (e.g, liver).
  • lipid nanoparticle refers to a nanoscopic particle composed of lipids having a size measured in nanometers (e.g., 1-5,000 nm).
  • the lipids comprised in the lipid nanoparticles comprise cationic lipids and/or ionizable lipids.
  • Any suitable cationic lipids and/or ionizable lipids known in the art can be used to formulate LNPs for delivery of gRNA and Cas endonuclease to the cells.
  • Exemplary cationic lipids include one or more amine group(s) bearing positive charge.
  • the cationic lipids are ionizable such that they can exist in a positively charged or neutral from depending on pH.
  • the cationic lipid of the lipid nanoparticle comprises a protonatable tertiary amine head group that shows positive charge at low pH.
  • the lipid nanoparticles can further comprise one or more neutral lipids (e.g., Distearoylphosphatidylcholine (DSPC), l,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-Dimyristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), 1,2-Dipalmitoyl-sn- glycero-3 -phosphoryl ethanolamine (DPPE) etc. as a helper lipid), charged lipids, steroids, and polymers conjugated lipids.
  • the LNP can comprise cholesterol.
  • the LNP can comprise a polyethylene glycol (PEG) lipid.
  • the lipid nanoparticles can comprise varying concentration of constituent lipids.
  • the molar percent of an ionizable lipid in the total lipid of a lipid nanoparticle is about, at least, at least about, at most or at most about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or a number or range between any two of these values.
  • the molar percent of an ionizable lipid in a lipid nanoparticle is in a range between about 40-70% (e.g, about 60%).
  • the lipid nanoparticle can further comprise a helper lipid (e.g., DSPC), a sterol lipid (e.g, cholesterol), and PEG lipid or a phospholipid PEG conjugate.
  • a helper lipid e.g., DSPC
  • a sterol lipid e.g, cholesterol
  • PEG lipid or a phospholipid PEG conjugate e.g., PEG lipid or a phospholipid PEG conjugate.
  • the molar percent of a helper lipid in a lipid nanoparticle is about 5%-20% (e.g., about 10.5%)
  • the molar percent of a sterol lipid is about 10%- 40% (e.g, about 21%)
  • the molarpercent of aPEGlipid is about 0.5%-10% (e.g, about 8.5%).
  • the LNP uptake into hepatocytes can be mediated by the Apolipoprotein E- low density lipoprotein receptor (ApoE-LDLR) or the A-Acetyl- - galactosamine/asialoglycoprotein receptor pathway (GalNAc-ASGPR) (Sato et al., 2020, Journal of Controlled Release, 322, 217-226.).
  • Apolipoprotein E- low density lipoprotein receptor Apolipoprotein E- low density lipoprotein receptor (ApoE-LDLR) or the A-Acetyl- - galactosamine/asialoglycoprotein receptor pathway (GalNAc-ASGPR) (Sato et al., 2020, Journal of Controlled Release, 322, 217-226.).
  • the LNP herein described for delivery of gRNA and Cas endonuclease to the cells can be formulated to follow the ApoE-LDLR uptake pathway.
  • the LNP herein described for delivery of gRNA and Cas endonuclease to the cells can be formulated to follow the GalNAc-ASGPR uptake pathway.
  • the LNP formulations herein described can be used to treat a subject with a disease or disorder that presents as heterozygous (HeFH) or homozygous (HoFH) for the loss of low density lipoprotein receptor (LDLR).
  • HeFH heterozygous
  • HoFH homozygous
  • LDLR low density lipoprotein receptor
  • the lipid nanoparticles comprise N- Acetylgalactosamine (GalNAc), an amino sugar derivative of galactose.
  • GalNAc is present in the LNP in a molar percentage of about, at least, at least about, at most, or at most about 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, or 6.0%.
  • GalNAc is present in the LNP in a molar percentage of about 2.5%.
  • the lipid nanoparticles disclosed herein, in some embodiments, do not comprise GalNAc.
  • the lipid nanoparticles comprise GalNAc in a molar percentage of no more than about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, or less.
  • the concentration of the nanoparticles in the compositions disclosed herein is about 58.2 mg/mL (e.g., of total lipids), and the nanoparticles are complexed with a total of about 2 mg/mL of nucleic acid of (a) the AGT gRNA and (b) the Cas9 mRNA.
  • the concentration of the plurality of nanoparticles is about 58.2 mg/mL, and the nanoparticles are complexed with (a) the AGT gRNA at about 1.5 mg/mL, and (b) the Cas9 mRNA at about 0.5 mg/mL.
  • the relative amount of the total RNA ((a) the AGT gRNA or a nucleic acid encoding a gRNA that targets AGT gene, and (b) a nucleic acid encoding a RNA-guided endonuclease) and the total lipid in the nanoparticles can vary in different embodiments.
  • the nanoparticles can have the total lipid and the total RNA at a weight ratio of about 15:1, 16:1, 17:1, 18: 1, 19:1, 20:1, 21:1, 22: 1, 23: 1, 24:1, 25:1, 26: 1, 27: 1, 28:1, 29:1, or 30: 1.
  • the nanoparticles can have the total lipid and the total RNA at a weight ratio of about 30: 1. In some embodiments, the nanoparticles can have the total lipid and the total RNA at a molar ratio of about 30: 1, 31 :1, 32:1, 33: 1, 34:1, 35:1, 36: 1, 37:1, 38: 1, 39: 1, 40:1, 41: 1, 42:1, 43:1, 44:1, 45: 1, 46:1, 47:1, 48: 1, 49:1 or 50:1. In some embodiments, the nanoparticles can have the total lipid and the total RNA at a molar ratio of about 40: 1.
  • the concentration of the nanoparticles in the compositions disclosed herein is about, at least, at least about, at most or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 mg/m
  • the RNA in the nanoparticles is formulated at a concentration of about, at least, at least about, at most, or at most about 50, 75, 100, 200, 400, 600, 800, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 pg/ml or a number or a range between any two of these values.
