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WO2025162435A1 - Compositions et méthodes de traitement d'une maladie hépatique - Google Patents

Compositions et méthodes de traitement d'une maladie hépatique

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Publication number
WO2025162435A1
WO2025162435A1 PCT/CN2025/075417 CN2025075417W WO2025162435A1 WO 2025162435 A1 WO2025162435 A1 WO 2025162435A1 CN 2025075417 W CN2025075417 W CN 2025075417W WO 2025162435 A1 WO2025162435 A1 WO 2025162435A1
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Prior art keywords
composition
seq
sequence
nos
guide rna
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Application number
PCT/CN2025/075417
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English (en)
Inventor
Songyuan Li
Leqi LIAO
Wenhu CAO
Huanle LIU
Han QIU
Feifei DUAN
Ye Chen
Fuxin Shi
Jialin TAO
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Accuredit Therapeutics Suzhou Co Ltd
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Accuredit Therapeutics Suzhou Co Ltd
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Publication of WO2025162435A1 publication Critical patent/WO2025162435A1/fr
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Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01062—17Beta-estradiol 17-dehydrogenase (1.1.1.62)

Definitions

  • This disclosure relates to compositions and methods for the treatment of nonalcoholic steatohepatitis (NASH) .
  • NASH nonalcoholic steatohepatitis
  • Nonalcoholic steatohepatitis is a severe form of nonalcoholic fatty liver disease (NAFLD) , a condition in which fat builds up in the liver. NASH causes the liver to swell and become damaged. NASH tends to develop in people who are overweight or obese, or have diabetes, high cholesterol or high triglycerides. However, some people have NASH even if they do not have any risk factors. Progression of NASH may lead to complications such as cirrhosis, liver cancer, liver failure, or cardiovascular disease.
  • NASH nonalcoholic fatty liver disease
  • NASH The primary characteristic of NASH is inflammation due to the accumulation of lipids in the liver, largely in the form of triglycerides.
  • steatosis along with varied signs of liver injury: either lobular or portal inflammation (aform of liver injury) or ballooning degeneration.
  • NASH can further include histological features such as portal inflammation, polymorphonuclear cell infiltrates, Mallory bodies, apoptotic bodies, clear vacuolated nuclei, microvesicular steatosis, megamitochondria, and perisinusoidal fibrosis.
  • Hepatocyte death via apoptosis or necroptosis and inflammation are hallmarks of NASH.
  • HSD17 ⁇ 13 Loss-of-function mutations and single nucleotide variants (SNVs) of the 17 ⁇ -hydroxysteroid dehydrogenase type 13 (HSD17 ⁇ 13) gene have been identified as protective against development of chronic liver disease, e.g., NASH (Abul-Husn NS, et al. A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease. N Engl J Med. 2018 Mar 22; 378 (12) : 1096-1106. ) . HSD17 ⁇ 13 inhibition represents a potential approach to treat liver disease including NASH.
  • the HSD17 ⁇ 13 gene encodes the hepatic lipid droplet protein 17 ⁇ -hydroxysteroid dehydrogenase type 13 (HSD17 ⁇ 13) .
  • This disclosure relates to compositions and methods to reduce the expression of the HSD17B13 gene in cells using a CRISPR/Cas gene editing or base editing system, thereby substantially reducing or eliminating the production of 17 ⁇ -hydroxysteroid dehydrogenase type 13 protein in tissues, for example, in the liver.
  • This disclosure is based, at least in part, on the findings that novel guide RNAs (gRNA) with high editing efficiency can knockout or knock down HSD17B13 gene expression when used with a nuclease, e.g., Cas9 or a base editor, e.g., an adenine base editor (ABE) or a cytosine base editor (CBE) , thereby offering a long-lasting treatment for NASH.
  • a nuclease e.g., Cas9
  • a base editor e.g., an adenine base editor (ABE) or a cytosine base editor (CBE)
  • the disclosure provides a guide RNA comprising: (a) a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199; (b) at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199; or (c) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199.
  • the disclosure provides a vector comprising one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs comprise: (a) one or more sequences selected from SEQ ID NOs: 1-88, 89-111, or 149-199; (b) at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of one or more sequences selected from SEQ ID NOs: 1-88, 89-111, or 149-199; or (c) one or more sequences that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199.
  • the disclosure provides a composition comprising: (i) a nucleic acid, or a vector comprising the nucleic acid encoding a guide RNA, wherein the guide RNA comprises: (a) a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199; (b) at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199; or (c) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199; and (ii) a programmable nucleotide binding domain, a nucleic acid encoding a programmable nucleotide binding domain, or a vector comprising the nucleic acid encoding a programmable nucleo
  • the programmable nucleotide binding domain comprises a Cas nuclease or a Cas nickase.
  • the nucleic acid encoding the programmable nucleotide binding domain comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the polynucleotide sequence set forth in SEQ ID NO: 238.
  • the programmable nucleotide binding domain is a Cas9 comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequence set forth in SEQ ID NO: 237.
  • the Cas nuclease is a Class 2 Cas nuclease. In some embodiments, the Cas nuclease is Cas9, Cpf1, C2c1, C2c2, and C2c3, or a modified protein thereof. In some embodiments, the Cas nuclease is an S. pyogenes or an S. aureus Cas9 nuclease or a modified protein thereof. In some embodiments, the Cas nuclease is from a Type-II CRISPR/Cas system. In some embodiments, the programmable nucleotide binding domain is an adenine base editor (ABE) .
  • ABE adenine base editor
  • the ABE comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequence set forth in any one of SEQ ID NOs: 112-121.
  • the programmable nucleotide binding domain is a cytosine base editor (CBE) .
  • the CBE comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequence set forth in any one of SEQ ID NOs: 122-132.
  • the composition is used for editing of the 17 ⁇ -hydroxysteroid dehydrogenase type 13 (HSD17B13) gene.
  • the editing is calculated as a percentage of a population of cells that is edited (percent editing) . In some embodiments, between about 30%and 99%of the population of cells are edited. In some embodiments, the percent editing is between 30%and 35%, 35%and 40%, 40%and 45%, 45%and 50%, 50%and 55%, 55%and 60%, 60%and 65%, 65%and 70%, 70%and 75%, 75%and 80%, 80%and 85%, 85%and 90%, 90%and 95%, or 95%and 99%of the population of cells.
  • the composition reduces the levels of 17 ⁇ -hydroxysteroid dehydrogenase type 13 expressed in hepatocytes in a subject. In some embodiments, the level of 17 ⁇ -Hydroxysteroid dehydrogenase type 13 in hepatocytes of the subject is determined 8 weeks after administration of the composition. In some embodiments, the level of 17 ⁇ -Hydroxysteroid dehydrogenase type 13 in hepatocytes of the subject is compared to a negative control or a level determined in the subject before administration of the composition.
  • the level of 17 ⁇ -Hydroxysteroid dehydrogenase type 13 in hepatocytes of the subject is reduced by at least 20%relative to that in a corresponding negative control or a level determined in the subject before administration of the composition.
  • the composition is administered or delivered at least once. In some embodiments, the administration or delivery occurs at an interval of: (a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days; or (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks; or (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months; or (d) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • the guide RNA is at least partially complementary to a target sequence present in the human HSD17B13 gene.
  • the target sequence is in exon 1, 2, 3, 4, 5, 6, or 7 of the human HSD17B13 gene.
  • the guide RNA sequence is complementary to a target sequence in the positive strand of the HSD17B13 gene.
  • the guide RNA sequence is complementary to a target sequence in the negative strand of HSD17B13.
  • the guide RNA comprises a crRNA and further comprises a tracrRNA or a portion thereof, wherein the tracrRNA comprises the nucleotide sequence set forth in SEQ ID NO: 224 wherein the tracrRNA is operably linked to the crRNA.
  • the guide RNA is a single guide (sgRNA) .
  • guide RNA comprises at least one modification.
  • the at least one modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide, a phosphorothioate (PS) bond between nucleotides, or a 2'-fluoro (2'-F) modified nucleotide.
  • the at least one modification comprises a modification at one or more of the first five nucleotides at the 5' end of the guide RNA and/or one or more of the last five nucleotides at the 3' end of the guide RNA.
  • the at least one modification comprises a modification of at least 50%of the nucleotides of the guide RNA.
  • the sgRNA comprises a guide sequence that is at least 90%identical to a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199. In some embodiments, the sgRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-88, 89-111, or 149-199. In some embodiments, the guide RNA is associated with a lipid nanoparticle (LNP) . In some embodiments, the composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable carrier.
  • the composition reduces or prevents symptoms of non-alcoholic steatohepatitis (NASH) in the subject.
  • administering the composition leads to a deletion or insertion of one or more nucleotide (s) in the HSD17B13 gene.
  • administering the composition leads to the editing of an adenine (A) nucleobase to a guanine (G) nucleobase in the HSD17B13 gene.
  • administering the composition leads to the editing of a cytidine (C) nucleobase to a thymine (T) nucleobase in the HSD17B13 gene.
  • the deletion or insertion of a nucleotide (s) induces a frameshift or nonsense mutation in the HSD17B13 gene.
  • a frameshift or nonsense mutation is induced in the HSD17B13 gene of at least about 20%of cells.
  • the cells are liver cells.
  • a deletion or insertion of a nucleotide (s) occurs in the HSD17B13 gene at least 50-fold or more than in off-target sites.
  • the levels of 17 ⁇ -Hydroxysteroid dehydrogenase type 13 are measured in the blood of the subject.
  • the subject has NASH.
  • the subject exhibits symptoms of NASH.
  • after administration the subject exhibits an improvement, stabilization, or slowing of change in symptoms of NASH.
  • the composition or pharmaceutical formulation is administered via a viral vector.
  • the composition or pharmaceutical formulation is administered via lipid nanoparticles.
  • the disclosure provides methods of modifying the HSD17B13 gene and/or inducing a double-stranded break (DSB) within the HSD17B13 gene, comprising administering the composition to a cell, wherein the composition recognizes and cleaves a HSD17B13 target sequence.
  • DSB double-stranded break
  • the disclosure provides methods of reducing 17 ⁇ -Hydroxysteroid dehydrogenase type 13 levels in hepatocytes and/or treating NASH in a subject, comprising administering the composition to the subject in need thereof, wherein the composition recognizes and cleaves a HSD17B13 target sequence, thereby reducing 17 ⁇ -Hydroxysteroid dehydrogenase type 13 levels in hepatocytes and/or treating NASH in a subject.
  • the disclosure provides methods of modifying the HSD17B13 gene and/or inducing a double-stranded break (DSB) within the HSD17B13 gene, comprising administering a composition comprising: (i) a nucleic acid, or a vector comprising the nucleic acid encoding a guide RNA, wherein the guide RNA comprises: (a) a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199; (b) at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199; or (c) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199; and (ii) a programmable nucleotide binding domain, a
  • the disclosure provides methods of reducing 17 ⁇ -Hydroxysteroid dehydrogenase type 13 levels in hepatocytes and/or treating NASH in a subject, comprising administering a composition comprising: (i) a nucleic acid, or a vector comprising the nucleic acid encoding a guide RNA, wherein the guide RNA comprises: (a) a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199; (b) at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199; or (c) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199; and (ii) a programmable nucleotide binding domain,
  • the programmable nucleotide binding domain comprises a Cas nuclease or a Cas nickase. In some embodiments, the programmable nucleotide binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequence set forth in SEQ ID NO: 237.
  • the Cas nuclease is a Class 2 Cas nuclease.
  • the Cas nuclease is Cas9, Cpf1, C2c1, C2c2, and C2c3, or a modified protein thereof.
  • the Cas nuclease is an S. pyogenes or an S. aureus Cas9 nuclease or a modified protein thereof.
  • the Cas nuclease is from a Type-II CRISPR/Cas system.
  • the programmable nucleotide binding domain is an adenine base editor (ABE) .
  • the ABE comprises the amino acid sequence set forth in any one of SEQ ID NOs: 112-121.
  • the programmable nucleotide binding domain is an cytosine base editor (CBE) .
  • the CBE comprises the amino acid sequence set forth in any one of SEQ ID NOs: 122-132.
  • the methods are used for editing of the HSD17B13 gene.
  • the editing is calculated as a percentage of a population of cells that is edited (percent editing) . In some embodiments, between about 30%and 99%of the population of cells are edited. In some embodiments, the percent editing is between 30%and 35%, 35%and 40%, 40%and 45%, 45%and 50%, 50%and 55%, 55%and 60%, 60%and 65%, 65%and 70%, 70%and 75%, 75%and 80%, 80%and 85%, 85%and 90%, 90%and 95%, or 95%and 99%of the population of cells.
  • the composition reduces the abundance of 17 ⁇ -Hydroxysteroid dehydrogenase type 13 in the cells of at least one tissue or organ. In some embodiments, the at least one tissue or organ includes the liver.
  • 17 ⁇ -Hydroxysteroid dehydrogenase type 13 levels are determined 8 weeks after administration of the composition. In some embodiments, 17 ⁇ -Hydroxysteroid dehydrogenase type 13 levels are compared to a negative control or a level determined in the subject before administration of the composition. In some embodiments, 17 ⁇ -Hydroxysteroid dehydrogenase type 13 levels are reduced by at least 10%relative to that in a corresponding negative control or a level determined in the subject before administration of the composition.
  • the composition is administered or delivered at least once. In some embodiments, the administration or delivery occurs at an interval of: (a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days; or (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks; or (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months; or (d) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • the guide RNA is at least partially complementary to a target sequence present in the human HSD17B13 gene.
  • the target sequence is in exon 1, 2, 3, 4, 5, 6, or 7 of the human HSD17B13 gene.
  • the guide RNA sequence is complementary to a target sequence in the positive strand of the HSD17B13 gene.
  • the guide RNA sequence is complementary to a target sequence in the negative strand of HSD17B13.
  • the guide RNA comprises a crRNA and further comprises a tracrRNA or a portion thereof, wherein the tracrRNA comprises the nucleotide sequence set forth in SEQ ID NO: 224 wherein the tracrRNA is operably linked to the crRNA.
