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WO2025137646A1 - Méthodes d'édition de gènes et compositions pour le traitement de la fibrose kystique - Google Patents

Méthodes d'édition de gènes et compositions pour le traitement de la fibrose kystique Download PDF

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Publication number
WO2025137646A1
WO2025137646A1 PCT/US2024/061579 US2024061579W WO2025137646A1 WO 2025137646 A1 WO2025137646 A1 WO 2025137646A1 US 2024061579 W US2024061579 W US 2024061579W WO 2025137646 A1 WO2025137646 A1 WO 2025137646A1
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Prior art keywords
lnp
nucleic acid
cftr
subject
sort
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Inventor
Daniel J. Siegwart
Sumanta Chatterjee
Yehui Sun
Sakya MOHAPATRA
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University of Texas System
Recode Therapeutics Inc
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University of Texas System
Recode Therapeutics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • 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
    • C12N15/1138—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 against receptors or cell surface proteins
    • 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
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04002—Adenine deaminase (3.5.4.2)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4712—Cystic fibrosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/335—Modified T or U

Definitions

  • the present disclosure relates in some aspects to methods and uses of lipid nanoparticles comprising nucleic acids encoding a base editor and a guide RNA, for treating subjects with cystic fibrosis, and related methods, uses, and articles of manufacture.
  • Cystic Fibrosis is a genetic disorder that impacts tens of thousands of people worldwide. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an ion channel. These mutations result in dysregulated chloride and bicarbonate transport across the membrane of epithelial cells, causing cells to secrete thick, viscous mucus. This leads to inflammation, infections, tissue damage, respiratory issues, and organ failure, and result in patients having shortened life expectancy. Many CF- causing mutations in CFTR have been identified, but the most common is the gene variant F508del, in which the deletion of three base pairs cause the loss of phenylalanine at position 508.
  • LNPs lipid nanoparticles
  • ABEs adenine base editors
  • the present application provides methods of treating a subject with cystic fibrosis by administering to the subject a composition comprising a lipid nanoparticle (LNP) that comprises a gene editing system.
  • LNP lipid nanoparticle
  • a method of treating a subject with cystic fibrosis comprising administering to the subject a composition comprising a lipid nanoparticle (LNP) that comprises a gene editing system, wherein the gene editing system comprises: (i) a first nucleic acid encoding a base editor; and (ii) a second nucleic acid encoding a guide RNA (gRNA), and wherein the composition treats the cystic fibrosis in the subject.
  • the CFTR gene of the subject comprises a R553X stop codon mutation.
  • the administration of the composition results in an increase in the expression of the full-length cystic fibrosis transmembrane conductance regulator (CFTR) protein in the subject, as compared to a subject with cystic fibrosis and whose CFTR gene comprises a R553X stop codon mutation, and that is not administered the composition.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the administration of the composition results in an increase in the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in the subject, as compared to a subject with cystic fibrosis and whose CFTR gene comprises a R553X stop codon mutation, and that is not administered the composition.
  • the nucleic acid encoding the base editor is RNA.
  • the base editor is an adenine base editor (ABE).
  • the ABE comprises a tRNA adenosine deaminase (TadA) protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • the ABE is ABE8e.
  • the base editor is a cytosine base editor (CBE).
  • the CBE comprises a cytidine deaminase protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • the ABE or the CBE further comprises a Cas9 enzyme that does not have any nuclease activity.
  • the LNP comprises an ionizable cationic lipid, a zwitterionic phospholipid, a cholesterol, and a PEG lipid.
  • the LNP comprises 5A2-SC8, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, DMG-PEG, and one or more selective organ targeting (SORT) molecules.
  • DOPE l,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • SORT selective organ targeting
  • a method of delivering a gene editing system to a lung cell type in a subject comprising administering to the subject a composition comprising a lipid nanoparticle (LNP) that comprises a gene editing system, wherein the gene editing system comprises (i) a first nucleic acid encoding an endonuclease or a base editor; and (ii) a second nucleic acid encoding a guide RNA (gRNA), and wherein the gene editing system is delivered to a lung cell type in a subject.