  • the amount e.g., relative amount of (a) the AGT gRNA or a nucleic acid encoding a gRNA that targets AGT gene, and (b) a nucleic acid encoding a RNA-guided endonuclease (e.g., a mRNA encoding a Cas protein (e.g., a Cas9 mRNA)) in the nanoparticles can vary.
  • a RNA-guided endonuclease e.g., a mRNA encoding a Cas protein (e.g., a Cas9 mRNA)
  • the nanoparticles can have the nucleic acid encoding the RNA-guided endonuclease (e.g., a SpCas9 mRNA) and the AGT gRNA in a 1:5, 1 :4.5, 1:4, 1 :3.5, 1:3, 1 :2.5, 1:2, 1 :1.5, 1 :1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4: 1, 4.5: 1, or 5:1 ratio (by weight).
  • the nanoparticles can have the nucleic acid encoding the RNA-guided endonuclease and the AGT gRNA in a 3 : 1 ratio (by weight).
  • the plurality of nanoparticles is administered to the subject at a dose of about 0.05-10 mg/kg (determined by the total nucleic acids (e.g., the total of AGT gRNA and Cas9 mRNA)) per administration.
  • a single dose or each dose of the plurality of nanoparticles administrated to the subject can be nanoparticles complexed with 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg, 0.175 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg
  • the plurality of nanoparticles is administered to the subject at a dose of, or a dose about, 0.1 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 1 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg or 3 mg/kg (determined by the total of AGT gRNA and SpCas9 mRNA).
  • the lipid nanoparticles can have a mean diameter of, e.g., about, at least, at least about, at most or at most about 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, or a number or a range between any of these values.
  • the lipid nanoparticle particle size is about 50 to about 100 nm in diameter, or about 70 to about 90 nm in diameter, or about 55 to about 95 nm in diameter.
  • the compounds of the composition described herein are encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle.
  • the encapsulation can be full encapsulation, partial encapsulation, or both.
  • the nucleic acid and/or polypeptides are fully or substantially encapsulated e.g., greater than 90% of the RNA) in the lipid nanoparticle.
  • one or more compounds herein described are associated with a liposome or lipid nanoparticle via a covalent bond or non-covalent bond.
  • any of the compounds in the composition can be separately or together contained in a liposome or lipid nanoparticle.
  • a recombinant adeno-associated virus (AAV) vector can be used for delivery.
  • Techniques to produce rAAV particles, in which an AAV genome to be packaged that includes the polynucleotide to be delivered, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV typically requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i. e. , not in) the rAAV genome, and helper virus functions.
  • the AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived, and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes described herein. Production of pseudotyped rAAV is disclosed in, for example, international patent application publication number WO 2001/83692.
  • AAV particles packaging polynucleotides encoding compositions of the disclosure may comprise or be derived from any natural or recombinant AAV serotype.
  • the AAV particles may utilize or be based on a serotype selected from any of the following serotypes, and variants thereof including but not limited to AAV1, AAV10, AAV106.1/hu.37, AAV11, AAV114.3/hu.4O, AAV12, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV16.12/hu. l l, AAV16.3, AAV16.8/hu,10, AAV161.1O/hu.6O,
  • AAV42-8 AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2/hu.28, AAV46.6/hu.29, AAV4- 8/rl l.64, AAV4-8/rh.64, AAV4-9/rh.54, AAV5, AAV52.1/hu.2O, AAV52/hu.
  • the AAV serotype is, or has, a mutation in the AAV9 sequence as described by N Pulichla et al. (Molecular Therapy 19(6): 1070- 1078 (2011)), such as but not limited to, AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84.
  • the AAV serotype is, or has, a sequence as described in U.S. Patent No. 6,156,303, such as, but not limited to, AAV3B (SEQ ID NO: 1 and 10 of US Patent No. 6,156,303), AAV6 (SEQ ID NO: 2, 7 and 11 of US Patent No. 6,156,303), AAV2 (SEQ ID NO: 3 and 8 of US Patent No. 6,156,303), AAV3A (SEQ ID NO: 4 and 9 of US Patent No. 6,156,303), or derivatives thereof.
  • AAV3B SEQ ID NO: 1 and 10 of US Patent No. 6,156,303
  • AAV6 SEQ ID NO: 2, 7 and 11 of US Patent No. 6,156,303
  • AAV2 SEQ ID NO: 3 and 8 of US Patent No. 6,156,303
  • AAV3A SEQ ID NO: 4 and 9 of US Patent No. 6,156,303
  • the serotype may be AAVDJ or a variant thereof, such as AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology 82(12): 5887- 5911 (2008).
  • the amino acid sequence of AAVDJ8 may comprise two or more mutations in order to remove the heparin binding domain (HBD).
  • HBD heparin binding domain
  • 7,588,772 may comprise two mutations: (1) R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gin) and (2) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).
  • R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gin)
  • R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr
  • K406R where lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg)
  • R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gin)
  • R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).
  • the AAV serotype is, or has, a sequence as described in International Publication No. W02015121501, such as, but not limited to, true type AAV (ttAAV) (SEQ ID NO: 2 of W02015121501), "UPenn AAV10” (SEQ ID NO: 8 of W02015/12I501), "Japanese AAV10” (SEQ ID NO: 9 of W02015/121501), or variants thereof.
  • true type AAV ttAAV
  • UPenn AAV10 SEQ ID NO: 8 of W02015/12I501
  • Japanese AAV10 Japanese AAV10
  • AAV capsid serotype selection or use can be from a variety of species.
  • the AAV is an avian AAV (AAAV).
  • the AAAV serotype can be, or have, a sequence as described in U.S. Patent No. 9,238,800, such as, but not limited to, AAAV (SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, and 14 of US 9,238,800), or variants thereof.
  • the AAV is a bovine AAV (BAAV).
  • BAAV serotype can be, or have, a sequence as described in U.S. Patent No. 9,193,769, such as, but not limited to, BAAV (SEQ ID NO: 1 and 6 of U.S. 9,193,769), or variants thereof.