  • the guide RNA is a single guide (sgRNA) .
  • the guide RNA comprises at least one modification.
  • the at least one modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide, a phosphorothioate (PS) bond between nucleotides, or a 2'-fluoro (2'-F) modified nucleotide.
  • the at least one modification comprises a modification at one or more of the first five nucleotides at the 5' end of the guide RNA and/or one or more of the last five nucleotides at the 3' end of the guide RNA.
  • the at least one modification comprises a modification of at least 50%of the nucleotides of the guide RNA.
  • the sgRNA comprises a guide sequence that is at least 90%identical to a sequence selected from SEQ ID NOs: 1-88, 89-111, or 149-199.
  • the guide RNA is associated with a lipid nanoparticle (LNP) .
  • the composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable carrier.
  • administering the composition leads to a deletion or insertion of one or more nucleotide (s) in the HSD17B13 gene.
  • administering the composition leads to the editing of an adenine (A) nucleobase to a guanine (G) nucleobase in the HSD17B13 gene.
  • administering the composition leads to the editing of a cytidine (C) nucleobase to a thymine (T) nucleobase in the HSD17B13 gene.
  • the deletion or insertion of a nucleotide (s) induces a frameshift or nonsense mutation in the HSD17B13 gene.
  • a frameshift or nonsense mutation is induced in the HSD17B13 gene of at least 20%of cells.
  • the cells are liver cells, kidney cells, intestinal epithelial cells, or vascular epithelial cells.
  • a deletion or insertion of a nucleotide (s) occurs in the HSD17B13 gene at least 50-fold or more than in off-target sites.
  • the subject has NASH. In some embodiments, the subject exhibits symptoms of NASH. In some embodiments, after administration, the subject exhibits an improvement, stabilization, or slowing of change in symptoms of NASH. In some embodiments, the composition or pharmaceutical formulation is administered via a viral vector or via lipid nanoparticles.
  • a guide RNA that includes (a) a sequence selected from SEQ ID NOs: 94, 348, 349, 82, 307, 65, 263, 1-64, 66-81, 83-88, 89-93, 95-111, 149-199, 244-262, 264-306, 308-318, 332-347, or 350; (b) at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 94, 348, 349, 82, 307, 65, 263, 1-64, 66-81, 83-88, 89-93, 95-111, 149-199, 244-262, 264-306, 308-318, 332-347, or 350; or (c) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a sequence selected from SEQ ID NOs: 94, 348
  • the disclosure provides a vector comprising one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs include (a) one or more sequences selected from SEQ ID NOs: 94, 348, 349, 82, 307, 65, 263, 1-64, 66-81, 83-88, 89-93, 95-111, 149-199, 244-262, 264-306, 308-318, 332-347, or 350; (b) at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of one or more sequences selected from SEQ ID NOs: 94, 348, 349, 82, 307, 65, 263, 1-64, 66-81, 83-88, 89-93, 95-111, 149-199, 244-262, 264-306, 308-318, 332-347, or 350; or (c) one or more sequences that is at least 90%, 91%, 92%, 93%, 94%, 9
  • the disclosure provides a composition
  • a composition comprising (i) a nucleic acid, or a vector comprising the nucleic acid encoding a guide RNA, wherein the guide RNA includes (a) a sequence selected from SEQ ID NOs: 94, 348, 349, 82, 307, 65, 263, 1-64, 66-81, 83-88, 89-93, 95-111, 149-199, 244-262, 264-306, 308-318, 332-347, or 350; (b) at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 94, 348, 349, 82, 307, 65, 263, 1-64, 66-81, 83-88, 89-93, 95-111, 149-199, 244-262, 264-306, 308-318, 332-347, or 350; or (c) a sequence that is at least 90%, 91%, 92%, 93%,
  • the programmable nucleotide binding domain comprises a Cas nuclease or a Cas nickase.
  • the nucleic acid encoding the programmable nucleotide binding domain comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the polynucleotide sequence set forth in SEQ ID NO: 238.
  • the programmable nucleotide binding domain is a Cas9 comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequence set forth in SEQ ID NO: 237.
  • the Cas nuclease is a Class 2 Cas nuclease.
  • the Cas nuclease is Cas9, Cpf1, C2c1, C2c2, and C2c3, or a modified protein thereof.
  • the Cas nuclease is an S. pyogenes or an S. aureus Cas9 nuclease or a modified protein thereof.
  • the Cas nuclease is from a Type-II CRISPR/Cas system.
  • the programmable nucleotide binding domain is an adenine base editor (ABE) .
  • the ABE comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequence set forth in any one of SEQ ID NOs: 112-121, or 319-321.
  • the programmable nucleotide binding domain is a cytosine base editor (CBE) .
  • the CBE comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequence set forth in any one of SEQ ID NOs: 122-132.
  • any one of the compositions disclosed herein are used in editing of the 17 ⁇ -hydroxysteroid dehydrogenase type 13 (HSD17B13) gene.
  • the editing is calculated as a percentage of a population of cells that is edited (percent editing) . In some embodiments, between about 30%and 99%of the population of cells are edited. In some embodiments, the percent editing is between 30%and 35%, 35%and 40%, 40%and 45%, 45%and 50%, 50%and 55%, 55%and 60%, 60%and 65%, 65%and 70%, 70%and 75%, 75%and 80%, 80%and 85%, 85%and 90%, 90%and 95%, or 95%and 99%of the population of cells.
  • the composition reduces the levels of 17 ⁇ -hydroxysteroid dehydrogenase type 13 expressed in hepatocytes in a subject. In some embodiments, the level of 17 ⁇ -Hydroxysteroid dehydrogenase type 13 in hepatocytes of the subject is determined 8 weeks after administration of the composition. In some embodiments, the level of 17 ⁇ -Hydroxysteroid dehydrogenase type 13 in hepatocytes of the subject is compared to a negative control or a level determined in the subject before administration of the composition.
  • the level of 17 ⁇ -Hydroxysteroid dehydrogenase type 13 in hepatocytes of the subject is reduced by at least 20%relative to that in a corresponding negative control or a level determined in the subject before administration of the composition.
  • the composition is administered or delivered at least once. In some embodiments, the administration or delivery occurs at an interval of (a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days; or (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks; or (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months; or (d) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • the guide RNA is at least partially complementary to a target sequence present in the human HSD17B13 gene.
  • the target sequence is in exon 1, 2, 3, 4, 5, 6, or 7 of the human HSD17B13 gene.
  • the guide RNA sequence is complementary to a target sequence in the positive strand of the HSD17B13 gene.
  • the guide RNA sequence is complementary to a target sequence in the negative strand of HSD17B13.
  • the guide RNA comprises a crRNA and further comprises a tracrRNA or a portion thereof, wherein the tracrRNA comprises the nucleotide sequence set forth in SEQ ID NO: 224 wherein the tracrRNA is operably linked to the crRNA.
  • the guide RNA is a single guide (sgRNA) .
  • the guide RNA comprises at least one modification.
  • the at least one modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide, a phosphorothioate (PS) bond between nucleotides, or a 2'-fluoro (2'-F) modified nucleotide.
  • the at least one modification comprises a modification at one or more of the first five nucleotides at the 5' end of the guide RNA and/or one or more of the last five nucleotides at the 3' end of the guide RNA.
  • the at least one modification comprises a modification of at least 50%of the nucleotides of the guide RNA.
  • the sgRNA comprises a guide sequence that is at least 90%identical to a sequence selected from SEQ ID NOs: 94, 348, 349, 82, 307, 65, 263, 1-64, 66-81, 83-88, 89-93, 95-111, 149-199, 244-262, 264-306, 308-318, 332-347, or 350.
  • the sgRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 94, 348, 349, 82, 307, 65, 263, 1-64, 66-81, 83-88, 89-93, 95-111, 149-199, 244-262, 264-306, 308-318, 332-347, or 350.
  • the guide RNA is associated with a lipid nanoparticle (LNP) .
  • the composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable carrier.
  • the composition reduces or prevents symptoms of non-alcoholic steatohepatitis (NASH) in the subject.
  • NASH non-alcoholic steatohepatitis
  • administering the composition leads to a deletion or insertion of one or more nucleotide (s) in the HSD17B13 gene.
  • administering the composition leads to the editing of an adenine (A) nucleobase to a guanine (G) nucleobase in the HSD17B13 gene.
  • administering the composition leads to the editing of a cytidine (C) nucleobase to a thymine (T) nucleobase in the HSD17B13 gene.
  • the deletion or insertion of a nucleotide (s) induces a frameshift or nonsense mutation in the HSD17B13 gene.
  • a frameshift or nonsense mutation is induced in the HSD17B13 gene of at least about 20%of cells.
  • the cells are liver cells.
  • a deletion or insertion of a nucleotide (s) occurs in the HSD17B13 gene at least 50-fold or more than in off-target sites.
  • the levels of 17 ⁇ -Hydroxysteroid dehydrogenase type 13 are measured in the blood of the subject.
  • the subject has NASH.
  • the subject exhibits symptoms of NASH.
  • after administration the subject exhibits an improvement, stabilization, or slowing of change in symptoms of NASH.
  • the composition or pharmaceutical formulation is administered via a viral vector. In some embodiments, the composition or pharmaceutical formulation is administered via lipid nanoparticles.
  • the disclosure provides a method of modifying the HSD17B13 gene and/or inducing a double-stranded break (DSB) within the HSD17B13 gene, the method comprising administering a composition that includes (i) a nucleic acid, or a vector comprising the nucleic acid encoding a guide RNA, wherein the guide RNA comprises (a) a sequence selected from SEQ ID NOs: 94, 348, 349, 82, 307, 65, 263, 1-64, 66-81, 83-88, 89-93, 95-111, 149-199, 244-262, 264-306, 308-318, 332-347, or 350; (b) at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 94, 348, 349, 82, 307, 65, 263, 1-64, 66-81, 83-88, 89-93, 95-111, 149-199, 244-2
  • the disclosure provides a method of reducing 17 ⁇ -Hydroxysteroid dehydrogenase type 13 levels in hepatocytes and/or treating NASH in a subject, the method comprising administering a composition that includes (i) a nucleic acid, or a vector comprising the nucleic acid encoding a guide RNA, wherein the guide RNA comprises (a) a sequence selected from SEQ ID NOs: 94, 348, 349, 82, 307, 65, 263, 1-64, 66-81, 83-88, 89-93, 95-111, 149-199, 244-262, 264-306, 308-318, 332-347, or 350; (b) at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 94, 348, 349, 82, 307, 65, 263, 1-64, 66-81, 83-88, 89-93, 95-111, 149-
  • any one of the methods provided herein are for use in editing of the HSD17B13 gene.
  • the programmable nucleotide binding domain comprises a Cas nuclease or a Cas nickase. In some embodiments, the programmable nucleotide binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequence set forth in SEQ ID NO: 237.
  • the Cas nuclease is a Class 2 Cas nuclease.
  • the Cas nuclease is Cas9, Cpf1, C2c1, C2c2, and C2c3, or a modified protein thereof.
  • the Cas nuclease is an S. pyogenes or an S. aureus Cas9 nuclease or a modified protein thereof.
  • the Cas nuclease is from a Type-II CRISPR/Cas system.
  • the programmable nucleotide binding domain is an adenine base editor (ABE) .
  • the ABE comprises the amino acid sequence set forth in any one of SEQ ID NOs: 112-121, or 319-321.
  • the programmable nucleotide binding domain is a cytosine base editor (CBE) .
  • the CBE comprises the amino acid sequence set forth in any one of SEQ ID NOs: 122-132.
  • any one of the methods provided herein are for use in editing of the HSD17B13 gene.
  • the editing is calculated as a percentage of a population of cells that is edited (percent editing) . In some embodiments, between about 30%and 99%of the population of cells are edited. In some embodiments, the percent editing is between 30%and 35%, 35%and 40%, 40%and 45%, 45%and 50%, 50%and 55%, 55%and 60%, 60%and 65%, 65%and 70%, 70%and 75%, 75%and 80%, 80%and 85%, 85%and 90%, 90%and 95%, or 95%and 99%of the population of cells.
  • the composition reduces the abundance of 17 ⁇ -Hydroxysteroid dehydrogenase type 13 in the cells of at least one tissue or organ.
  • the at least one tissue or organ includes the liver.
  • 17 ⁇ -Hydroxysteroid dehydrogenase type 13 levels are determined 8 weeks after administration of the composition.
  • 17 ⁇ -Hydroxysteroid dehydrogenase type 13 levels are compared to a negative control or a level determined in the subject before administration of the composition.
  • 17 ⁇ -Hydroxysteroid dehydrogenase type 13 levels are reduced by at least 10%relative to that in a corresponding negative control or a level determined in the subject before administration of the composition.
  • the composition is administered or delivered at least once. In some embodiments, the administration or delivery occurs at an interval of (a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days; or (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks; or (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months; or (d) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • the guide RNA is at least partially complementary to a target sequence present in the human HSD17B13 gene.
  • the target sequence is in exon 1, 2, 3, 4, 5, 6, or 7 of the human HSD17B13 gene.
  • the guide RNA sequence is complementary to a target sequence in the positive strand of the HSD17B13 gene.
  • the guide RNA sequence is complementary to a target sequence in the negative strand of HSD17B13.
  • the guide RNA comprises a crRNA and further comprises a tracrRNA or a portion thereof, wherein the tracrRNA comprises the nucleotide sequence set forth in SEQ ID NO: 224 wherein the tracrRNA is operably linked to the crRNA.
  • the guide RNA is a single guide (sgRNA) .
  • the guide RNA comprises at least one modification.
  • the at least one modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide, a phosphorothioate (PS) bond between nucleotides, or a 2'-fluoro (2'-F) modified nucleotide.
  • the at least one modification comprises a modification at one or more of the first five nucleotides at the 5' end of the guide RNA and/or one or more of the last five nucleotides at the 3' end of the guide RNA.