  • LNP lipid nanoparticle
  • gRNA guide RNA
  • the lung cell type is an endothelial cell or an epithelial cell. In some of any embodiments, the lung cell type is an immune cell. In some embodiments, the lung cell type is a stem cell.
  • the endonuclease is a Cas nuclease of the CRISPR-Cas system. In other embodiments, the Cas nuclease is a Cas9 nuclease, a Cas 12 nuclease, or a Cas 13 nuclease.
  • the nucleic acid encoding the endonuclease is DNA. In some embodiments, the nucleic acid encoding the endonuclease is RNA. In some embodiments, the nucleic acid encoding the base editor is DNA. In some of any embodiments, the nucleic acid encoding the base editor is RNA.
  • the base editor is an adenine base editor (ABE).
  • the ABE comprises a tRNA adenosine deaminase (TadA) protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • the ABE is ABE8e.
  • the base editor is a cytosine base editor (CBE).
  • the CBE comprises a cytidine deaminase protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • the ABE or the CBE further comprises a Cas9 enzyme that does not have any nuclease activity.
  • the ratio of the first nucleic acid to the second nucleic acid is 1:1 on a molecule:molecule basis. In some embodiments, the ratio of the first nucleic acid to the second nucleic acid is 1: 1 on a weight basis. In some embodiments, the ratio of the first nucleic acid to the second nucleic acid is 2:1 on a molecule: molecule basis. In some embodiments, the ratio of the first nucleic acid to the second nucleic acid is 2:1 on a weight basis.
  • the LNP comprises an ionizable cationic lipid, a zwitterionic phospholipid, a cholesterol, and a PEG lipid.
  • the LNP comprises 5A2- SC8, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, DMG-PEG, and one or more selective organ targeting (SORT) molecules.
  • DOPE l,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • SORT selective organ targeting
  • the one or more SORT molecules comprises permanently positively charged moiety.
  • the one or more SORT molecules is selected from the group consisting of 18:1 DOTMA (DOTMA); DORI, DC-6-14; 12:0 EPC (Chloride Salt); 14:0 EPC (Chloride Salt); 16:0 EPC (Chloride Salt); 18:0 EPC (Chloride Salt); 18:1 EPC (Chloride Salt); 16:0-18:1 EPC (Chloride Salt); 14:1 EPC (Triflate Salt); 18:0 DDAB (Dimethyldioctadecylammonium (Bromide Salt)); 14:0 TAP; 16:0 TAP; 18:0 TAP; 18:1 TAP (DOTAP); 18:1 TAP (DOTAP, MS Salt); 18:1 DODAP, or 18:1 PA (l,2-dioleoyl-sn-glycero-3-phosphate (sodium salt)) (18PA).
  • DOTMA DOTMA
  • PA l,2-dioleoyl-sn-gly
  • the one or more SORT molecule comprises DOTAP (1,2- dioleoyl-3-trimethylammonium propane). In some embodiments, the one or more SORT molecule comprises 18PA. In some embodiments, the one or more SORT molecule comprises DODAP. In some embodiments, the DODAP comprises about 20% molar ratio of the total lipids. In some embodiments, the DOTAP comprises about 50% molar ratio of the total lipids. In some embodiments, the 18PA comprises about 10% molar ratio of the total lipids. In some of any embodiments, the one or more SORT molecule comprises DOTMA. In some embodiments, the LNP comprises a ratio of DOPE:DOTMA between 3:1 and 1:3. In embodiments, the ratio of DOPE:DOTMA is about 3:1. In some embodiments, the ratio of DOPE:DOTMA is about 1:1.
  • the SORT molecule comprises from about 5% to about 60% molar percentage of the LNP. In some embodiments, the SORT molecule comprises about 40% molar percentage of the LNP. In some embodiments, the SORT molecule comprises about 50% molar percentage of the LNP. In some embodiments, the LNP binds vitronectin.