  • the BAAV serotype can be or have a sequence as described in U.S. Patent No. 7,427,396, such as, but not limited to, BAAV (SEQ ID NO: 5 and 6 of US7427396), or variants thereof.
  • the AAV is a caprine AAV.
  • the caprine AAV serotype can be, or have, a sequence as described in U.S. Patent No. 7427396, such as, but not limited to, caprine AAV (SEQ ID NO: 3 of US7427396), or variants thereof.
  • the AAV is engineered as a hybrid AAV from two or more parental serotypes.
  • the AAV is AAV2G9 which comprises sequences from AAV2 and AAV9.
  • the AAV2G9 AAV serotype can be, or have, a sequence as described in US2016/0017005.
  • the AAV is a serotype generated by the AAV9 capsid library with mutations in amino acids 390-627 (VP1 numbering) as described by Pulichla et al. (Molecular Therapy 19(6): 1070-1078 (2011).
  • the serotype and corresponding nucleotide and amino acid substitutions can be, but is not limited to, AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G, C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G1203A, G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S), AAV9.14 (
  • the AAV is a serotype comprising at least one AAV capsid CD8+ T-cell epitope.
  • the serotype can be AAV1, AAV2 or AAV8.
  • the AAV may be a variant, such as PHP. A or PHP.B as described in Deverman. 2016. Nature Biotechnology. 34(2): 204-209.
  • AAV vector serotypes can be matched to target cell types.
  • the following exemplary cell types can be transduced by the indicated AAV serotypes among others.
  • viral vectors include, but are not limited to, lentivirus, alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, Epstein Barr virus, papovavirus, poxvirus, vaccinia virus, and herpes simplex virus.
  • Cas9 mRNA, sgRNA targeting one or two loci in AGT gene, and donor DNA can each be separately formulated into lipid nanoparticles, or are all coformulated into one lipid nanoparticle.
  • Cas9 mRNA can be formulated in a lipid nanoparticle, while sgRNA and donor DNA can be delivered in an AAV vector.
  • Options are available to deliver the Cas9 nuclease as a DNA plasmid, as mRNA or as a protein.
  • the guide RNA can be expressed from the same DNA, or can also be delivered as an RNA.
  • the RNA can be chemically modified to alter or improve its half-life, or decrease the likelihood or degree of immune response
  • the endonuclease protein can be complexed with the gRNA prior to delivery.
  • Viral vectors allow efficient delivery; split versions of Cas9 and smaller orthologs of Cas9 can be packaged in AAV, as can donors for HDR.
  • a range of non- viral delivery methods also exist that can deliver each of these components, or non- viral and viral methods can be employed in tandem.
  • nanoparticles can be used to deliver the protein and guide RNA, while AAV can be used to deliver a donor DNA.
  • a composition described above can further have one or more additional reagents, where such additional reagents are selected from a buffer, a buffer for introducing a polypeptide or polynucleotide into a cell, a wash buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for in vitro production of the polypeptide from DNA, adaptors for sequencing and the like.
  • a buffer can be a stabilization buffer, a reconstituting buffer, a diluting buffer, or the like.
  • a composition can also include one or more components that can be used to facilitate or enhance the on-target binding or the cleavage of DNA by the endonuclease, or improve the specificity of targeting.
  • RNA compositions are generally formulated to achieve a physiologically compatible pH, and range from a pH of about 3 to a pH of about 11, about pH 3 to about pH 7, depending on the formulation and route of administration. In some embodiments, the pH is adjusted to a range from about pH 5 to about pH 8.
  • Suitable excipients can include, for example, carrier molecules that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.
  • Other exemplary excipients include antioxidants (for example and without limitation, ascorbic acid), chelating agents (for example and without limitation, EDTA), carbohydrates (for example and without limitation, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (for example and without limitation, oils, water, saline, glycerol and ethanol), wetting or emulsifying agents, pH buffering substances, and the like.
  • Physiologically tolerable carriers are well known in the art.
  • Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline.
  • Aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclusion of water Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • the amount of an active compound used in the cell compositions that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • stable or “stability” as used herein can refer to the ability of the compounds herein described (e.g., an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and/or gRNA) to maintain therapeutic efficacy (e.g. , all or the maj ority of its intended biological activity and/or physiochemical integrity) over extended periods of time.
  • therapeutic efficacy e.g. , all or the maj ority of its intended biological activity and/or physiochemical integrity
  • the stability of one or more of the compounds described herein can be 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, or more than 3 years.
  • the temperature of storage can vary.
  • the storage temperature can be, can be about, can be at least, or can be at least about -80°C, -65°C, -20°C, 5°C, or a number or range between any two of these values.
  • the storage temperature is less than or equal to -65°C.
  • the compounds herein described e.g., an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and/or gRNA
  • a composition can be delivered via transfection such as calcium phosphate transfection, DEAE- dextran mediated transfection, cationic lipid-mediated transfection, electroporation, electrical nuclear transport, chemical transduction, electrotransduction, Lipofectamine-mediated transfection, Effectene-mediated transfection, lipid nanoparticle (LNP)-mediated transfection, or any combination thereof.
  • the composition is introduced to the cells via lipid-mediated transfection using a lipid nanoparticle.
  • compositions herein described can be administered to a subject in need thereof to treat an AGT-associated condition.
  • the present disclosure also provides a gene therapy approach for treating an AGT-associated condition in a subject by editing the AGT gene of the subject.
  • the AGT gene of relevant cells in the subject e.g., hepatocytes
  • the materials and methods described herein which uses RNA-guided endonuclease, such as Cas9, to edit a target sequence from a genome thereby resulting in reduced expression of AGT in the liver, thereby providing a long-term or permanent cure for the AGT- associated condition by permanently reducing the levels of AGT protein and its downstream angiotensin peptides.
  • associated indicates a relation between the two items such that the occurrence of an item (e.g., AGT protein level) is accompanied by the occurrence of the other item (e.g., a disease or condition), which includes but is not limited to a cause-effect relation and sign/symptom- disease relation.