  • the at least one modification comprises a modification of at least 50%of the nucleotides of the guide RNA.
  • the sgRNA comprises a guide sequence that is at least 90%identical to a sequence selected from SEQ ID NOs: 94, 348, 349, 82, 307, 65, 263, 1-64, 66-81, 83-88, 89-93, 95-111, 149-199, 244-262, 264-306, 308-318, 332-347, or 350.
  • the guide RNA is associated with a lipid nanoparticle (LNP) .
  • the composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable carrier.
  • administering the composition leads to a deletion or insertion of one or more nucleotide (s) in the HSD17B13 gene.
  • administering the composition leads to the editing of an adenine (A) nucleobase to a guanine (G) nucleobase in the HSD17B13 gene.
  • administering the composition leads to the editing of a cytidine (C) nucleobase to a thymine (T) nucleobase in the HSD17B13 gene.
  • the deletion or insertion of a nucleotide (s) induces a frameshift or nonsense mutation in the HSD17B13 gene.
  • a frameshift or nonsense mutation is induced in the HSD17B13 gene of at least 20%of cells.
  • the cells are liver cells, kidney cells, intestinal epithelial cells, or vascular epithelial cells.
  • a deletion or insertion of a nucleotide (s) occurs in the HSD17B13 gene at least 50-fold or more than in off-target sites.
  • the subject has NASH. In some embodiments, the subject exhibits symptoms of NASH. In some embodiments, after administration, the subject exhibits an improvement, stabilization, or slowing of change in symptoms of NASH. In some embodiments, the composition or pharmaceutical formulation is administered via a viral vector or via lipid nanoparticles.
  • FIGs. 1A-1E show gene editing efficiency of LNP formulated modified gRNA H3-h-82 with SpCas9 mRNA in PHH.
  • FIGs. 2A-2D show gene editing efficiency of LNP formulated modified gRNA H3-h-65 with SpCas9 mRNA in PHH.
  • FIG. 3 shows gene editing efficiency of LNP formulated mutated SpCas9 mRNA with gRNA H3-h-82-seq24 in PHH.
  • FIG. 4 shows base editing efficiency in PHH Using LNP-Formulated TadA-Optimized T8.4 ABEs and gRNA E3_SD_97.
  • FIG. 5 shows base editing efficiency of mRNA-Optimized FF-725 ABE with gRNA E3_SD_97 in PHH Lipo-transfection.
  • FIG. 6 shows base editing efficiency of LNP-Formulated, mRNA-Optimized FF-725 ABE with gRNA E3_SD_97 in PHH.
  • FIG. 7 shows base editing efficiency of LNP-Formulated TadA and mRNA-Optimized T8.4 ABE with gRNA E3_SD_97 in PHH.
  • FIG. 8 shows base editing efficiency of LNP formulated modified gRNA E3_SD_97 with FF-1051 in PHH (ON*stands for on-target editing, OT*stands for maximum off-target editing) .
  • compositions and methods for editing the human 17 ⁇ -hydroxysteroid dehydrogenase type 13 (HSD17B13) gene are provided.
  • the compositions and methods described herein are for treating subjects having nonalcoholic steatohepatitis (NASH) associated with HSD17B13 levels.
  • NASH nonalcoholic steatohepatitis
  • gRNAs Guide RNAs
  • the guide RNAs used in the disclosed methods and compositions can comprise a guide sequence targeting the HSD17B13 gene.
  • Exemplary guide sequences targeting the HSD17B13 gene are shown in Table 3 at SEQ ID NOs: 1-88, Table 11 at SEQ ID NOs: 89-111, and Table 20 at SEQ ID NOs: 149-199.
  • Guide sequences useful in the guide RNA compositions and methods described herein are shown in Table 3, Table 11, Table 20, and throughout the application.
  • Each of the guide sequences in Table 3, Table 11, Table 20 may further comprise additional nucleotides to form a crRNA, e.g., with the following exemplary nucleotide sequence following the guide sequence at its 3' end: GUU UUA GAG CUA UGC UGU UUU G (SEQ ID NO: 223) .
  • the guide sequences of Table 3, Table 11, or Table 20 may further comprise additional nucleotides to form a sgRNA, e.g., with the following exemplary nucleotide sequence following the 3' end of the guide sequence, wherein the sgRNA has a custom-designed short crRNA component followed by the trans-activating crispr RNA (tracrRNA) component: GUU UUA GAG CUA GAA AUA GCA AGU UAA AAU AAG GCU AGU CCG UUA UCA ACU UGA AAA AGU GGC ACC GAG UCG GUG CUU UU (SEQ ID NO: 224) in the 5' to 3' orientation.
  • SEQ ID NO: 224 can be operable connected to the 3' end of the guide sequence in the 5' to 3' orientation.
  • the sgRNA is modified.
  • the gRNA sequence has the modification pattern described in WO2016164356 and WO2016089433, each of which is incorporated herein in its entirety.
  • the gRNA comprises a guide sequence that direct an RNA-guided DNA binding agent, which can be a nuclease (e.g., a Cas nuclease such as Cas9) , to a target DNA sequence in HSD17B13.
  • the gRNA includes a crRNA having a guide sequence shown in any of Table 3, Table 11, or Table 20.
  • the gRNA includes a guide sequence having at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of any one of the guide sequences of SEQ ID NOs: 1-88 shown in Table 3, SEQ ID NOs: 89-111 shown in Table 11, or SEQ ID NOs: 149-199 shown in Table 20.
  • the gRNA comprises a guide sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to at least 16, 17, 18, 19, or 20 contiguous nucleotides of any one of the guide sequences of SEQ ID NOs: 1-88 shown in Table 3, SEQ ID NOs: 89-111 shown in Table 11, or SEQ ID NOs: 149-199 shown in Table 20.
  • the gRNA may further comprise a tracrRNA.
  • the crRNA and tracrRNA may be associated as a single RNA (sgRNA) , or may be on separate RNAs as a dual guide RNA (dgRNA) .
  • sgRNA single RNA
  • dgRNA dual guide RNA
  • the crRNA and tracrRNA components may be covalently linked, e.g., via a phosphodiester bond or other covalent bond.
  • the guide RNA may comprise two RNA molecules as a dgRNA.
  • the dgRNA can comprise a first RNA molecule comprising a crRNA having, e.g., a guide sequence shown in Table 3, Table 11, or Table 20, and a second tracrRNA molecule.
  • the first and second RNA molecules are not covalently linked, but may form a RNA duplex via the base pairing between portions of the crRNA and the tracrRNA.
  • the guide RNA may comprise a sgRNA.
  • the sgRNA may comprise a crRNA (or a portion thereof) having a guide sequence shown in Table 3, Table 11, or Table 20 covalently linked to a tracrRNA.
  • the sgRNA may comprise at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of any one of the guide sequences of SEQ ID NOs: 1-88 shown in Table 3, SEQ ID NOs: 89-111 shown in Table 11, or SEQ ID NOs: 149-199 shown in Table 20.
  • the crRNA and the tracrRNA are covalently linked via a linker.
  • the sgRNA forms a stem-loop structure via the base pairing between portions of the crRNA and the tracrRNA.
  • the crRNA and the tracrRNA are covalently linked via one or more bonds that are not a phosphodiester bond.
  • the tracrRNA may comprise all or a portion of a tracrRNA sequence derived from a naturally occurring CRISPR/Cas system.
  • the tracrRNA comprises a truncated or modified wild type tracrRNA.
  • the length of the tracrRNA can depend on the CRISPR/Cas system used.
  • the tracrRNA comprises or consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides.
  • the tracrRNA may comprise certain secondary structures, such as, for example, one or more hairpin or stem-loop structures, or one or more bulge structures.
  • the composition comprises a gRNA that comprises a guide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to at least 16, 17, 18, 19, or 20 contiguous nucleotides of any one of the guide sequences of SEQ ID NOs: 1-88 shown in Table 3, SEQ ID NOs: 89-111 shown in Table 11, or SEQ ID NOs: 149-199 shown in Table 20.
  • the composition includes a guide RNA having a guide sequence selected from SEQ ID NOs: 1-88 shown in Table 3, SEQ ID NOs: 89-111 shown in Table 11, or SEQ ID NOs: 149-199 shown in Table 20.
  • the guide RNA having a guide sequence selected from SEQ ID NOs: 1-88 shown in Table 3, SEQ ID NOs: 89-111 shown in Table 11, or SEQ ID NOs: 149-199 shown in Table 20 may be a chemically modified sgRNA, such as an end modified RNA.
  • the guide RNA having a guide sequence selected from SEQ ID NOs: 1-88 shown in Table 3, SEQ ID NOs: 89-111 shown in Table 11, or SEQ ID NOs: 149-199 shown in Table 20 may be dgRNA, such as a chemically modified dgRNA.
  • the composition comprises at least one, e.g., at least two gRNAs having guide sequences selected from any two or more of the guide sequences of SEQ ID NOs: 1-88 shown in Table 3, SEQ ID NOs: 89-111 shown in Table 11, or SEQ ID NOs: 149-199 shown in Table 20.
  • the composition comprises at least two gRNAs that each comprise a guide sequence at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any of the nucleic acids of SEQ ID NOs: 1-88 shown in Table 3, SEQ ID NOs: 89-111 shown in Table 11, or SEQ ID NOs: 149-199 shown in Table 20.
  • the guide RNAs provided herein can be useful for recognizing (e.g., hybridizing to) a target sequence in the HSD17B13 gene.
  • the HSD17B13 target sequence may be recognized and cleaved by a Cas nuclease having a guide RNA.
  • an RNA-guided DNA binding agent such as a Cas nuclease
  • the selection of the one or more guide RNAs is determined based on target sequences within the HSD17B13 gene or within its regulatory region.
  • the one or more guide RNAs is based on target sequences within any one of Exons 1-7, the 5’ regulatory region, the 3’ regulatory region, or exon-intron boundaries of the HSD17B13 gene.
  • mutations e.g., frameshift mutations resulting from indels occurring as a result of a nuclease-mediated double-stranded break (DSB)
  • DSB nuclease-mediated double-stranded break
  • the location of a DSB is an important factor in the amount or type of protein knockdown that may result.
  • a gRNA complementary or having complementarity to a target sequence within HSD17B13 is used to direct the RNA-guided DNA binding agent to a particular location in the HSD17B13 gene.
  • gRNAs are designed to have guide sequences that are complementary or have complementarity to target sequences in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of HSD17B13.
  • a frameshift or nonsense mutation is induced in the HSD17B13 gene of about 10%, about 15%, about 20%, about 25%, about 30%of cells to about 35%of the cells.
  • the gRNA is chemically modified.
  • a gRNA having one or more modified nucleosides or nucleotides is called a “modified” gRNA or “chemically modified” gRNA, to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues.
  • a gRNA comprises a hybrid DNA-RNA guide, in which one or more DNA nucleotides replaces one or more RNA nucleotides in the polynucleotide sequence of the gRNA.
  • a modified gRNA is synthesized with a non-canonical nucleoside or nucleotide, is here called “modified. ”
  • Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification) ; (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar (an exemplary sugar modification) ; (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification) ; (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification) ;
  • modified gRNAs having nucleosides and nucleotides (collectively “residues” ) that can have two, three, four, or more modifications.
  • a modified residue can have a modified sugar and/or a modified nucleobase.
  • every base of a gRNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group.
  • all, or substantially all, of the phosphate groups of an gRNA molecule are replaced with phosphorothioate groups.
  • modified gRNAs comprise at least one modified residue at or near the 5' end of the RNA.
  • modified gRNAs comprise at least one modified residue at or near the 3' end of the RNA.
  • the gRNA comprises one, two, three or more modified residues.
  • at least 5% e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%
  • modified nucleosides or nucleotides are modified nucleosides or nucleotides.
  • Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum.
  • nucleases can hydrolyze nucleic acid phosphodiester bonds.
  • the gRNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases.
  • the modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells (e.g., in vivo and ex vivo) .
  • the term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.
  • the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent.
  • the modified residue e.g., modified residue present in a modified nucleic acid
  • the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.
  • modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroami dates, alkyl or aryl phosphonates and phosphotriesters.
  • the phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral.
  • the stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp) .
  • the phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications.
  • the charged phosphate group can be replaced by a neutral moiety.
  • moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxy methyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.
  • Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications.
  • the nucleobases can be tethered by a surrogate backbone. Examples include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.
  • the modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e., at sugar modification.
  • the 2' hydroxyl group (OH) can be modified, e.g., replaced with a number of different “oxy” or “deoxy” substituents.
  • modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion.
  • Examples of 2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar) ; polyethyleneglycols (PEG) , O (CH2CH2O) n CH2CH2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20) .
  • R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar
  • the 2' hydroxyl group modification can be 2'-O-Me. In some embodiments, the 2' hydroxyl group modification can be a 2'-fluoro modification, which replaces the 2' hydroxyl group with a fluoride.
  • the 2' hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O- (CH2) n-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylened
  • the 2' hydroxyl group modification can include “unlocked” nucleic acids (UNA) in which the ribose ring lacks the C2'-C3' bond.
  • the 2' hydroxyl group modification can include the methoxy ethyl group (MOE) , (OCH2CH2OCH3, e.g., a PEG derivative) .
  • “Deoxy” 2' modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA) ; halo (e.g., bromo, chloro, fluoro, or iodo) ; amino (wherein amino can be, e.g., NEE; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid) ; NH (CH2CH2NH) nCH2CH2-amino (wherein amino can be, e.g., as described herein) , -NHC (0) R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar) , cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and
  • the sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a modified nucleic acid can include nucleotides containing, e.g, arabinose, as the sugar.
  • the modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms.
  • the modified nucleic acids can also include one or more sugars that are in the L form, e.g., L-nucleosides.
  • the modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a nucleobase.
  • a modified base also called a nucleobase.
  • nucleobases include, but are not limited to, adenine (A) , guanine (G) , cytosine (C) , and uracil (U) . These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids.
  • the nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog.