  • the guide RNA comprises a circular RNA. In some of any embodiments, the guide RNA comprises a linear RNA. In some embodiments, the guide RNA is a single guide RNA (sgRNA). In some embodiments, the guide RNA comprises a target sequence that is complementary with a target sequence of a cystic fibrosis transmembrane conductance regulator (CFTR) gene. In some embodiments, the nucleotide sequence of the guide RNA is AAGTAAAACCTCTACAAATG (SEQ ID NO: 1) or TTGCTCATTGACCTCCACTC (SEQ ID NO: 2).
  • the composition comprises a pharmaceutically acceptable carrier. In some embodiments, the composition is administered intravenously. In some of any embodiments, the subject is a human. In some embodiments, the subject has cystic fibrosis.
  • a method of modifying the nucleic acid sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a lung cell type, wherein the CFTR gene comprises a R553X stop codon mutation comprising: (a) contacting the lung cell type with a composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises (i) a first nucleic acid encoding a base editor; and (ii) a second nuclec acid encoding a guide RNA; (b) determining the nucleic acid sequence of the CFTR gene in the lung cell type, wherein the nucleic acid sequence of the CFTR gene in the lung cell type is modified to remove the R553X stop codon mutation.
  • the modification comprises the replacing of the thymine at 1789 base in exon 11 of the CFTR gene with cytosine.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • a CFTR gene in the lung cell type comprises a R553X mutation
  • the method comprising: (a) contacting the lung cell type with a composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises (i) a first nucleic acid encoding a base editor; and (ii) a second nucleic acid encoding a guide RNA; (b) determining the expression of full-length CFTR protein in the lung cell type, wherein the expression of full-length CFTR protein is increased in the lung cell type, as compared to a lung cell type comprising a CFTR gene comprising a R553X mutation, and that is not contacted with the composition.
  • LNP lipid nanoparticle
  • a method of modulating the activity of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in a lung cell type, wherein the CFTR gene in the lung cell type comprises a R553X mutation comprising: (a) contacting the lung cell type with a composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises (i) a first nucleic acid encoding a base editor; and (ii) a second nucleic acid encoding a guide RNA; (b) determining the activity of the CFTR protein in the lung cell type, wherein the activity of the CFTR protein is modulated in the lung cell type, as compared to a lung cell type comprising a CFTR gene comprising a R553X mutation, and that is not contacted with the composition.
  • LNP lipid nanoparticle
  • expression of the CFTR protein is determined in a lung cell type in a subject, wherein the subject has been administered the composition, and wherein the expression is determined by one or more bioassays comprising sweat chloride concentration assay, ⁇ -adrenergic sweat assay, and nasal potential difference assay.
  • expression of the CFTR protein is determined by analysis of chloride levels in the sweat of the subject.
  • the chloride levels in the sweat of the subject after being administered the composition are decreased as compared to the chloride levels in the sweat of the subject before being administered the composition.
  • the expression is measured using western blotting, immunoprecipitation, and anti-CFTR antibodies.
  • the activity of the CFTR protein is increased in the lung cell type, as compared to a lung cell type comprising a CFTR gene comprising a R553X mutation, and that is not contacted with the composition.
  • the lung cell type is an endothelial cell or an epithelial cell. In some embodiments, the lung cell type is an immune cell. In some embodiments, the lung cell type is a stem cell.
  • the nucleic acid encoding the base editor is DNA. In some embodiments, the nucleic acid encoding the base editor is RNA. In some embodiments, the base editor is an adenine base editor (ABE). In some embodiments, the ABE comprises a tRNA adenosine deaminase (TadA) protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence. In some embodiments, the base editor is a cytosine base editor (CBE).
  • ABE adenine base editor
  • the CBE comprises a cytidine deaminase protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • the ABE or the CBE further comprises a Cas9 enzyme that does not have any nuclease activity.