  • an item e.g., AGT protein level
  • the other item e.g., a disease or condition
  • angiotensinogen-associated disease or “AGT- associated disease” or “RAAS-pathway-associated disease is a disease or disorder that is caused by, or associated with renin-angiotensin-aldosterone system (RAAS) activation, or a disease or disorder the symptoms of which or progression of which responds to RAAS inactivation.
  • RAAS renin-angiotensin-aldosterone system
  • AGT-associated disease includes a disease, disorder or condition that would benefit from reduction in AGT expression. Such diseases are typically associated with high blood pressure.
  • angiotensinogen-associated diseases include hypertension, e.g., borderline hypertension (also known as prehypertension), primary hypertension (also known as essential hypertension or idiopathic hypertension), secondary hypertension (also known as inessential hypertension), hypertensive emergency (also known as malignant hypertension), hypertensive urgency, isolated systolic or diastolic hypertension, pregnancy-associated hypertension (e.g., preeclampsia, eclampsia, and post-partum preelampsia), diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension (also known as renal hypertension), Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, labile hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arterios
  • the AGT-associated disease can be, e.g., hypertension, hypertensive heart disease, hypertensive nephropathy, pregnancy-associated hypertension, atherosclerosis, arteriosclerosis, chronic kidney disease, glomerulosclerosis, coarctation of the aorta, aortic aneurism, ventricular fibrosis, Cushing's syndrome, and other glucocorticoid excess states including chronic steroid therapy, pheochromocytoma, primary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid/parathyroid disease, heart failure, myocardial infarction, stroke, diabetes mellitus, renal failure, and systemic sclerosis.
  • hypertension hypertensive heart disease
  • hypertensive nephropathy pregnancy-associated hypertension
  • atherosclerosis arteriosclerosis
  • chronic kidney disease chronic kidney disease
  • glomerulosclerosis coarctation of the aorta, aortic
  • an AGT-associated disease is resistant hypertension.
  • “Resistant hypertension” is blood pressure that remains above goal (e.g., 140/90 mmHg) despite use of three antihypertensive medications of different classes, including diuretic.
  • an AGT-associated disease is refractory hypertension.
  • “Refractory hypertension” is blood pressure that is uncontrolled despite using five or more antihypertensive medications of different classes, including a long-acting thiazide diuretic and a mineralocorticoid receptor antagonist (MRA) at maximal or maximally tolerated doses.
  • MRA mineralocorticoid receptor antagonist
  • an AGT-associated disease is pregnancy-associated hypertension (e.g., pregnancy -induced hypertension, preeclampsia, and eclampsia).
  • pregnancy-associated hypertension e.g., pregnancy -induced hypertension, preeclampsia, and eclampsia.
  • the nanoparticles e.g., LNPs comprising ionizable lipids
  • the nanoparticles complexed with (a) a guide RNA (gRNA) or a nucleic acid encoding a gRNA that targets AGT gene, and (b) a nucleic acid encoding an RNA-guided endonuclease (e.g., Cas9 mRNA) is administered to a subject in need via IV infusion.
  • the administration can be, for example, a single dose, or two or more doses.
  • the nanoparticles can be, for example, rapidly distributed to, e.g., liver of the subject, and the nanoparticles can enter hepatocytes of the subject (e.g., via endocytosis).
  • ionizable lipid disruption of endosome can break the nanoparticles, thereby releasing the nucleic acid encoding the RNA-guided endonuclease (e.g., Cas9 mRNA) from the nanoparticles.
  • the RNA-guided endonuclease e.g., Cas9
  • the RNA-guided endonuclease (e.g., Cas9) can be synthesized and form endonuclease-gRNA RNP complex to achieve gene-editing.
  • endogenous DNA repair through non-homologous end joining results in introduction of indels into AGT gene, leading to frameshift mutations that prevent production of functional AGT protein.
  • the methods disclosed herein result in modulation (e.g., reduction) in AGT expression.
  • robust on-target editing of AGT gene can be achieved with minimal or no off-target editing.
  • Disclosed herein include methods for treating an AGT-associated or RAAS- pathway-associated disease or disorder in a subject in need thereof.
  • the method comprises administering to the subject any one of the compositions disclosed herein, thereby treating the AGT-associated or RAAS-pathway-associated disease or disorder in the subject.
  • Disclosed herein include methods for treating a subject that has or is suspected of having hypertension or preeclampsia.
  • the method comprises administering to the subject any one of the compositions disclosed herein, thereby treating the hypertension or preeclampsia.
  • the method comprises administering to the subject a composition comprising a plurality of nanoparticles complexed with: (a) a guide RNA (gRNA) that targets an AGT genomic locus, comprising a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, and 127-129 or a portion thereof, or a nucleic acid encoding the gRNA; and (b) a Cas9 endonuclease or a nucleic acid encoding a Cas9 endonuclease, thereby treating the AGT- associated or RAAS-pathway-associated disease or disorder in the subject.
  • gRNA guide RNA
  • the method comprises administering to the subject a composition comprising a plurality of nanoparticles complexed with: (a) a gRNA that targets an AGT genomic locus, comprising a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, and 127-129 or a portion thereof, or a nucleic acid encoding the gRNA; and (b) a Cas9 endonuclease or a nucleic acid encoding a Cas9 endonuclease, thereby treating the hypertension.
  • the method comprises administering to the subject a composition comprising a plurality of nanoparticles complexed with: (a) a gRNA that targets an AGT genomic locus, comprising a spacer sequence comprising any one of the sequences of SEQ ID NOs: 64-71, 73, 75-84, and 127-129 or a portion thereof, or a nucleic acid encoding the gRNA; and (b) a Cas9 endonuclease or a nucleic acid encoding a Cas9 endonuclease, thereby treating the preeclampsia.
  • the Cas9 endonuclease can be, for example, . pyogenes Cas9, A aureus Cas9, N. meningitides Cas9, A. thermophilus CRISPR1 Cas9, A. thermophilus CRISPR 3 Cas9, or T. denticola Cas9.