  • the nucleobase can include, for example, naturally occurring and synthetic derivatives of a base.
  • each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA and/or tracr RNA.
  • one or more residues at one or both ends of the sgRNA may be chemically modified, or the entire sgRNA may be chemically modified.
  • Certain embodiments comprise a 5' end modification.
  • Certain embodiments comprise a 3' end modification.
  • one or more or all of the nucleotides in single stranded overhang of a guide RNA molecule are deoxynucleotides.
  • a gRNA can have one or more modifications.
  • the modification includes a 2'-O-methyl (2'-O-Me) modified nucleotide.
  • the modification includes a phosphorothioate (PS) bond between nucleotides.
  • mA, ” “mC, ” “mU, ” or “mG” may be used to denote a nucleotide that has been modified with 2’-O-Me.
  • the guide RNA includes a sgRNA having a guide sequence selected from SEQ ID NOs: 1-88 shown in Table 3, SEQ ID NOs: 89-111 shown in Table 11, or SEQ ID NOs: 149-199 shown in Table 20 and the nucleotides of SEQ ID NO: 224, wherein the nucleotides of SEQ ID NO: 224 are on the 3' end of the guide sequence, and wherein the guide sequence may be modified as shown in SEQ ID NO: 225.
  • gRNA modifications are shown in e.g., WO2020198697, WO2016164356, and WO2016089433, incorporated by reference herein in its entirety.
  • the PAM also known as the protospacer adjacent motif, is a short specific sequence complementary to a portion of the gRNA, following the target DNA sequence that is essential for cleavage by Cas nuclease.
  • the PAM is about 2-8 nucleotides downstream of the DNA sequence targeted by the guide RNA and the Cas cuts 3-4 nucleotides upstream of it.
  • PAM sequences are exemplified below in Tables 1-2.
  • a PAM in the context of this disclosure can be any one of the sequences in Tables 1-2 or any other sequence known in the art.
  • PAM of synthetic SpCas9 variants N is A, G, C or T.
  • R is A or G.
  • N is A, G, C or T.
  • R is A or G.
  • nucleic acid having an open reading frame encoding a programmable nucleotide binding domain may be combined in a composition or method with any of the gRNAs disclosed herein.
  • the nucleic acid having an open reading frame encoding a programmable nucleotide binding domain is administered as a DNA or an mRNA.
  • the programmable nucleotide binding domain is administered in its amino acid form, i.e., as a protein.
  • the nucleic acid encoding the programmable nucleotide binding domain is part of a vector described herein.
  • the nucleic acid encoding the programmable nucleotide binding domain may have any of the characteristics described in WO2020198697, incorporated by reference herein in its entirety.
  • the programmable nucleotide binding domain for use in the compositions and methods described herein is a Class 2 Cas nuclease.
  • the programmable nucleotide binding domain has double-strand endonuclease activity.
  • the programmable nucleotide binding domain comprises a Cas nuclease, such as a Class 2 Cas nuclease (which may be, e.g., a Cas nuclease of Type II, V, or VI) .
  • Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins and modifications thereof.
  • Cas9 nucleases examples include those of the type II CRISPR systems of S. pyogenes, S. aureus, and other prokaryotes (see, e.g., the list in the next paragraph) , and modified (e.g., engineered or mutant) versions thereof. See, e.g., US2016/0312198 A1; US 2016/0312199 A1.
  • Other examples of Cas nucleases include a Csm or Cmr complex of a type III CRISPR system or the Cas 10, Csml, or Cmr2 subunit thereof; and a Cascade complex of a type I CRISPR system, or the Cas3 subunit thereof.
  • the Cas nuclease may be from a Type-IIA, Type-11B, or Type-IIC system
  • a Type-IIA Type-11B
  • Type-IIC Type-IIC system
  • the RNA-guided DNA binding agent is a Cas nickase, e.g. a Cas9 nickase.
  • the RNA-guided DNA binding agent is an S. pyogenes Cas9 nuclease.
  • Wild type Cas9 has two nuclease domains: RuvC and HNH.
  • the RuvC domain cleaves the non-target DNA strand
  • the HNH domain cleaves the target strand of DNA.
  • the Cas9 nuclease comprises more than one RuvC domain and/or more than one HNH domain.
  • the Cas9 nuclease is a wild type Cas9.
  • the Cas9 is capable of inducing a double strand break in target DNA.
  • the Cas nuclease can cleave one or both strands of dsDNA.
  • the Cas nuclease can cleave a single strand of DNA.
  • the Cas nuclease may or may not have DNA nickase activity.
  • An exemplary Cas9 amino acid sequence is provided as SEQ ID NO: 237.
  • An exemplary Cas9 mRNA ORF sequence which includes start and stop codons, is provided as SEQ ID NO: 238.
  • An exemplary Cas9 mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 239.
  • the programmable nucleotide binding domain is a part of a base editor or a base editing system.
  • a programmable nucleotide binding domain of a base editor can itself comprise one or more domains.
  • a polynucleotide programmable nucleotide binding domain can comprise one or more nuclease domains.
  • the nuclease domain of a polynucleotide programmable nucleotide binding domain can comprise an endonuclease or an exonuclease.
  • exonuclease refers to a protein or polypeptide capable of digesting a nucleic acid (e.g., RNA or DNA) from free ends
  • exonuclease refers to a protein or polypeptide capable of catalyzing (e.g., cleaving) internal regions in a nucleic acid (e.g., DNA or RNA) .
  • an endonuclease can cleave a single strand of a double-stranded nucleic acid.
  • Any DNA destabilizing molecule can be used in the compositions described herein in any combination, including but not limited to a Cas9 or Cas12 nickase, a Cas9 or Cas12 protein (e.g., dCas) operably linked to a single guide RNA (sgRNA) , any RNA programmable system, a zinc finger nuclease nickase (ZFN nickase) , a TALEN nickase, and/or one or more nucleotides (e.g., one or more peptide nucleic acids (PNAs) , locked nucleic acids (LNAs) and/or bridged nucleic acids (BNAs) ) .
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • BNAs bridged nucleic acids
  • the base editing composition comprises more than one DNA destabilizing molecule, for example one or more proteins (e.g., nickases, etc. ) and/or one or more nucleotides.
  • the composition comprises a ZFN nickase and one or more additional proteins and/or nucleotide DNA destabilizing molecules (e.g., one or more nucleotides as described herein) .
  • the base editing composition does not comprise a Cas9 protein, but may comprise other Cas protein (e.g, non-Cas9 RNA programmable systems) .
  • the DNA-destabilizing molecule comprises a zinc finger nuclease (ZFN) nickase.
  • the nuclease is a zinc finger nuclease (ZFN) or TALE DNA binding domain-nuclease fusion (TALEN) .
  • ZFNs and TALENs comprise a DNA binding domain (zinc finger protein or TALE DNA binding domain) that has been engineered to bind to a target site in a gene of choice and cleavage domain or a cleavage half domain.
  • the at least one zinc finger protein (ZFP) DNA-binding domain of the base editing composition can be operably linked to one or more of the other components of the base editing composition, for example to one or more of the DNA destabilizing molecules (e.g., to Cas9 nickase, dCas9, etc. ) and/or to the at least one adenine or cytosine deaminase.
  • at least one ZFP DNA-binding domain is operably linked to the adenine or cytosine deaminase.
  • the base editing composition comprises first and second ZFP DNA-binding domains, wherein the first ZFP DNA-binding domain is operably linked to the Cas9 nickase.
  • the ZFP DNA-binding domain can comprise 3, 4, 5, 6 or more fingers and may bind to a target site on either side (5' or 3') of the targeted base to be edited.
  • the ZFP binds to a target site that is 1 to 100 (or any number therebetween) nucleotides on either side of the targeted base.
  • the ZFP binds to a target site that is 1 to 50 (or any number therebetween) nucleotides on either side of the targeted base.
  • an endonuclease can cleave both strands of a double-stranded nucleic acid molecule.
  • a polynucleotide programmable nucleotide binding domain can be a deoxyribonuclease.
  • a polynucleotide programmable nucleotide binding domain can be a ribonuclease.
  • a nuclease domain of a polynucleotide programmable nucleotide binding domain can cut zero, one, or two strands of a target polynucleotide.
  • the polynucleotide programmable nucleotide binding domain can comprise a nickase domain.
  • nickase refers 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 duplexed nucleic acid molecule (e.g., DNA) .
  • a nickase can be derived from a fully catalytically active (e.g., natural) form of a polynucleotide programmable nucleotide binding domain by introducing one or more mutations into the active polynucleotide programmable nucleotide binding domain.
  • 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.
  • the residue H840 retains catalytic activity and can thereby cleave a single strand of the nucleic acid duplex.
  • a Cas9-derived nickase domain can comprise an H840A mutation, while the amino acid residue at position 10 remains a D.
  • a nickase can be derived from a fully catalytically active (e.g., natural) form of a polynucleotide programmable nucleotide binding domain by removing all or a portion of a nuclease domain that is not required for the nickase activity.
  • a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9
  • the Cas9-derived nickase domain can comprise a deletion of all or a portion of the RuvC domain or the HNH domain.
  • a base editor comprising a polynucleotide programmable nucleotide binding domain comprising a nickase domain is thus able to generate a single-strand DNA break (nick) at a specific polynucleotide target sequence (e.g., determined by the complementary sequence of a bound guide nucleic acid) .
  • the strand of a nucleic acid duplex target polynucleotide sequence that is cleaved by a base editor comprising a nickase domain is the strand that is not edited by the base editor (i.e., the strand that is cleaved by the base editor is opposite to a strand comprising a base to be edited) .
  • a base editor comprising a nickase domain can cleave the strand of a DNA molecule which is being targeted for editing. In such embodiments, the non-targeted strand is not cleaved.
  • base editors comprising a polynucleotide programmable nucleotide binding domain which is catalytically dead (i.e., incapable of cleaving a target polynucleotide sequence) .
  • catalytically dead and nuclease dead are used interchangeably to refer to a polynucleotide programmable nucleotide binding domain which has one or more mutations and/or deletions resulting in its inability to cleave a strand of a nucleic acid while retaining its ability, and specificity, to bind to a target polynucleotide.
  • a catalytically dead polynucleotide programmable nucleotide binding domain base editor can lack nuclease activity as a result of specific point mutations in one or more nuclease domains.
  • the Cas9 can comprise both a D10A mutation and an H840A mutation. Such mutations inactivate both nuclease domains, thereby resulting in the loss of nuclease activity.
  • a catalytically dead polynucleotide programmable nucleotide binding domain can comprise one or more deletions of all or a portion of a catalytic domain (e.g., RuvCl and/or HNH domains) .
  • a catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) as well as a deletion of all or a portion of a nuclease domain.
  • fusion proteins comprising domains that act as polynucleotide-programmable DNA binding proteins, which can be used to guide a protein, such as a base editor, to a specific nucleic acid (e.g., DNA or RNA) sequence.
  • a fusion protein comprises a nucleic acid programmable DNA binding protein domain and one or more deaminase domains.
  • Non-limiting examples of polynucleotide-programmable DNA binding proteins include, Cas9 (e.g., dCas9 and nCas9) , Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, and Cas12i.
  • Cas9 e.g., dCas9 and nCas9
  • Cas12a/Cpf1 Cas12b/C2c1
  • Cas12c/C2c3 Cas12d/CasY
  • Cas12e/CasX Cas12g, Cas12h, and Cas12i.
  • Cas enzymes include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csnl or Csxl2) , Casl0, Casl0d, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, Csy1 , Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cm
  • nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, although they may not be specifically listed in this disclosure. See, e.g., Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here? “ CRISPR J. 2018 Oct; 1 : 325-336. doi: 10.1089/crispr. 2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4; 363 (6422) : 88-91. doi: 10. l 126/science. aav7271, the entire contents of each are hereby incorporated by reference.
  • the disclosure provides a fusion protein comprising a type V CRISPR/Cas effector protein.
  • Type V CRISPR/Cas effector proteins are a subtype of Class 2 CRISPR/Cas effector proteins.
  • Cas12 family proteins such as Cas12a
  • Examples include, but are not limited to: Cas12 family (Cas12a, Cas12b, Cas12c) , C2c4, C2c8, C2c5, C2c10, and C2c9; as well as CasX (Cas12e) and CasY (Cas12d) . Also see, e.g., Koonin et al., Curr Opin Microbial. 2017 June; 37: 67-78: "Diversity, classification and evolution of CRISPR-Cas systems. " In some embodiments, the CBEs disclosed herein comprise a type V CRISPR/Cas effector protein.
  • the disclosure provides TALE base editors, which can comprise a TALE domain, a deaminase domain and/or cofactor protein (e.g., FokI endonuclease) domain that comprise fusion proteins having the general structure NH2- [TALE] - [deaminase domain] -COOH, NH2- [deaminase domain] - [TALE] -COOH, NH2- [TALE] - [deaminase domain] - [cofactor protein] -COOH, NH2- [cofactor protein] - [deaminase domain] - [TALE] -COOH, NH2- [cofactor protein] -[TALE] - [deaminase] -COOH or NH2- [deaminase domain] - [TALE] - [cofactor protein] -COOH; wherein each instance of "] - [" comprises an optional linker, e.g. a peptid
  • the disclosed methods involve transducing (e.g., via transfection) cells with a plurality of complexes each comprising a fusion protein comprising a TAL effector domain and a deaminase domain and a cofactor protein, wherein each cofactor protein localizes the fusion protein to a distinct target sequence.
  • a plurality of complexes each comprising a fusion protein comprising a TAL effector domain and a deaminase domain and a cofactor protein, wherein each cofactor protein localizes the fusion protein to a distinct target sequence.
  • the methods involve the transfection of nucleic acid constructs (e.g., plasmids) that each (or together) encode the components of a plurality of complexes of a TALE base editor comprising a TALE domain and a deaminase domain, and a cofactor protein.
  • the disclosed fusion proteins comprise a cofactor protein domain, i.e., the domain is incorporated into the fusion protein construct.