  • the ratio of the first nucleic acid to the second nucleic acid is 1:1 on a molecule:molecule basis. In some embodiments, the ratio of the first nucleic acid to the second nucleic acid is 1: 1 on a weight basis. In some embodiments, the ratio of the first nucleic acid to the second nucleic acid is 2:1 molecule: molecule basis. In some embodiments, the ratio of the first nucleic acid to the second nucleic acid is 2:1 on a weight basis.
  • the LNP comprises an ionizable cationic lipid, a zwitterionic phospholipid, a cholesterol, and a PEG lipid.
  • the LNP comprises 5A2- SC8, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, DMG-PEG, and one or more selective organ targeting (SORT) molecules.
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • SORT selective organ targeting
  • the one or more SORT molecules comprises permanently positively charged moiety.
  • the one or more SORT molecule is selected from the group consisting of 18:1 DOTMA (DOTMA); DORI, DC-6-14; 12:0 EPC (Chloride Salt); 14:0 EPC (Chloride Salt); 16:0 EPC (Chloride Salt); 18:0 EPC (Chloride Salt); 18:1 EPC (Chloride Salt); 16:0-18:1 EPC (Chloride Salt); 14:1 EPC (Triflate Salt); 18:0 DDAB (Dimethyldioctadecylammonium (Bromide Salt)); 14:0 TAP; 16:0 TAP; 18:0 TAP; 18:1 TAP (DOTAP); 18:1 TAP (DOTAP, MS Salt); 18:1 DODAP, or 18:1 PA (l,2-dioleoyl-sn-glycero-3-phosphate (sodium salt)) (18PA).
  • DOTMA DOTMA
  • PA l,2-dioleoyl-sn-g
  • the one or more SORT molecules comprises DOTAP (1,2- dioleoyl-3-trimethylammonium propane). In some embodiments, the one or more SORT molecules comprises 18PA. In some embodiments, the one or more SORT molecules comprises DODAP. In some embodiments, the DODAP comprises about 20% molar ratio of the total lipids. In some embodiments, the DOTAP comprises about 50% molar ratio of the total lipids. In some embodiments, the 18PA comprises about 10% molar ratio of the total lipids.
  • the SORT molecule comprises DOTMA.
  • the LNP comprises a ratio of DOPE:DOTMA of between 3:1 and 1:3. In some embodiments, the ratio of DOPE:DOTMA is about 3:1. In some embodiments, the ratio of DOPE:DOTMA is about 1:1. In some embodiments, the SORT molecule comprises from about 5% to about 60% molar percentage of the LNP. In some embodiments, the SORT molecule comprises about 40% molar percentage of the LNP. In some embodiments, the SORT molecule comprises from 50% molar percentage of the LNP. In some embodiments, the LNP binds vitronectin.
  • the guide RNA comprises a circular RNA. In some of any embodiments, the guide RNA comprises a linear RNA. In some embodiments, the guide RNA is a single guide RNA (sgRNA). In some embodiments, the guide RNA comprises a target sequence that is complementary with a target sequence of a cystic fibrosis transmembrane conductance regulator (CFTR) gene. In some embodiments, the nucleotide sequence of the guide RNA is AAGTAAAACCTCTACAAATG (SEQ ID NO: 1) or TTGCTCATTGACCTCCACTC (SEQ ID NO: 2).
  • the function of the CFTR protein is determined by one or more bioassays comprising sweat chloride concentration assay, ⁇ -adrenergic sweat assay, and nasal potential difference assay. In some embodiments, the function of the CFTR protein is determined by analysis of chloride levels in the sweat of the subject. In some embodiments, the chloride levels in the sweat of the subject after being administered the composition are decreased as compared to the chloride levels in the subject before being administered the composition.
  • the composition comprises a pharmaceutically acceptable carrier.
  • the subject is a human.
  • the administration of the composition to the subject is by intravenous administration.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • a composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises (i) a first nucleic acid encoding a base editor; and (ii) a second nucleic acid encoding a guide RNA; (b) determining the function of the CFTR gene in the subject, wherein the function of the CFTR gene is restored in the subject.