  • the plurality of nanoparticles can be lipid nanoparticles.
  • the lipid nanoparticles can comprise one or more neutral lipids, charged lipids, ionizable lipids, steroids, and polymers conjugated lipids.
  • the lipid nanoparticles can comprise cholesterol, a polyethylene glycol (PEG) lipid, or both.
  • the method can comprise administering to the subject the composition at a single dose of about 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, or 1.0 mg/kg of total nucleic acids of (a) and (b).
  • a single dose or each dose of the plurality of nanoparticles administrated to the subject can be nanoparticles complexed with 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6
  • the plurality of nanoparticles is administered to the subject at a dose of, or a dose about, 0.1 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 1 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg or 3 mg/kg (determined by the total of AGT gRNA and SpCas9 mRNA).
  • the method can comprise a single administration of the composition to the subject.
  • the composition described herein e.g., LNPs comprising A GT gRNA) or a nucleic acid encoding an AGT gRNA; and a nucleic acid encoding an RNA-guided endonuclease
  • LNPs comprising A GT gRNA
  • AGT gRNA a nucleic acid encoding an AGT gRNA
  • a nucleic acid encoding an RNA-guided endonuclease can be administered to the subject in need thereof one or more times, for example once, twice, three times, four times, five times, or six times.
  • any of the two administrations can be, for example, one day to one year part.
  • the first administration can be, or be about, 1 to 21 days apart (e.g., one day, two days, three days, four days, five days, six days, seven days, ten days, two weeks, three weeks, or a value or a range between any two of these values) apart from the second administration.
  • the second administration can be, or be about, 1 day to one year (e.g., one day, two days, three days, four days, five days, six days, seven days, two weeks, three weeks, four weeks, five weeks, six weeks, two months, three months, six months, a year, or a value or a range between any two of these values) apart from the third administration.
  • the interval between any of the two adjacent administrations can be the same or different in length.
  • the first administration is about a week (e g., 7 days) apart from the second administration
  • the second administration is about five weeks (e.g., 35 days) apart from the third administration.
  • the method described herein does not comprise regular on-schedule administration of the composition, e.g., every two days, every three days, every five days, weekly, biweekly, monthly, bimonthly, quarterly, biquarterly, yearly, or biyearly administration.
  • the method described herein does not comprise any administration of the composition three months, six months, nine months, a year, two years, or longer, after the first, second, or third administration of the composition. In some embodiments, the method described herein does not comprise any administration of the composition after the second or third administration of the composition.
  • the method described herein can be effective, in some embodiments, that the subject does not need to receive any additional treatment for conditions related to AGT (e.g., hypertension) in the life time after the one-time treatment using the composition described herein.
  • the expression of AGT in the subject can be reduced in the subject.
  • the expression of AGT can be reduced in the liver of the subject. The reduction can be relative to (a) the AGT expression of the subject prior to being administered the composition; (b) the AGT expression in one or more untreated subjects; and/or (3) a reference level of AGT expression of healthy subjects.
  • the expression of AGT in the subject can be reduced by at least 20% after the administration.
  • the expression of AGT is reduced in the subject by about, by at least, or by at least about 20% in the subject after administration (e.g., 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%
  • the genetic modification of the AGT gene results in a significantly reduced AGT protein or mRNA in liver.
  • the AGT protein or mRNA level is reduced by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 9
  • the methods described herein can decrease the AGT protein or mRNA level in the liver by about, at least or at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or a number or a range between any two of these values.
  • the genetic modification of the AGT gene results in a significantly reduced level of blood pressure in a subject.
  • the blood pressure level is reduced by about, at least, at least about, at most, or at most about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or a number or a range between any two of these values.
  • the subject has a blood pressure of above 140/90 mmHg or above 180/120 mmHg and the genetic modification of the AGT ene can reduce the blood pressure in the subject below 140/90 mmHg.
  • the genetic modification of the AGT gene can reduce the blood pressure in the subject to a normal level, e g., between 90/60 mmHg and 120/80 mmHg.
  • the genetic modification of the AGT gene results in a significant reduction of angiotensin I and/or angiotensin II levels in the plasma of the subject (e. , mammal, NHP, a human subject).
  • the gene editing methods described herein can reduce the angiotensin I and/or angiotensin II levels by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%
  • the angiotensin I and/or angiotensin II level is reduced by about, at least or at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values.
  • the levels of angiotensin I and/or angiotensin II in the subject are reduced following administration of the composition relative to (a) the angiotensin I and/or angiotensin II levels of the subject prior to being administered the composition; (b) the angiotensin I and/or angiotensin II levels in one or more untreated subjects; and/or (3) a reference level of angiotensin I and/or angiotensin II of healthy subjects.
  • the AGT protein, AGT mRNA, angiotensin I and angiotensin II levels in a genetically modified subject are about, less than or less than about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%
  • the reduction of the blood pressure the reduction of the blood pressure
  • AGT protein, AGT mRNA, angiotensin I and/or angiotensin II levels; and/or the reduction of angiotensin I and/or angiotensin II levels in the plasma of the subject can be for at least two weeks, at least three weeks, at least four weeks, or at least a month.
  • the method can comprise administering to the subject a therapeutically effective amount of at least one additional therapeutic agent to the subject.
  • the additional therapeutic agent can be, or comprise, ACE inhibitors (e.g., enalapril, lisinopril, perindopril, ramipril, captopril, banezepril, quinapril, trandolapril, enalapril, and fosinopril), angiotensin-2 receptor blockers (e.g., candesartan, irbesartan, losartan, valsartan, azilsartan, telmisartan, and olmesartan), calcium channel blockers (e.g., amlodipine, felodipine, nifedipine, diltiazem, and verapamil), diuretics (e.g, indapamide, chlorothizaide, hydrochlorothiazide, chlorthalidone, meto
  • the additional treatment is administered to the subject 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or more prior to the administration of the plurality of nanoparticles to the subject.