  • the TALE base editor comprises a TALE domain and a deaminase domain, and the cofactor protein is introduced into the cell separately from the base editor.
  • the constructs that encode the TALE base editors are transfected into the cell separately from the constructs that encode the cofactor proteins.
  • these components are encoded on a single construct and transfected together.
  • these single constructs encoding the TALE base editor and cofactor proteins can be transfected into the cell iteratively, with each iteration associated with a subset of target sequences.
  • these single constructs can be transfected into the cell over a period of days. In other embodiments, they can be transfected into the cell over a period of weeks.
  • the disclosure provides compositions for the modification of the HSD17B13 gene, the composition including a nuclease at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid set for in SEQ ID NO: 237 and an sgRNA comprising any one of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, and SEQ ID NOs: 149-199.
  • the nuclease comprises the amino acid sequence of SEQ ID NO: 237 or a variant thereof and the sgRNA comprises SEQ ID NO: 18.
  • the nuclease comprises the amino acid sequence of SEQ ID NO: 237 or a variant thereof and the sgRNA comprises SEQ ID NO: 31. In some embodiments, the nuclease comprises the amino acid sequence of SEQ ID NO: 237 or a variant thereof and the sgRNA comprises SEQ ID NO: 39. In some embodiments, the nuclease comprises the amino acid sequence of SEQ ID NO: 237 or a variant thereof and the sgRNA comprises SEQ ID NO: 41. In some embodiments, the nuclease comprises the amino acid sequence of SEQ ID NO: 237 or a variant thereof and the sgRNA comprises SEQ ID NO: 42.
  • the nuclease comprises the amino acid sequence of SEQ ID NO: 237 or a variant thereof and the sgRNA comprises SEQ ID NO: 65. In some embodiments, the nuclease comprises the amino acid sequence of SEQ ID NO: 237 or a variant thereof and the sgRNA comprises SEQ ID NO: 66. In some embodiments, the nuclease comprises the amino acid sequence of SEQ ID NO: 237 or a variant thereof and the sgRNA comprises SEQ ID NO: 74. In some embodiments, the nuclease comprises the amino acid sequence of SEQ ID NO: 237 or a variant thereof and the sgRNA comprises SEQ ID NO: 79. In some embodiments, the nuclease comprises the amino acid sequence of SEQ ID NO: 237 or a variant thereof and the sgRNA comprises SEQ ID NO: 83.
  • the disclosure provides compositions for the modification of the HSD17B13 gene, the composition including an ABE including an adenosine deaminase at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid set for in any one of SEQ ID NOs: 112-121 and an sgRNA comprising any one of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, and SEQ ID NOs: 149-199.
  • the adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 114 or a variant thereof and the sgRNA comprises SEQ ID NO: 94.
  • the adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 114 or a variant thereof and the sgRNA comprises SEQ ID NO: 95. In some embodiments, the adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 119 or a variant thereof and the sgRNA comprises SEQ ID NO: 94. In some embodiments, the adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 119 or a variant thereof and the sgRNA comprises SEQ ID NO: 95.
  • the adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 118 or a variant thereof and the sgRNA comprises SEQ ID NO: 94. In some embodiments, the adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 118 or a variant thereof and the sgRNA comprises SEQ ID NO: 95. In some embodiments, the adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 125 or a variant thereof and the sgRNA comprises SEQ ID NO: 94. In some embodiments, the adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 125 or a variant thereof and the sgRNA comprises SEQ ID NO: 95.
  • the disclosure provides compositions for the modification of the HSD17B13 gene, the composition including a CBE including a cytidine deaminase at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid set for in any one of SEQ ID NOs: 122-132 and an sgRNA comprising any one of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, and SEQ ID NOs: 149-199.
  • the cytidine deaminase comprises the amino acid sequence of SEQ ID NO: 125 or a variant thereof and the sgRNA comprises SEQ ID NO: 94.
  • the cytidine deaminase comprises the amino acid sequence of SEQ ID NO: 125 or a variant thereof and the sgRNA comprises SEQ ID NO: 95.
  • the programmable nucleotide binding domain comprises an adenosine deaminase.
  • the adenosine deaminases provided herein are capable of deaminating adenine.
  • the adenosine deaminases provided herein are capable of deaminating adenine in a deoxyadenosine residue of DNA.
  • the term “adenosine deaminase” refers to a deaminase that can deaminate adenine in a deoxyadenosine residue of DNA, and in some cases, cytosine in a deoxyadenosine residue of DNA.
  • the adenosine deaminases provided herein are capable of deaminating cytosine.
  • the adenosine deaminases provided herein are capable of deaminating cytosine in a deoxyadenosine residue of DNA.
  • the adenosine deaminase can be derived from any suitable organism (e.g., E. coli) .
  • the adenine deaminase is a naturally occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein.
  • One of skill in the art will be able to identify the corresponding residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. Accordingly, one of skill in the art would be able to generate mutations in any naturally occurring adenosine deaminase that corresponds to any of the mutations described herein.
  • the adenosine deaminase is from a prokaryote.
  • the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus injluenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli.
  • the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to any one of the amino acid sequences set forth in any one of SEQ ID NOs: 112-121, or to any of the adenosine deaminases provided herein. It should be appreciated that any of the adenosine deaminases provided herein can include one or more mutations (e.g., any of the mutations provided herein) .
  • the disclosure provides any deaminase domains with a certain percent identify plus any of the mutations or combinations thereof described herein.
  • the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 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 more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs: 112-121, or any of the adenosine deaminases provided herein.
  • the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NOs: 112-121, or any of the adenosine deaminases provided herein.
  • the deaminases provided herein are capable of deaminating adenine. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine in a deoxyadenosine residue of DNA.
  • the deaminases provided herein are capable of deaminating cytosine or 5-methylcytosine to uracil or thymine. In some embodiments, the deaminases provided herein are capable of deaminating cytosine in DNA.
  • compositions and methods disclosed herein include nucleobase editors, e.g., adenosine base editors, for editing, modifying or altering a target nucleotide sequence of a polynucleotide.
  • nucleobase editors comprising a programmable nucleotide binding domain, for example, a polynucleotide programmable nucleotide binding domain (e.g., Cas9) or zinc finger protein DNA binding domain or TALE DNA binding domain and at least one nucleobase editing domain, e.g., an adenosine deaminase.
  • a polynucleotide programmable nucleotide binding domain (e.g., Cas9 or Cas12)
  • 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
  • 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
  • base editing activity is assessed by efficiency of editing.
  • Base editing can be determined by any suitable means, for example, by Sanger sequencing or next generation sequencing.
  • base editing efficiency is measured by percentage of total sequencing reads with nucleobase conversion effected by the base editor, for example, percentage of total sequencing reads with target A-T base pair converted to a G-C base pair.
  • base editing efficiency is measured by percentage of total cells with nucleobase conversion effected by the base editor, when base editing is performed in a population of cells.
  • a base editor system refers to the components required for editing a nucleobase of a target nucleotide sequence.
  • a base editor system comprises (1) a polynucleotide programmable nucleotide binding domain (e.g., Cas9) ; (2) a deaminase domain (e.g. an adenosine deaminase and/or cytidine deaminase; see PCT/US2019/044935, PCT/US2020/016288, each of which is incorporated herein by reference for its entirety) for deaminating said nucleobase; and (3) one or more guide polynucleotide (e.g., guide RNA) .
  • a polynucleotide programmable nucleotide binding domain e.g., Cas9
  • a deaminase domain e.g. an adenosine deaminase and/or cytidine deaminas
  • a polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain.
  • a base editor is an adenine or adenosine base editor (ABE) .
  • a base editor system can comprise more than one base editing component.
  • a base editor system can include more than one deaminase.
  • a base editor system can include one or more adenosine deaminases.
  • a single guide polynucleotide can be utilized to target different deaminases to a target nucleic acid sequence.
  • a pair of guide polynucleotides can be utilized to target different deaminases to a target nucleic acid sequence.
  • the deaminase domain and the polynucleotide programmable nucleotide binding component of a base editor system can be associated with each other covalently or noncovalently, or any combination of associations and interactions thereof.
  • a deaminase domain can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain.
  • a polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain.
  • a polynucleotide programmable nucleotide binding domain can target a deaminase domain to a target nucleotide sequence by noncovalently interacting with or associating with the deaminase domain.
  • the deaminase domain can comprise an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with, an additional heterologous portion or domain that is part of a polynucleotide programmable nucleotide binding domain.
  • the additional heterologous portion can be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion can be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion can be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion can be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion can be capable of binding to a polynucleotide linker. The additional heterologous portion can be a protein domain.
  • the additional heterologous portion can be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a steril alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or a RNA recognition motif.
  • KH K Homology
  • a base editor system can further include a guide polynucleotide component. It should be appreciated that components of the base editor system can be associated with each other via covalent bonds, noncovalent interactions, or any combination of associations and interactions thereof. In some embodiments, a deaminase domain can be targeted to a target nucleotide sequence by a guide polynucleotide.
  • the deaminase domain can comprise an additional heterologous portion or domain (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting with, associating with, or capable of forming a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide.
  • the additional heterologous portion or domain e.g., polynucleotide binding domain such as an RNA or DNA binding protein
  • the additional heterologous portion can be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion can be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion can be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion can be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion can be capable of binding to a polynucleotide linker. The additional heterologous portion can be a protein domain.
  • the additional heterologous portion can be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or a RNA recognition motif.
  • KH K Homology
  • a base editor described herein can comprise a deaminase domain which includes an adenosine deaminase.
  • 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.
  • Adenosine deaminase is capable of deaminating (i.e., removing an amine group) adenine of a deoxyadenosine residue in deoxyribonucleic acid (DNA) .
  • the nucleobase editors provided herein can be made by fusing together one or more protein domains, thereby generating a fusion protein.
  • the fusion proteins provided herein comprise one or more features that improve the base editing activity (e.g., efficiency, selectivity, and specificity) of the fusion proteins.
  • the fusion proteins provided herein can comprise a Cas9 domain that has reduced nuclease activity.
  • the fusion proteins provided herein can have a Cas9 domain that does not have nuclease activity (dCas9) , or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9) .
  • dCas9 nuclease activity
  • nCas9 nickase Cas9 nickase
  • an A-to-G base editor further comprises an inhibitor of inosine base excision repair, for example, an uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine specific nuclease.
  • UMI uracil glycosylase inhibitor
  • 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.
  • a deaminated adenosine residue e.g., inosine
  • a base editor comprising an adenosine deaminase can act on any polynucleotide, including DNA, RNA and DNA-RNA hybrids.
  • a base editor comprising an adenosine deaminase can deaminate a target A of a polynucleotide comprising RNA.
  • the base editor can comprise an adenosine deaminase domain capable of deaminating a target A of an RNA polynucleotide and/or a DNA-RNA hybrid polynucleotide.
  • an adenosine deaminase incorporated into a base editor comprises all or a portion of adenosine deaminase acting on RNA (ADAR, e.g., ADAR1 or ADAR2) .
  • adenosine deaminase incorporated into a base editor comprises all or a portion of adenosine deaminase acting on tRNA (ADAT) .
  • a base editor comprising an adenosine deaminase domain can also be capable of deaminating an A nucleobase of a DNA polynucleotide.
  • an adenosine deaminase domain of a base editor comprises all or a portion of an ADAT comprising one or more mutations which permit the ADAT to deaminate a target A in DNA.
  • the adenosine deaminase can be derived from any suitable organism (e.g., E. coli) .
  • the adenine deaminase is a naturally occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein.
  • the adenosine deaminase comprises a polypeptide sequence set forth in any one of SEQ ID NOs: 112-121.
  • the fusion proteins disclosed herein can comprise a cytidine deaminase.
  • the cytidine deaminases provided herein are capable of deaminating cytosine or 5-methylcytosine to uracil or thymine.
  • the cytidine deaminases provided herein are capable of deaminating cytosine in DNA.
  • the cytidine deaminase may be derived from any suitable organism.
  • the cytidine deaminase is from a prokaryote.
  • the cytidine deaminase is from a bacterium.
  • the cytidine deaminase is from a mammal (e.g., human) .
  • the cytidine deaminase is a naturally occurring (e.g., wild type) cytidine deaminase. In some embodiments, the cytidine deaminase is a naturally occurring cytidine deaminase that includes one or more mutations corresponding to any of the mutations provided herein.
  • One of skill in the art will be able to identify the corresponding residue in any homologous protein, e.g., by sequence alignment and determination of homologous residues. Accordingly, one of skill in the art would be able to generate mutations in any naturally occurring cytidine deaminase that corresponds to any of the mutations described herein.
  • the cytidine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%identical to any one of the cytidine deaminase amino acid sequences set forth in SEQ ID NOs 122-132. It should be appreciated that cytidine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein) .
  • the disclosure provides any deaminase domains with a certain percent identity plus any of the mutations or combinations thereof described herein.
  • the cytidine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 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 more mutations compared to a reference sequence, or any of the cytidine deaminases provided herein.
  • the cytidine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences known in the art or described herein.
  • a cytidine deaminase of a base editor can comprise all or a 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 APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D ( “APOBEC3E” now refers to this) , APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and Activation-induced (cytidine) deaminase (AID) .
  • a deaminase incorporated into a base editor comprises all or a portion of an APOBEC1 deaminase.
  • a deaminase incorporated into a base editor comprises all or a portion of APOBEC2 deaminase.
  • a deaminase incorporated into a base editor comprises all or a portion of is an APOBEC3 deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of an APOBEC3A deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3B deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3C deaminase.
  • a deaminase incorporated into a base editor comprises all or a portion of APOBEC3D deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3E deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3F deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3G deaminase.
  • a deaminase incorporated into a base editor comprises all or a portion of APOBEC3H deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC4 deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of activation-induced deaminase (AID) . In some embodiments a deaminase incorporated into a base editor comprises all or a portion of cytidine deaminase 1 (CDA1) .