  • LNP lipid nanoparticle
  • 5% to about 95% of the function of the CFTR gene is restored.
  • the restoring of the function of the CFTR gene is determined by the increase of CFTR protein expression.
  • expression of the CFTR protein is determined by one or more bioassays comprising sweat chloride concentration assay, ⁇ - adrenergic sweat assay, and nasal potential difference assay.
  • expression of the CFTR protein is determined by analysis of chloride levels in the sweat of the subject.
  • chloride levels in the sweat of the subject after being administered the composition are decreased as compared to levels in a subject before being administered the composition.
  • the expression is measured using western blotting, immunoprecipitation, and anti-CFTR antibodies.
  • the nucleic acid encoding the base editor is DNA. In some embodiments, the nucleic acid encoding the base editor is RNA. In some embodiments, the base editor is an adenine base editor (ABE). In some embodiments, the base editor is ABE8e. In some embodiments, the ABE comprises a tRNA adenosine deaminase (TadA) protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence. In some embodiments, the base editor is a cytosine base editor (CBE).
  • ABE adenine base editor
  • the CBE comprises a cytidine deaminase protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • the ABE or the CBE further comprises a Cas9 enzyme that does not have any nuclease activity.
  • the ratio of the first nucleic acid to the second nucleic acid is 1:1 on a molecule:molecule basis. In some embodiments, the ratio of the first nucleic acid to the second nucleic acid is 1: 1 on a weight basis. In some embodiments, the ratio of the first nucleic acid to the second nucleic acid is 2:1 molecule: molecule basis. In some embodiments, the ratio of the first nucleic acid to the second nucleic acid is 2:1 on a weight basis.
  • the LNP comprises an ionizable cationic lipid, a zwitterionic phospholipid, a cholesterol, and a PEG lipid.
  • the LNP comprises 5A2- SC8, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, DMG-PEG, and one or more selective organ targeting (SORT) molecules.
  • DOPE l,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • SORT selective organ targeting
  • the one or more SORT molecules comprises permanently positively charged moiety.
  • the one or more SORT molecule is selected from the group consisting of 18:1 DOTMA (DOTMA); DORI, DC-6-14; 12:0 EPC (Chloride Salt); 14:0 EPC (Chloride Salt); 16:0 EPC (Chloride Salt); 18:0 EPC (Chloride Salt); 18:1 EPC (Chloride Salt); 16:0-18:1 EPC (Chloride Salt); 14:1 EPC (Triflate Salt); 18:0 DDAB (Dimethyldioctadecylammonium (Bromide Salt)); 14:0 TAP; 16:0 TAP; 18:0 TAP; 18:1 TAP (DOTAP); 18:1 TAP (DOTAP, MS Salt); 18:1 DODAP, or 18:1 PA (l,2-dioleoyl-sn-glycero-3-phosphate (sodium salt)) (18PA).
  • DOTMA DOTMA
  • PA l,2-dioleoyl-sn-g
  • the one or more SORT molecules comprises DOTAP (1,2- dioleoyl-3-trimethylammonium propane). In some embodiments, the one or more SORT molecules comprises 18PA. In some embodiments, the one or more SORT molecules comprises DODAP. In some embodiments, the DODAP comprises about 20% molar ratio of the total lipids. In some embodiments, the DOTAP comprises about 50% molar ratio of the total lipids. In some embodiments, the 18PA comprises about 10% molar ratio of the total lipids. In some embodiments, the one or more SORT molecules comprises DOTMA. In some embodiments, the LNP comprises a ratio of DOPE:DOTMA of between 3:1 and 1:3.
  • the ratio of DOPE:DOTMA is about 3:1. In some embodiments, the ratio of DOPE:DOTMA is about 1:1. In some embodiments, the one or more SORT molecules comprises from about 5% to about 60% molar percentage of the LNP. In some embodiments, the one or more SORT molecules comprises about 40% molar percentage of the LNP. In some embodiments, the SORT molecule comprises from 50% molar percentage of the LNP. In some embodiments according to any one of the methods described above, the LNP binds vitronectin. [0038] In some embodiments, the guide RNA comprises a circular RNA. In some embodiments, the guide RNA comprises a linear RNA. In some embodiments, the guide RNA is a single guide RNA (sgRNA).