  • the additional treatment is administered to the subject at most 2 hours prior to administration of the plurality of nanoparticles.
  • the additional treatment and the plurality of nanoparticles are administered simultaneously.
  • the method can comprise administering to the subject a therapeutically effective amount of at least one additional therapeutic agent to treat one or more symptoms of hypertension e.g., refractory hypertension or resistant hypertension).
  • the at least one additional therapeutic agent can comprise ACE inhibitors, angiotensin-2 receptor blockers, calcium channel blockers, diuretics, beta blockers, or a combination thereof.
  • the at least one additional therapeutic agent can comprise enalapril, lisinopril, perindopril, ramipril, candesartan, irbesartan, losartan, valsartan and olmesartan, amlodipine, felodipine, nifedipine, diltiazem, verapamil, indapamide bendroflumethiazide, atenolol, bisoprolol, or a combination thereof.
  • the additional treatment is an siRNA therapy.
  • various tests can be used to diagnose an AST-associated disease or disorder in a subject and/or evaluate state of the disease or disorder in the subject.
  • the subject can have an elevated blood pressure (e.g., equal to or greater than 130/80 mmHg) as compared to a reference value.
  • the target tissue for the compositions and methods described herein is liver tissue.
  • the target cells for the compositions and methods described herein is hepatocyte.
  • the pharmaceutical composition thereof can be administered by aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intraci sternal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, and/or intradermal injection, or any combination thereof.
  • the administration can be local or systemic.
  • the systemic administration includes enteral and parenteral administration. In some embodiments, more than one administration can be employed to achieve the desired level of gene expression over a period of various intervals, e.g., daily, weekly, monthly, or yearly.
  • the pharmaceutical composition thereof can be administered to a subject in need thereof at a pharmaceutically effective amount.
  • pharmaceutically effective amount means that the amount of the pharmaceutical composition that will elicit a desired therapeutic effect and/or biological or medical responses of a tissue, system, animal or human.
  • the administration can result in a desired reduction in the expression of the AGT gene such as a desired reduction in the levels of the AGT protein, other downstream angiotensin peptides in the RAAS pathway, and/or the blood pressure in the subject.
  • PHL primary human hepatocyte
  • PMH primary monkey hepatocyte
  • Materials used in primary human hepatocyte (PHH) and primary monkey hepatocyte (PMH) plating protocol include INVITROGRO CP (plating) medium (Vendor Cat. No. BioIVT Z99029), TORPEDO Antibiotic Mix (Vendor Cat. No. BioIVT Z99000), primary human hepatocytes, primary monkey hepatocytes, and plates.
  • INVITROGRO medium was prepared by combining 45 mL INVITROGRO CP Medium (Z99029) and 1.0 mL of TORPEDO Antibiotic Mix (Z99000). The vial of PHH or PMH from liquid nitrogen was thawed in a 37° C water bath until most of the cells are thawed as liquid. Its content was transferred into the pre-warmed 10 mL complete medium. Cells were resuspended by gently inverting the tube and centrifuged at 100g for 8 minutes. After spinning, supernatant was removed without disrupting pellet, leaving about 200 pL of complete medium.
  • Opti-MEM ThermoFisher 31985062, Lipofectamine MessengerMAX (ThermoFisher LMRNA), 96-well flat-bottom plates, 96-well PCR plate and temperature resistant plate cover, sgRNA, and Cas9 mRNA.
  • lipofectamine MessengerMAX was equilibrated to room temperature. Guide RNAs and/or Cas9 mRNA were thawed on ice. Stock sgRNA and Cas9 mRNA was diluted to 1 pg/pL working concentration with nuclease-free water. Number of gRNA reactions was counted to calculate the volume of Cas9 mRNA and OptiMEM needed.
  • gRNA-Cas9-MessengerMAX-OptiMEM reaction was prepared using the following calculations per well per guide and scaled up as needed.
  • Tube 1 and Tube 2 were combined at 1 : 1 volume ratio and incubated for 15 minutes at room temperature. Plates with PHH and/or PMH were removed from an incubator and 50 pl of a reaction mix was added per well. Plates were returned to a CO2 incubator. Media was replaced on day 3 post-transfection and cells continued to culture for additional two days until samples harvesting (day 5 post-transfection). To harvest the samples, plates were removed from incubator, media was collected and frozen for AGT Elisa assay. Attached cells were lysed for DNA extraction.
  • the PCR mix plate was set up in a thermal cycler using the following cycling conditions.
  • ELISA was conducted for total Angiotensinogen Rat and Human/primate.
  • Materials used in the ELISA include human total AGT kit (Vendor: IBL, Cat. No.: 27412) and rat total AGT (Vendor: IBL, Cat. No.: 27104).
  • wash buffer was diluted 40-fold with deionized water.
  • Antibody was diluted 30-fold.
  • Standard was reconstituted with deionized water to stock concentration 40 ng/mL and serial dilution was made for the standard curve.
  • Samples were diluted with EIA buffer: cell supernatants were diluted 1 :20 and rat serum was diluted 1 :2.
  • FIG. 2A displays non-limiting exemplary data depicting on-target editing efficiencies of four selected gRNAs comprising the indicated spacer sequences in primary human and monkey hepatocytes. DNA editing and total AGT secreted in the media are shown as percentage of un-edited cells. The data is representative of 3-4 independent experiments for PHH donors and 2 technical replicates for PMH donor.
  • FIG. 2B displays non-limiting exemplary data depicting on-target editing efficiencies of selected gRNAs comprising the indicated spacer sequences in primary human hepatocytes. DNA editing and total AGT secreted in the media are shown as percentage of unedited cells.
  • FIG. 3 displays non-limiting exemplary data depicting on-target editing efficiencies of selected gRNAs comprising the indicated spacer sequences in primary monkey hepatocytes. DNA editing and total AGT secreted in the media are shown as percentage of unedited cells.