  • CDA1 cytidine deaminase 1
  • the cytidine deaminase is (a) a cytidine deaminase enzyme from Burkholderia cenocepacia, (b) a SCP1.201-like deaminase enzyme from Streptacidiphilus jeojiense, (c) an Imm1 family immunity protein enzyme from Brevilactibacter coleopterorum, (d) a DUF6531 domain-containing protein enzyme from Pseudomonas koreensis, (e) a PAAR domain-containing protein enzyme from Yersinia similis, (f) an RHS repeat protein enzyme from Salmonella enterica, (g) a PAAR domain-containing protein enzyme from Pseudomonas, (h) an APOBEC-1 enzyme from Tupaia chinensis, (i) an APOBEC-1-like enzyme from Suncus etruscus, (j) an APOBEC-1 enzyme from Fukomys damarensis,
  • the cytidine deaminase is an enzyme having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs 122-132.
  • Some aspects of the present disclosure are based on the recognition that modulating the deaminase domain catalytic activity of any of the fusion proteins described herein, for example by making point mutations in the deaminase domain, can affect the processivity of the fusion proteins (e.g., base editors) .
  • mutations that reduce, but do not eliminate, the catalytic activity of a deaminase domain within a base editing fusion protein can make it less likely that the deaminase domain will catalyze the deamination of a residue adjacent to a target residue, thereby narrowing the deamination window.
  • the ability to narrow the deamination window can prevent unwanted deamination of residues adjacent to specific target residues, which can decrease or prevent off-target effects.
  • compositions and methods disclosed herein include cytidine base editors, for editing, modifying or altering a target nucleotide sequence of a polynucleotide.
  • nucleobase editors comprising a programmable nucleotide binding domain, for example, a polynucleotide programmable nucleotide binding domain (e.g., Cas9) or zinc finger protein DNA binding domain or TALE DNA binding domain and at least one nucleobase editing domain, e.g., a cytidine deaminase.
  • a polynucleotide programmable nucleotide binding domain (e.g., Cas9 or Cas12)
  • 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
  • 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
  • the compositions and methods disclosed herein include a cytidine base editor (CBE) .
  • fusion proteins provided herein comprise one or more nucleic acid editing domains.
  • a base editor disclosed herein comprises a fusion protein comprising a 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.
  • the uridine base can then be substituted with a thymidine base (e.g., by cellular repair machinery) to cause a C:G to a T: Atransition.
  • deamination of a C to U in a nucleic acid by a base editor may or may not 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 or deaminase 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 an 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
  • 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 catalyzes a C nucleobase that is positioned in the context of a single-stranded portion of a polynucleotide.
  • the entire polynucleotide comprising a target C can be single-stranded.
  • a cytidine deaminase incorporated into the base editor can deaminate a target C in a single-stranded RNA polynucleotide.
  • a base editor comprising a cytidine deaminase domain can act on a double-stranded polynucleotide, but the target C can be positioned in a portion of the polynucleotide which at the time of the deamination reaction is in a single-stranded state.
  • a base editor comprising a cytidine deaminase domain
  • the target C can be positioned in a portion of the polynucleotide which at the time of the deamination reaction is in a single-stranded state.
  • a Cas9 domain several nucleotides can be left unpaired during formation of the Cas9-gRNA-target DNA complex, resulting in formation of a Cas9 "R-loop complex" .
  • These unpaired nucleotides can form a bubble of single-stranded DNA that can serve as a substrate for a single-strand specific nucleotide deaminase enzyme (e.g., cytidine deaminase) .
  • a single-strand specific nucleotide deaminase enzyme e.g., cytidine deaminase
  • the efficacy of a gRNA is determined when delivered together with other components, e.g., a nucleic acid encoding an RNA-guided DNA binding agent such as any of those described herein. In some embodiments, the efficacy of a combination of a gRNA and a nucleic acid encoding an RNA-guided DNA binding agent is determined.
  • RNA-guided DNA nuclease and a guide RNA disclosed herein can lead to double-stranded breaks in the DNA, which can produce errors in the form of insertion/deletion (indel) mutations upon repair by cellular machinery.
  • Indel insertion/deletion
  • Many mutations due to indels alter the reading frame or introduce premature stop codons and, therefore, produce a non-functional protein.
  • the efficacy of particular gRNAs or combinations is determined based on in vitro models.
  • the in vitro model is HEK293 cells.
  • the in vitro model is HUH7 human hepatocarcinoma cells.
  • the in vitro model is HepG2 cells.
  • the in vitro model is primary human hepatocytes.
  • the in vitro model is primary rodent hepatocytes.
  • the in vitro model is primary cynomolgus hepatocytes. With respect to using primary human hepatocytes, commercially available primary human hepatocytes can be used to provide greater consistency between experiments.
  • the number of off-target sites at which a deletion or insertion occurs in an in vitro model is determined, e.g., by analyzing genomic DNA from primary human hepatocytes transfected in vitro with Cas9 mRNA and the guide RNA.
  • such a determination comprises analyzing genomic DNA from primary human hepatocytes transfected in vitro with Cas9 mRNA and the guide RNA. Exemplary procedures for such determinations are provided in the working examples below.
  • the efficacy of particular gRNAs or combinations is determined across multiple in vitro cell models for a gRNA selection process.
  • a cell line comparison of data with selected gRNAs is performed.
  • cross screening in multiple cell models is performed.
  • the efficacy of particular gRNAs or combinations is determined based on in vivo models.
  • the in vivo model is a rodent model.
  • the rodent model is a mouse, which expresses a human HSD17B13 gene, which may be a mutant human HSD17B13 gene.
  • the in vivo model is a non-human primate, for example, a cynomolgus monkey.
  • the efficacy of a guide RNA or combination is measured by percent editing of HSD17B13.
  • the percent editing of HSD17B13 is compared to the percent editing necessary to achieve knockdown of HSD17B13 protein, e.g., in the cell culture media in the case of an in vitro model or in serum or tissue in the case of an in vivo model.
  • the percent editing is between 30 and 99%of the population of cells.
  • the percent editing is between 30%and 35%, 35%and 40%, 40%and 45%, 45%and 50%, 50%and 55%, 55%and 60%, 60%and 65%, 65%and 70%, 70%and 75%, 75%and 80%, 80%and 85%, 85%and 90%, 90%and 95%, or 95%and 99%of the population of cells. In some embodiments, the percent editing is between 30%-95%, 40%-90%, or 50%-85%, 30%-60%, 40%-80%, 50%-75%, or 60%-90%.
  • the efficacy of a guide RNA or combination is measured by the number and/or frequency of indels at off-target sequences within the genome of the target cell type.
  • efficacious guide RNAs and combinations are provided which produce indels at off target sites at very low frequencies (e.g., ⁇ 5%) in a cell population and/or relative to the frequency of indel creation at the target site.
  • the disclosure provides for guide RNAs which do not exhibit off-target indel formation in the target cell type (e.g., a hepatocyte) , or which produce a frequency of off-target indel formation of ⁇ 5%in a cell population and/or relative to the frequency of indel creation at the target site.
  • the disclosure provides guide RNAs and combinations which do not exhibit any off target indel formation in the target cell type (e.g., hepatocyte) .
  • guide RNAs and combinations are provided which produce indels at less than 5 different off-target sites, e.g., as evaluated by one or more methods described herein. In some embodiments, guide RNAs and combinations are provided which produce indels at less than or equal to 4, 3, 2, or 1 off-target site (s) , e.g., as evaluated by one or more methods described herein. In some embodiments, the off-target site (s) does not occur in a protein coding region in the target cell (e.g., hepatocyte) genome.
  • detecting gene editing events such as the formation of insertion/deletion ( “indel” ) mutations and homology directed repair (HDR) events in target DNA utilize linear amplification with a tagged primer and isolating the tagged amplification products (herein after referred to as "LAM-PCR, " or “Linear Amplification (LA) " method) , as described in WO2018/067447 or Schmidt et al., Nature Methods 4: 1051-1057 (2007) .
  • detecting gene editing events such as the formation of insertion/deletion ( "indel” ) mutations and homology directed repair (HDR) events in target DNA, further comprises sequencing the linear amplified products or the further amplified products.
  • Sequencing may comprise any method known to those of skill in the art, including, next generation sequencing, and cloning the linear amplification products or further amplified products into a plasmid and sequencing the plasmid or a portion of the plasmid. Exemplary next generation sequencing methods are discussed, e.g., in Shendure et al., Nature 26: 1135-1145 (2008) .
  • detecting gene editing events such as the formation of insertion/deletion ( “indel” ) mutations and homology directed repair (HDR) events in target DNA
  • detecting gene editing events further comprises performing digital PCR (dPCR) or droplet digital PCR (ddPCR) on the linear amplified products or the further amplified products, or contacting the linear amplified products or the further amplified products with a nucleic acid probe designed to identify DNA having homology-directed repair (HDR) template sequence and detecting the probes that have bound to the linear amplified product (s) or further amplified product (s) .
  • the method further comprises determining the location of the HDR template in the target DNA.
  • the method further comprises determining the sequence of an insertion site in the target DNA, wherein the insertion site is the location where the HDR template incorporates into the target DNA, and wherein the insertion site may include some target DNA sequence and some HDR template sequence.
  • the amount of HSD17B13 in cells measures efficacy of a gRNA or combination. In some embodiments, the amount of HSD17B13 in cells is measured using western blot. In some embodiments, the cell used is HUH7 cells. In some embodiments, the cell used is a primary human hepatocyte. In some embodiments, the cell used is a primary cell obtained from an animal. In some embodiments, the amount of HSD17B13 is compared to the amount of glyceraldehyde 3-phosphate dehydrogenase GAPDH (ahousekeeping gene) to control for changes in cell number.
  • GAPDH housekeeping gene
  • the amount of HSD17B13 is reduced by between 30%and 35%, 35%and 40%, 40%and 45%, 45%and 50%, 50%and 55%, 55%and 60%, 60%and 65%, 65%and 70%, 70%and 75%, 75%and 80%, 80%and 85%, 85%and 90%, 90%and 95%, or 95%and 99%of the ALAS1 in cells detected in the subject before administration of the composition.
  • the amount of HSD17B13 is reduced by between 30%-95%, 40%-90%, or 50%-85%, 30%-60%, 40%-80%, 50%-75%, or 60%-90%of the HSD17B13 in cells detected in the subject before administration of the composition.
  • the disclosure provides a method of treating NASH which includes administering a composition including a guide RNA having any one or more of the guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199. In some embodiments, the disclosure provides a method of treating NASH which includes administering a composition including an sgRNA comprising any one or more of the guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199.
  • the guide RNA can be administered together with a nucleic acid or vector described herein encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or as a component of a base editing system, e.g., an ABE or a CBE system.
  • a nucleic acid or vector described herein encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or as a component of a base editing system, e.g., an ABE or a CBE system.
  • the disclosure provide a method of treating NASH which includes administering a composition including an sgRNA comprising any one or more of the guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199 administered together with an ABE comprising an adenosine deaminase having an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to any one of the amino acid sequences set forth in any one of SEQ ID NOs: 112-121.
  • the disclosure provide a method of treating NASH which includes administering a composition including an sgRNA comprising any one or more of the guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199 administered together with a CBE comprising a cytidine deaminase having an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%identical to any one of the cytidine deaminase amino acid sequences set forth in SEQ ID NOs 122-132.
  • the RNA-guided DNA nuclease may be an S. pyogenes Cas9.
  • the guide RNA is chemically modified.
  • the guide RNA and the nucleic acid encoding an RNA-guided DNA nuclease or the base editing system are administered in an LNP described herein, such as an LNP having a CCD lipid (e.g., an amine lipid, such as lipid A) , a helper lipid (e.g., cholesterol) , a stealth lipid (e.g., a PEG lipid, such as PEG2k-DMG) , and optionally a neutral lipid (e.g., DSPC) .
  • CCD lipid e.g., an amine lipid, such as lipid A
  • a helper lipid e.g., cholesterol
  • a stealth lipid e.g., a PEG lipid, such as PEG2k-DMG
  • the disclosure provides a method of inducing a double-stranded break (DSB) within the HSD17B13 gene including administering a composition having a guide RNA as described herein, e.g., having any one or more guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199.
  • gRNAs such as any one or more of the guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199 are administered to recognize and bind to the HSD17B13 gene.
  • the guide RNA can be administered together with a nucleic acid (e.g., mRNA) or vector described herein encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) , or together with a base editing system e.g., an ABE or a CBE system.
  • a nucleic acid e.g., mRNA
  • vector described herein encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9)
  • a base editing system e.g., an ABE or a CBE system.
  • the RNA-guided DNA nuclease may be an S. pyogenes Cas9.
  • the guide RNA is chemically modified.
  • a method of inducing a double-stranded break (DSB) within the HSD17B13 gene comprising administering a composition comprising a guide RNA, such as a chemically modified guide RNA, comprising any one or more guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199.
  • a guide RNA such as a chemically modified guide RNA, comprising any one or more guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199.
  • any one or more of the gRNAs comprising any one or more of the guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199 are administered to induce a DSB in the HSD17B13 gene.
  • the guide RNA can be administered together with a nucleic acid or vector described herein encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) .
  • the RNA-guided DNA nuclease may be an S. pyogenes Cas9.
  • the guide RNA is chemically modified.
  • the guide RNA and the nucleic acid encoding an RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP comprising a CCD lipid (e.g., an amine lipid, such as lipid A) , a helper lipid (e.g., cholesterol) , a stealth lipid (e.g., a PEG lipid, such as PEG2k-DMG) , and optionally a neutral lipid (e.g., DSPC) .
  • a CCD lipid e.g., an amine lipid, such as lipid A
  • helper lipid e.g., cholesterol
  • a stealth lipid e.g., a PEG lipid, such as PEG2k-DMG
  • a neutral lipid e.g., DSPC
  • a method of modifying the HSD17B13 gene comprising administering a composition comprising a guide RNA as described herein, e.g., having any one or more of the guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199.