  • a Batch 2 of 9 guides (B2 guides, all cross-reacting human and monkey gRNAs) and a Batch 3 of 7 guides (B3 guides, 4 cross-reacting human and monkey and 3 human only gRNAs) were tested in PHH and PMH.
  • B2 guides were tested in PHH donors FGL and VFB and PMH donor VDU.
  • B2 guides had high editing in PHH, and in PMH (FIG. 8).
  • B3 guides were tested in PHH donors FGL and PMH donor VDU. B3 guides also had higher editing efficiency in PHH, and in PMH (FIG. 9).
  • a dose ranging study was performed in the spontaneous hypertensive rat model (SHR) to evaluate editing AGT in liver hepatocytes and the resulting decrease of AGT expression and blood pressure.
  • the test material was an LNP with a Cas9 mRNA and guide RNA payload.
  • Test material was delivered by systemic administration (bolus injection into the tail vein) at a dose of 0, 0.5, 1.0, or 2.0 mg/kg when the animals reached an age of 13 weeks.
  • Each dose group consisted of 10 males and 2 females. Two males and two females from each group were euthanized 14 days following test material administration and the percentage of editing in the hepatocytes was determined using TIDE analysis.
  • Blood pressure was measured monthly using a noninvasive tail cuff method on the remaining eight male rats from each dose group. Blood sera was collected from each animal to determine the amount of AGT using a commercially available ELISA kit.
  • test material was LNPs with a Cas9 mRNA and either SpAgRl (spacer sequence SEQ ID NO: 129) or rAGT_E2_G21 (spacer sequence SEQ ID NO: 155) guide RNA payload.
  • Test materials were delivered by systemic administration (bolus inj ection into the tail vein) at a dose of 2.0 mg/kg when the animals reached an age of 9 weeks. Each group consisted of 3 males. Animals were euthanized 7 days following test material administration and the percentage of editing in the hepatocytes was determined using TIDE analysis. Blood plasma was collected from each animal to determine the amount of AGT using a commercially available ELISA kit.
  • Test material preparation To prepare test materials, LNP formulation with Cas9 mRNA and guide RNA to rat AGT stored at -80°C was thawed and diluted with PBS (GIBCO
  • Test material was systemically administered when the animals were 15 weeks of age. Rats were warmed for approximately 5 minutes using a heat lamp. Each rat was placed in a restraint device and the tail cleansed with an isopropanol wipe. Test material was administered into one of the lateral tail veins in a single bolus injection using a 1 mL syringe fitted with a 25 gauge needle. Hemostasis was achieved by applying direct pressure to the wound using a sterile gauze square. Once hemostasis was achieved, the animal was returned to its cage.
  • Blood pressure measurements Blood pressure was measured in conscious, manually restrained animals using a non-invasive tail cuff system (CODA: Kent Scientific Corp., Torrington, CT). Blood pressure was measured monthly for the duration of the study.
  • CODA Kent Scientific Corp., Torrington, CT.
  • Angiotensinogen ELISA The level of rat AGT was determined in the sera or plasma samples using an ELISA kit (catalog number: 27104; IBL America, Minneapolis, MN) according to manufacturer’s instructions.
  • FIG. 4A displays non-limiting exemplary data depicting editing efficiencies of gRNAs SpAgRl in SHR at a dose of 0, 0.5, 1.0, or 2.0 mg/kg.
  • FIG. 4B displays non-limiting exemplary data depicting serum AGT reduction.
  • FIG. 4C displays non-limiting data depicting blood pressure change following the administration.
  • FIG. 4A shows dose-dependent editing efficiency in hepatocytes up to 60% at 2.0 mg/kg. By Day 35, the serum AGT level is reduced by 66% at a dose of 1.0 mg/kg and 85% at a dose of 2.0 mg/kg (FIG. 4B).
  • FIG. 7 shows that rAGT_E2G21 gRNAs outperforms SpAgRl gRNAs 7 days after editing.
  • three SHR males per group were injected with 2 mg/kg of either SpAgRl LNP or rAGT_E2G21 LNP.
  • Liver DNA editing efficiency (%) was assessed by Sanger sequencing of the corresponding gRNA target site with subsequent TIDE analysis of DNA sequences.
  • rAGT_E2G21 achieved an editing efficiency of 61%, while the editing efficiency of SpAgRl is 55% (FIG. 7).
  • Total AGT protein in plasma of dosed animals was measured by Elisa. Protein knockdown is shown as percentage of protein decrease compared to pre-dose AGT plasma levels. Guide rAGT_E2G21 dosing resulted in more efficient AGT knockdown (92%) compared to the AGT knockdown achieved by SpAgRl (85%) (FIG. 7).
  • truncated guides with 17, 18, or 19 bases in length can reduce off-target activity compared to their 20-base counterparts.
  • the same experimental procedure used in Example 2 was used in this example to evaluate the editing efficiency of truncated gRNAs.
  • gRNAs comprising a full-length xhAGT_E2_G129 spacer (20 bp; SEQ ID NO: 64) and truncated counterparts (17 bp, 18 bp or 19 bp) were tested in PHH donor (FGL) and PMH donor (VDU).
  • gRNAs comprising a full length or truncated spacer (E2_G125, E2_G129, E2_G48 or E2G43) were tested in PHH donor FGL.
  • gRNAs comprising a full length or truncated spacer (E2_G43, E2_G48, or E2_G129) were tested in PHH donors FGL and 501.
  • the full-length and truncated guides can comprise chemical modification.
  • the E2_G125 spacer in different lengths can comprise the chemical modifications shown in Table 6
  • FIGS. 5A-5B display non-limiting exemplary data depicting editing efficiency and AGT protein reduction of gRNAs comprising truncated spacer sequences in comparison to their full-length counterparts in primary human hepatocytes.
  • Example 2 The same experimental procedure used in Example 2 was used in this example to evaluate the editing efficiency of three gRNAs formulated in LNPs (xhAGT_E2_g31 : SEQ ID NO: 127, xhAGT_E2_g42: SEQ ID NO: 75, xhAGT_E2_g48 : SEQ ID NO: 67).