  • gRNAs comprising any one or more of the guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199 are administered to modify the HSD17B13 gene.
  • the guide RNA can be administered together with a nucleic acid or vector described herein encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) .
  • a Cas nuclease e.g., Cas9
  • the RNA-guided DNA nuclease may be an S. pyogenes Cas9.
  • the guide RNA is chemically modified.
  • the guide RNA and the nucleic acid encoding an RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP comprising a CCD lipid (e.g., an amine lipid, such as lipid A) , a helper lipid (e.g., cholesterol) , a stealth lipid (e.g., a PEG lipid, such as PEG2k-DMG) , and optionally a neutral lipid (e.g., DSPC) .
  • a CCD lipid e.g., an amine lipid, such as lipid A
  • helper lipid e.g., cholesterol
  • a stealth lipid e.g., a PEG lipid, such as PEG2k-DMG
  • a neutral lipid e.g., DSPC
  • a method of treating NASH comprising administering a composition comprising a guide RNA as described herein, e.g., having any one or more of the guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199.
  • gRNAs comprising any one or more of the guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199 are administered to treat NASH.
  • the guide RNA can be administered together with a nucleic acid or vector described herein encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) .
  • a Cas nuclease e.g., Cas9
  • the RNA-guided DNA nuclease may be an S. pyogenes Cas9.
  • the guide RNA is chemically modified.
  • the guide RNA and the nucleic acid encoding an RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP comprising a CCD lipid (e.g., an amine lipid, such as lipid A) , a helper lipid (e.g., cholesterol) , a stealth lipid (e.g., a PEG lipid, such as PEG2k-DMG) , and optionally a neutral lipid (e.g., DSPC) .
  • a CCD lipid e.g., an amine lipid, such as lipid A
  • helper lipid e.g., cholesterol
  • a stealth lipid e.g., a PEG lipid, such as PEG2k-DMG
  • a neutral lipid e.g., DSPC
  • the disclosure provides a method of reducing HSD17B13 expression including administering a guide RNA as described herein, e.g. having any one or more of the guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199.
  • gRNAs comprising any one or more of the guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199 are administered to reduce HSD17B13 expression in hepatic tissue.
  • the gRNA can be administered together with a nucleic acid or vector described herein encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) .
  • a Cas nuclease e.g., Cas9
  • the RNA-guided DNA nuclease may be an S. pyogenes Cas9.
  • the guide RNA is chemically modified.
  • the guide RNA and the nucleic acid encoding an RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP comprising a CCD lipid (e.g., an amine lipid, such as lipid A) , a helper lipid (e.g., cholesterol) , a stealth lipid (e.g., a PEG lipid, such as PEG2k-DMG) , and optionally a neutral lipid (e.g., DSPC) .
  • a CCD lipid e.g., an amine lipid, such as lipid A
  • helper lipid e.g., cholesterol
  • a stealth lipid e.g., a PEG lipid, such as PEG2k-DMG
  • a neutral lipid e.g., DSPC
  • the disclosure features a method of reducing the levels of HSD17B13 protein in the tissues of a subject including comprising administering a composition comprising a guide RNA as described herein, e.g., having any one or more of the guide sequences of SEQ ID NOs: 1-88, SEQ ID NOs: 89-111, or SEQ ID NOs: 149-199.
  • the gRNA can be administered together with a nucleic acid or vector described herein encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) .
  • the RNA-guided DNA nuclease may be an S. pyogenes Cas9.
  • the guide RNA is chemically modified.
  • the guide RNA and the nucleic acid encoding an RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP comprising a CCD lipid (e.g., an amine lipid, such as lipid A) , a helper lipid (e.g., cholesterol) , a stealth lipid (e.g., a PEG lipid, such as PEG2k-DMG) , and optionally a neutral lipid (e.g., DSPC) .
  • a CCD lipid e.g., an amine lipid, such as lipid A
  • helper lipid e.g., cholesterol
  • a stealth lipid e.g., a PEG lipid, such as PEG2k-DMG
  • optionally a neutral lipid e.g., DSPC
  • the gRNA includes a guide sequence of Table 3, Table 11, or Table 20 together with an RNA-guided DNA nuclease such as a Cas nuclease to induce DSBs, and non-homologous ending joining (NHEJ) during repair leads to a mutation in the HSD17B13 gene.
  • NHEJ leads to a deletion or insertion of a nucleotide (s) , which induces a frameshift or nonsense mutation in the HSD17B13 gene.
  • administering the guide RNA and nucleic acid encoding an RNA-guided DNA binding agent reduces levels of HSD17B13 in hepatocytes of the subject.
  • the subject is mammalian. In some embodiments, the subject is human. In some embodiments, the subject is cow, pig, monkey, sheep, dog, cat, fish, or poultry. In some embodiments, the subject is a companion animal or a livestock animal.
  • RNA-guided DNA-binding agent e.g., an S. pyogenes Cas9.
  • the guide RNA is chemically modified.
  • the composition that includes the guide RNA and nucleic acid is administered intravenously. In some embodiments, the composition that includes the guide RNA and nucleic acid is administered into the hepatic circulation.
  • a single administration of a composition that includes a guide RNA and nucleic acid provided herein is sufficient to knock down expression of the target protein, for example, HSD17B13.
  • a single administration of a composition that includes a guide RNA and nucleic acid provided herein is sufficient to knock out expression of the target protein in a population of cells.
  • more than one administration of a composition that includes a guide RNA and nucleic acid provided herein may be beneficial to maximize editing via cumulative effects.
  • a composition provided herein can be administered 2, 3, 4, 5, or more times, such as 2 times.
  • Administrations can be separated by a period of time ranging from, e.g., 1 day to 2 years, such as 1 to 7 days, 7 to 14 days, 14 days to 30 days, 30 days to 60 days, 60 days to 120 days, 120 days to 183 days, 183 days to 274 days, 274 days to 366 days, 366 days to 2 years, 2 years to 5 years, or 5 years to 10 years.
  • 1 day to 2 years such as 1 to 7 days, 7 to 14 days, 14 days to 30 days, 30 days to 60 days, 60 days to 120 days, 120 days to 183 days, 183 days to 274 days, 274 days to 366 days, 366 days to 2 years, 2 years to 5 years, or 5 years to 10 years.
  • treatment slows, halts, or reverses disease progression.
  • efficacy of treatment is measured by increased survival time of the subject.
  • efficacy of treatment is measured by subject-reported outcomes.
  • subject-reports outcomes can be measured over the course of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 or more months after treatment begins.
  • HepG2 cell line The human hepatocellular carcinoma cell line HepG2 was cultured in DMEM media supplemented with 10%fetal bovine serum. Cells were plated at a density of 1,000, 000-1, 500, 000 cells/well in a 6-well plate or 8, 000-22, 000 cells/well in a 96-well plate 24 hours prior to electroporation. Cells were electroporated with Celetrix electroporator (Celetrix, CTX-1500A) per the manufacturer's protocol. Cells were electroporated with an RNP complex containing Cas9 Nuclease (5-50 pmol) , sgRNA (10-500 pmol) and Celetrix buffer.
  • Huh7 cell line The human hepatocellular carcinoma cell line Huh7 was cultured in DMEM media supplemented with 10%fetal bovine serum. Cells were plated at a density of 500, 000-1,500, 000 cells/well in a 6-well plate or 5, 000-15, 000 cells/well in 96-well plate 24 hours prior to electroporation. Cells were transfected with Lipofectamine MessengerMAX (ThermoFisher, Cat. LMRNA003) per the manufacturer's protocol. Cells per well were transfected with a lipoplex containing 1-500 ng Cas9 mRNA, 2-1, 000 ng sgRNA and Lipofectamine MessengerMAX.
  • Lipofectamine MessengerMAX ThermoFisher, Cat. LMRNA003
  • Primary hepatocytes Primary human hepatocytes (PHH) were cultured per the manufacturer's protocol. In brief, the cells were thawed and resuspended in hepatocyte thawing medium with supplements followed by centrifugation at 100 g for 10 minutes for concentration. The supernatant was discarded and the pelleted cells resuspended in hepatocyte plating medium plus supplement pack. Cells were counted and plated on Bio-coat collagen I coated plates (ThermoFisher, Cat. 877272) at a density of 132, 000 cells/well in a 48-well plate or 270, 000 cells/well in a 24-well plate. Plated cells were allowed to settle and adhere for 4 to 6 hours in a tissue culture incubator at 37°C and 5%CO2 atmosphere.
  • RNAiMax ThermoFisher, Cat. 13778150
  • Transfected cells were harvested post-transfection at 72 hours.
  • the genomic DNA was extracted from each well of a 6-well/24-well/96-well plate using QuickExtract DNA Extraction Solution (LGC Lucigen, Cat. QE09050) per manufacturer's protocol. All DNA samples were subjected to subsequent Sanger sequencing analyses or amplicon-seq using NGS, as described herein.
  • Sanger sequencing was utilized to identify the editing efficiency introduced by gene editing. Primers were designed around the target site within the gene of interest (e.g., HSD17B13) , and the genomic area of interest was amplified. Sanger sequencing was performed on 3730xl DNA Analyzer (ThermoFisher, Cat. 3730XL) per manufacturer's protocol. The raw sequencing files (. ab1) were analyzed for determining editing efficiency in online analysis tools (e.g., TIDE: tide. nki. nl/, ICE: ice. synthego. com/) .
  • online analysis tools e.g., TIDE: tide. nki. nl/, ICE: ice. synthego. com/
  • NGS Next-generation sequencing
  • sequencing was utilized to identify the presence of insertions and deletions introduced by gene editing.
  • Primers were designed around the target site within the gene of interest (e.g., HSD17B13) , and the genomic area of interest was amplified.
  • PCR was performed per the manufacturer's protocols (Illumina) to add chemistry for sequencing.
  • the amplicons were sequenced on an Illumina NovaSeq 6000 instrument.
  • the reads were aligned to a reference genome (e.g., the human reference genome (hg38) , the cynomologus reference genome (mf5) , the rat reference genome (rn6) , or the mouse reference genome (mm10) ) after eliminating those having low quality scores.
  • the resulting files containing the reads were mapped to the reference genome (BAM files) , where reads that overlapped the target region of interest were selected and the number of wild type reads versus the number of reads which contain an insertion, substitution, or deletion was calculated.
  • the editing percentage (e.g., the "editing efficiency” or “percent editing” or “indel frequency” ) is defined as the total number of sequence reads with insertions/deletions ( “indels” ) or substitutions over the total number of sequence reads, including wild type.
  • sgRNAs targeting HSD17B13 were designed for use with SpCas9 and are shown in Table 3 below.
  • sgRNAs targeting human HSD17B13 were delivered together with SpCas9 protein to HepG2 cells as described in the Materials and Methods above. Percent editing was determined for sgRNAs comprising each guide sequence and the guide sequences were then rank-ordered based on highest %edit. The editing data are listed below in Table 4.
  • Table 3 Selected sgRNAs for SpCas9 *MM0-4 columns show number of sites with 0-4 mismatches to selected sgRNA in human reference genome.
  • Table 4 HSD17B13 editing data in HepG2 cells with Cas9 protein and sgRNAs
  • sgRNAs targeting human HSD17B13 were delivered together with SpCas9 mRNA to Huh7 cells as described in the Materials and Methods. Experiments were performed in 96 well plate format and each well was transfected with 100 ng SpCas9 mRNA and 100 ng sgRNA. Percent editing was determined for sgRNAs comprising each guide sequence and the guide sequences were then rank-ordered based on highest %edit. The editing data are listed below in Table 5.
  • Selected sgRNAs targeting human HSD17B13 were delivered to primary human hepatocytes (PHH) from two different donors. Experiments were performed as described in the Materials and Methods in 24 well plate format and each well was transfected with 500 ng SpCas9 mRNA and 500 ng sgRNA. Percent editing was determined for sgRNAs comprising each guide sequence and the guide sequences were then rank-ordered based on highest %edit. The editing data are listed below in Table 6 and 7, respectively, for the two different donors.
  • Table 7 HSD17B13 editing data in PHH (donor #2) with Cas9 mRNA and sgRNAs
  • dsDNA double-stranded DNA
  • dsDNA insertion-based assay was used to screen for potential genomic off-target sites cleaved by Cas9 with the corresponding gRNA.
  • HepG2 cells were maintained in MEM (Gibco) supplemented with 10%FBS (OPCEL) at 37 °C and 5%CO 2 atmosphere. About 500, 000 HepG2 cells were electroporated in 4D-Nuclefector (LONZA, X-unit) with 1.6 ⁇ g of dsDNA, 1.8 ⁇ g of Cas9 mRNA and 1.8 ⁇ g of gRNA (Genscript) . After one week, genomic DNA was extracted and processed for next-generation sequencing (NGS) analysis using a NextSeq6000 sequencer.
  • NGS next-generation sequencing
  • the dsDNA incorporation efficiency for each potential off-target site was calculated as the number of reads at this site divided by the reads at the on-target site within the HSD17B13 gene.
  • the sum of reads from the top 30 off-target sites divided by that of on-target site (top 30 off/on) were used as semi-quantitative readouts for comparison of off-target potentials between different sgRNAs.
  • the number of detected total off target sites and the top five sites of highest dsODN incorporation efficiencies are listed in Table 8.
  • Genomic DNA obtained from cells described in Example 2 was further analyzed for off-target editing by NGS-based amplicon sequencing. Potential off-target sites for each sgRNA were identified and are listed in Table 8. The off-target site editing efficiency was divided by the on-target efficiency in the same experiment to normalize for different transfection efficiencies, with results shown in Tables 9 and 10.
  • Targeted editing efficiency is calculated as the percentage of reads containing single nucleotide mutation at sites directly disrupting splicing or introducing premature stop codons, relative to the total number of reads.
  • Max editing efficiency is calculated as the percentage of reads containing single nucleotide mutation at sites within sgRNA targeting region, relative to the total number of reads.
  • Indel editing efficiency is calculated as the percentage of reads containing insertions or deletions within sgRNA targeting region, relative to the total number of reads.