  • the full-length gRNAs were tested in PHH donor (FGL and VFB; FIGS. 6A-6B) and PMH donor (NZU; FIG. 6C).
  • FIGS. 6A-6C display non-limiting exemplary data depicting editing efficiency and AGT protein reduction of these gRNAs in comparison to that of ANGPTL3 gRNA T6.
  • Data points represent an average for two independent experiments.
  • the data demonstrates a dose-dependent gene editing and reduction of the AGT protein by these gRNAs.
  • about 55% reduction of AGT protein from baseline was observed in FGL donor treated with 0.5 ng/ul xhAGT_E2_g31 and about 84-89% reduction from baseline was observed in FGL donor treated with 2 ng/pl and 4 ng/pl xhAGT_E2_g31 (see, for example, FIG. 6A).
  • a dose ranging study of rAGT_E2_G21 guide in a LNP formulation was performed in the spontaneous hypertensive rat model (SHR) to evaluate AGT editing in the liver and the resulting decrease of blood pressure.
  • All animals were implanted with Data Science International transmitters (HD-S10) at least 2 weeks prior to study initiation and LNP dosing for collection of blood pressure and heart rate data.
  • the test material was an LNP with a Cas9 mRNA and guide RNA payload.
  • Test material was delivered by systemic administration (bolus injection into the tail vein) at a dose of 0, 0.0625, 0.125, 0.25, 0.5, 1.0, or 2.0 mg/kg.
  • Each dose group consisted of 5 males and 5 females. All the animals were euthanized 29 days following test material administration and the percentage of editing in the liver was determined using Amplicon Sequencing analysis. Blood pressure was recorded daily continuously for at least 22 hours from unanesthetized animals from each dose group.
  • FIG. 10A displays non-limiting exemplary data depicting editing efficiencies of gRNA rAGT_E2G21 in all SHRs at a dose of 0, 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg/kg.
  • FIG. 10B displays non-limiting exemplary data depicting editing efficiencies of the same gRNA in female and male SHRs at a dose of 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg/kg.
  • the data suggested that liver DNA was edited by the LNPs in a dose-dependent manner.
  • female rats appeared to demonstrate lower editing efficiency compared to male rates.
  • FIG. 11 displays non-limiting exemplary mean arterial pressure (MAP) of animals treated with the LNPs at a dose of 0, 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg/kg.
  • MAP mean arterial pressure
  • FIGS. 12A-B display non-limiting exemplary data depicting mean arterial pressure (MAP) following the LNP administration in male rates (FIG. 12A) and female rates (FIG. 12B).
  • Tables 9 and 10 provide the MAP results for male and female rats by Day 7 and Day 125. Concomitant decreases in blood pressure following rAGT_E2_G21 LNP administration were observed by Day 7 and sustained by Day 125.
  • FIGS. 13A-B display non-limiting exemplary data depicting serum AGT (FIG. 13A) and renin levels (FIG. 13B).
  • FIGS. 14A-B display non-limiting exemplary data depicting the percentage knockdown of AGT. Interim results indicated 66% AGT editing in liver, resulting in a sustained AGT reduction in gRNA-treated animals by >85% starting by Day 14 and lasting through D70.
  • FIG. 15 displays non-limiting exemplary data from clinical chemistry testing. No liver functional impairment or damage was detected after the administration (e.g., 15 days after the administration). There was a transient increase in AST, ALT, and ALP in treated males with no change in females. There was a transient decrease in ALB in LNP treated males and females.
  • FIG. 16A displays non-limiting exemplary data depicting editing efficiency and protein reduction percentage from baseline of four exemplary gRNAs xhAGT_E2_g30, xhAGT_E2_g48, xhAGT_E2_g83, and xhAGT_E2_glO3 in PHH donor 1 and PHH donor 2.
  • FIG. 16B displays non-limiting exemplary data depicting editing efficiency and protein reduction percentage from baseline of four exemplary gRNAs xhAGT_E2_g30, xhAGT_E2_g48, xhAGT_E2_g83, and xhAGT_E2_glO3 in PMH donor 1 and PMH donor 2. Dose-dependent AGT editing and protein reduction were observed in all four gRNAs.
  • FIG. 17 displays non-limiting exemplary data depicting liver gene editing efficiencies and AGT protein deduction in serum from baseline of four exemplary gRNAs xhAGT_E2_g30 (E2G30), xhAGT_E2_g48 (E2G48), xhAGT_E2_g83 (E2G83), and xhAGT_E2_glO3 (E2G103) in NHPs. All four gRNAs demonstrated substantial DNA editing efficiency in liver and significant AGT protein knockdown in serum. In particular, LNPs containing xhAGT_E2_g48 gRNA resulted in about 85% and 97% protein reduction in male and female animals.
  • FIG. 18 displays non-limiting exemplary data depicting the pre-dose and postdose AGT concentration in NHP serum.
  • the averaged AGT serum concentrations are also summarized in Table 13 below.
  • Characterization of off-target editing typically consists of nomination of potential off-target editing sites, followed by confirmation using deep sequencing.
  • nomination of sites used both computational and experimental methods.
  • confirmation of off-target editing at the sites was performed on PHHs, primary human spleen cells, and primary human adrenal gland cells. These cell types were chosen based on biodistribution of CRISPR-Cas9 on-target gene editing observed after LNP delivery (Gillmore et al., 2021). Cells were cultured in vitro and treated with concentrations of LNPs that resulted in on-target editing rates similar to or exceeding in vivo editing observed in associated tissues from the cynomolgus monkey GLP toxicity study.

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Abstract

La présente divulgation concerne des méthodes, des compositions et des kits destinés à traiter des états pathologiques qui sont liés à la modulation de l'expression du gène de l'angiotensinogène (AGT) par modification génique.
PCT/IB2025/052358 2024-03-05 2025-03-04 Modulation de l'expression d'un gène agt (angiotensinogène) Pending WO2025186726A1 (fr)

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