  • ABEs demonstrated editing efficiencies exceeding 75%at the targeted nucleotide, with several combinations surpassing the performance of editors ABE8.8 and ABE8e with results shown in Table 14.
  • CBEs several combinations exhibited editing efficiencies exceeding 75%at the targeted nucleotide.
  • CE11 and CE43 outperformed CBE4max across the majority of the selected sgRNAs, with results shown in Table 15.
  • PHHs Primary human liver hepatocytes (PHH) were cultured per the manufacturer's protocol. Cells were thawed and resuspended in hepatocyte thawing medium with supplements followed by centrifugation at 100 g for 10 minutes for concentration. The supernatant was discarded, and the pelleted cells resuspended in hepatocyte plating medium plus supplement. Cells were counted and plated on Bio-coat collagen I coated 24-well plates (ThermoFisher, Cat. 877272) at a density of 270, 000 cells/well.
  • RNA extracted from those samples were analyzed for HSD17B13 and GAPDH RNA expression by qRT-PCR using three pairs of primers (primer pair 6 and 7 for HSD17B13, ref primer pair for GAPDH) .
  • the RNA expression of HSD17B13 in each sample was subjected to normalization using GAPDH expression as a reference, and the relative HSD17B13 expression levels in edited samples were compared to untreated PHH samples, as shown in Table 17.
  • EXAMPLE 8 Evaluate potent base editors for precise base editing of HSD17B13 in Primary Human Hepatocytes (PHHs)
  • Selected base editors and sgRNAs were further validated in primary human hepatocytes.
  • 250 ng base editor mRNA and 250 ng end-modified sgRNA were transfected into PHHs following the procedure described in Example 7.
  • cells were seeded at a different density at 132, 000 cells/well in a 48 well plate for transfection.
  • 72 hours after transfection genomic DNA was extracted from cells and editing efficiency was analyzed by amplicon-seq using NGS. The editing efficiencies achieved in two PHH donors for each combination are shown in Table 18.
  • Non-NGG PAM base editors were tested with the sgRNAs disclosed herein for HSD17B13 base editing.
  • Base editor sgRNAs were designed to disrupt splicing sites or introduce premature stop codons at the HSD17B13 locus using NG or NNG as PAM (described in Table 20) .
  • off-target sites of selected sgRNAs were predicted using Cas-OFFinder, and the top three sites were selected as shown in Table 23.
  • HEK293T cells were transfected with 80 ng base editor plasmid and 40 ng sgRNA plasmid, and DNA from those samples were harvested after 72 hours as described in Example 9.
  • the on-target and off-target sites were then amplified and editing efficiencies were analyzed and shown in Table 24 and 25. All combinations showed robust editing at on-target sites with minimal off-target activities.
  • a dsDNA insertion-based assay was used to screen for potential genomic off-target sites cleaved by Cas9 with the corresponding gRNA.
  • HepG2 cells were maintained in MEM (Gibco) supplemented with 10%FBS (OPCEL) at 37°C and 5%CO2 atmosphere.
  • 0.5 million HepG2 cells were electroporated in 4D-Nuclefector (LONZA) with 1.6 ⁇ g of dsDNA, 1.8 ⁇ g of Cas9 mRNA and 1.8 ⁇ g of gRNA (Genscript) .
  • genomic DNA was extracted and processed for NGS analysis (See, e.g., Tsai et al., Nature Biotechnology 33, 187-197; 2015) in a NextSeq6000 sequencer.
  • the dsDNA incorporation efficiency for each potential off-target site was calculated as the number of reads at this site divided by the reads at the on-target site (HSD17B13) .
  • the sum of reads from the top 30 off target sites divided by that of on target site (top 30 off/on) were used as semi-quantitative readouts for comparison of off-target potentials between different sgRNA.
  • the number of total off target sites, and the top five sites of highest dsODN incorporation efficiencies are listed in Table 26.
  • Genomic DNA samples obtained from Example 3 were analyzed for on-target and off-target activities using the top sites identified in Example 11.
  • On target and top off target sites of selected DNA samples were amplified by PCR using Taq Pro Multiplex DNA Polymerase (Vazyme) .
  • PCR product was purified with VAHTS DNA Clean Beads (Vazyme) and sequenced by NGS. To mitigate the potential influence of transfection efficiencies, the editing efficiency of each off-target site was normalized against the editing efficiency of its corresponding on-target site. The results are shown in Table 27.
  • dsDNA double-stranded DNA
  • dsDNA insertion-based assay was used to screen for potential genomic off-target sites cleaved by Cas9 with the corresponding gRNA. Experiments were performed as described in the Materials and Methods in 24 well plate format and each well was transfected with 500 ng SpCas9 mRNA, 100 ng sgRNA, and 50 ng dsDNA. After three days, genomic DNA was extracted and processed for next-generation sequencing (NGS) analysis using a NovaSeq 6000 sequencing system. The dsDNA incorporation efficiency for each potential off-target site was calculated as the number of reads at this site divided by the reads at the on-target site within the HSD17B13 gene.
  • NGS next-generation sequencing
  • top 30 off-target sites The sum of reads from the top 30 off-target sites divided by that of on-target site (top 30 off/on) were used as semi-quantitative readouts for comparison of off-target potentials between different sgRNAs.
  • the number of detected total off target sites and the top five sites of highest dsDNA incorporation efficiencies are listed in Table 28.
  • LNP containing SpCas9 mRNA and selected sgRNAs targeting human HSD17B13 were delivered to primary human hepatocytes (PHH) from three different donors. Experiments were performed as described in the Materials and Methods in 48 well plate format and each well was transfected with different doses of LNP. Percent editing was determined for sgRNAs comprising each guide sequence and the guide sequences were then rank-ordered based on highest %edit. The Editing efficiency data are listed below in Table 29.
  • H3-h-82 gRNAs (listed in Table 32) were delivered together with SpCas9 mRNA to Huh7 cells as described in the Materials and Methods.
  • LNP containing SpCas9 mRNA and modified H3-h-82 gRNAs were delivered to primary human hepatocytes (PHH) from three different donors. Experiments were performed as described in the Materials and Methods in 48 well plate format and each well was transfected with different doses of LNP. Percent editing of on-target (ON) and off-target (OT) was determined for sgRNAs comprising different modifications. The Editing efficiency data are listed below in Tables 33, 34, 35, 36, 37, 38, and Figs 1A-1E.
  • Spacer modified H3-h-65 gRNAs (listed in Table 32) was delivered together with SpCas9 mRNA to Huh7 cells as described in the Materials and Methods.
  • LNP containing SpCas9 mRNA and modified H3-h-65 gRNAs were delivered to primary human hepatocytes (PHH) from three different donors. Experiments were performed as described in the Materials and Methods in 48 well plate format and each well was transfected with different doses of LNP. Percent editing of on-target (ON) and off-target (OT) was determined for sgRNAs comprising different modification. The Editing efficiency data are listed below in Tables 39, 40, 41, 42, 43, and Figs 2A-2D.
  • SpCas9 protein sequence SEQ ID NO: 237) to generate SpCas9 variants.
  • the mutated SpCas9 variants were delivered as mRNA alongside sgRNA to Huh7 cells according to protocols described in Materials and Methods.
  • LNP containing the mutated SpCas9 mRNA and sgRNA gPC spacer sequence listed in Table 32) were delivered to primary human hepatocytes (PHH) derived from three different donors.
  • the modified variants were evaluated in primary human hepatocytes (PHH) in combination with E3_SD_97 gRNA.
  • PHH primary human hepatocytes
  • the co-transfection of mRNA and gRNA was performed at a 1: 1 weight ratio following procedures described in Example 7.
  • Editing efficiencies were determined through next-generation sequencing (NGS) analysis of extracted genomic DNA. Enhanced editing efficiency, compared to the original ABE T8.4, was observed in several mutated variants (results shown in Table 47) .
  • the top-performing variants were subsequently formulated with E3_SD_97 gRNA into lipid nanoparticles (LNPs) according to methods described in patent WO/2023/185697A2, and their activity was further validated in PHH (Fig. 4) .
  • LNPs lipid nanoparticles
  • the mRNA coding sequence of a ABE T8.4 mutated variant FF-725 was optimized to improve expression, resulting in variants FF-891, FF-892, FF-893, and FF-1009 derived from the original sequence FF-725 mRNA (sequences provided in Table 49) . Editing activity testing of these optimized mRNAs was performed in PHH using established lipofection transfection methods followed by NGS analysis. Enhanced performance was demonstrated by the optimized mRNA sequences compared to the original sequence (Fig. 5) .
  • top-performing mRNA sequences was further validated in PHH following their formulation in LNPs (Fig. 6) .
  • the most effective mRNA coding sequences were subsequently combined with the top-performing ABE TadA variants to generate FF-1050 (FF-878 TadA in FF-892 mRNA) and FF-1051 (FF-903 TadA in FF-892 mRNA) (sequences provided in Table 49) .
  • FF-1050 FF-878 TadA in FF-892 mRNA
  • FF-1051 FF-903 TadA in FF-892 mRNA
  • E3_SD_97 gRNA 2'-fluoro modified ribonucleotides were introduced into the spacer sequence of E3_SD_97 gRNA to enhance ABE editing efficiency, resulting in variants E3_SD_97-seq01-15 (sequences provided in Table 50) .
  • Modified gRNAs and unmodified controls were lipo-transfected into Huh7 cells alongside ABE mRNA at two different doses-125 ng mRNA + 125 ng gRNA or 32.5 ng mRNA + 32.5 ng gRNA-and analyzed for editing efficiency using NGS.
  • the on-target (ON) or off-target (OFF) editing results were shown in Table 51.
  • Top enhanced modified sites were combined and generates modified gRNA variants E3_SD_97-seq21, E3_SD_97-seq22, and E3_SD_97-seq23 (sequences provided in Table 50) .
  • the modified gRNAs and unmodified controls were formulated with ABE FF-1051 mRNA in LNPs, and their on-target and top site off-target activities were evaluated in PHH using previously described procedures. Significant improvement in ABE editing efficiency was achieved through 2'-fluoro modifications at specific sites, while off-target activity remained below 0.5% (Fig. 8) .
  • compositions for delivery of the protein and nucleic acid components of CRISPR/Cas to a cell, such as a cell in a patient are needed.
  • compositions with useful properties for in vitro and in vivo delivery that can stabilize and deliver RNA components are of interest.
  • the LNP compositions comprise: an RNA component; and a lipid component, wherein the lipid component comprises: (1) 45-55 mol-%amine lipid; (2) 9-11 mol-%neutral lipid; and (3) 1-5 mol-%PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is 3-8.
  • Humanized HSD17B13 mice with C57BL/6J background were engineered such that a region of the endogenous murine hsd17b13 locus was deleted and replaced with an orthologous human HSD17B13 sequence so that the locus encodes a human HSD17B13 protein.
  • mice ranging 6-12 weeks of age were used in in vivo study. Animals were weighed and grouped according to body weight for preparing dosing solutions based on group average weight. LNPs were dosed via the tail vein in a volume of 10 mL/kg per animal. The animals were observed every day to monitor status. Blood samples were collected from saphenous vein or heart puncture at indicated time points. Liver tissues were collected from animals 5 ⁇ 7 days after dosing and were immediately put in -80°C for further NGS analysis.
  • LNP formulations were administered to animals at a 2: 1 weight ratio of SpCas9 mRNA or base editor mRNA (FF-725) to sgRNA, with specific dosing details provided in Table 52.
  • HSD17B13 gene editing efficiencies in liver tissue for each experimental group are presented in Table 52.
  • gRNA scaffold sequences were listed in Table 53. Successful editing of the HSD17B13 gene was demonstrated using multiple sgRNAs as specified.
  • sgRNA scaffold sequences *mA, mU, mC, mG indicates 2'O-methyl modifications to ribonucleotide; *stands for phosphorothioate modifications

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Abstract

L'invention concerne des ARN guides ciblant le gène HSD17B13. L'invention concerne également des vecteurs, des compositions et des méthodes de traitement de sujets atteints d'une maladie hépatique.
PCT/CN2025/075417 2024-02-02 2025-01-27 Compositions et méthodes de traitement d'une maladie hépatique Pending WO2025162435A1 (fr)

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Citations (4)

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US20180216104A1 (en) * 2017-01-23 2018-08-02 Regeneron Pharmaceuticals, Inc. HSD17B13 Variants And Uses Thereof
US20210222173A1 (en) * 2018-09-28 2021-07-22 Intellia Therapeutics, Inc. Compositions and Methods for Lactate Dehydrogenase (LDHA) Gene Editing
US20220220474A1 (en) * 2020-12-23 2022-07-14 Regeneron Pharmaceuticals, Inc. Treatment of Liver Diseases With Cell Death Inducing DFFA Like Effector B (CIDEB) Inhibitors
US20230265154A1 (en) * 2017-09-06 2023-08-24 Gracell Biotechnologies (Shanghai) Co., Ltd. Universal chimeric antigen receptor t-cell preparation technique

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US20180216104A1 (en) * 2017-01-23 2018-08-02 Regeneron Pharmaceuticals, Inc. HSD17B13 Variants And Uses Thereof
US20230265154A1 (en) * 2017-09-06 2023-08-24 Gracell Biotechnologies (Shanghai) Co., Ltd. Universal chimeric antigen receptor t-cell preparation technique
US20210222173A1 (en) * 2018-09-28 2021-07-22 Intellia Therapeutics, Inc. Compositions and Methods for Lactate Dehydrogenase (LDHA) Gene Editing
US20220220474A1 (en) * 2020-12-23 2022-07-14 Regeneron Pharmaceuticals, Inc. Treatment of Liver Diseases With Cell Death Inducing DFFA Like Effector B (CIDEB) Inhibitors

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DATABASE Protein 5 July 2022 (2022-07-05), ANONYMOUS : "tRNA adenosine(34) deaminase TadA [Intestinirhabdus alba]", XP093340944, retrieved from NCBI Database accession no. WP_155107477 *
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