  • sgRNA single guide RNA
  • the guide RNA comprises a target sequence that is complementary with a target sequence of a cystic fibrosis transmembrane conductance regulator (CFTR) gene.
  • the nucleotide sequence of the guide RNA is AAGTAAAACCTCTACAAATG (SEQ ID NO: 1) or TTGCTCATTGACCTCCACTC (SEQ ID NO: 2).
  • the composition comprises a pharmaceutically acceptable carrier.
  • the subject is a human.
  • the administration of the composition to the subject is by intravenous administration.
  • the LNP is localized to the lungs of the subject.
  • the LNP is capable of delivering the first and second nucleic acids to the lungs of the subject.
  • a lung cell type comprising a modified cystic fibrosis transmembrane conductance regulator (CFTR) gene, wherein the modification comprises the replacement of the thymine at 1789 base in exon 11 of the CFTR gene with cytosine.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • a method of treating cystic fibrosis in a subject comprising administering to the subject a composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises (i) a first nucleic acid encoding a base editor; and (ii) a second nucleic acid encoding a guide RNA, wherein the first nucleic acid and the second nucleic acid are delivered to a lung cell in the subject.
  • LNP lipid nanoparticle
  • FIG. 1A-1M shows that direct in vivo gene editing was achieved in mouse lungs that persisted for one year.
  • Mice were injected with LNP-Cre at 2 mg/kg total RNA (20:1, total lipid to RNA weight ratio) with two sequential doses, 48 hours apart. Mice treated with PBS were used as negative control (FIG. IB).
  • Ex vivo fluorescence imaging analyses of mouse lungs 2, 7, 21, 42, 60, 120 ,180, and 360 days after the last injection (FIG. 1C). Quantification analysis of ex vivo lung images was shown as average radiance (FIG. ID) and as total Flux (FIG. IE).
  • FIG. 2A-2G depicts durable in vivo gene editing in mouse lung with LNP-Cas9.
  • Lung SORT LNPs were used to co- deliver Cas9 mRNA and sgTOMl (LNP-Cas9).
  • Ngfr+ lung basal stem cells Ngfr+ lung basal stem cells
  • FIG. 2G Krt5+lung basal stem cells. Similar to the result of Cre editing, tdTom+ cells retained persistent expression across the lungs including 38.7% of endothelial cells, 32.5% of epithelial cells, 6.1% of immune cells, 16.7% of Ngfr + lung basal stem cells, and 7.2% of Krt5 + lung basal stem cells for up to 240 days.
  • FIG. 3A-3D depicts the minimal toxicity observed after LNP-Cas9 treatments.
  • FIG. 4A-4G depicts lung SORT LNP-mediated editing in tracheal and bronchus regions.
  • LNP-Cre were intravenously administered to Ai14 mice in two successive doses, each being 2 mg/kg total RNA, 48 hours apart.
  • the tracheas and bronchus regions were extracted 48 hours following the final injection, and tdTom expression (tdTom+) across various cell types was quantified using flow cytometry.
  • the composition of cells markedly differed between the trachea and bronchus regions of the lung (FIG. 4A).
  • the trachea harbored more immune cells (-55.8%) and fewer epithelial ( ⁇ 13.7%j and endothelial cells (-8.4%) compared to the bronchus (35.4% immune cells, 28.7% epithelial cells, and 23.5% endothelial cells).
  • Percentages of total edited cells, endothelial cells, immune cells, epithelial cells, Ngfr + basal stem cells, and Krt5 + basal stem cells in the trachea compared to the bronchus region (FIG. 4B-FIG. 4G).
  • FIG. 5A-5B depicts gene editing in mouse lung endothelial progenitor cells with LNP-Cre.
  • Ai14 mice were dosed with two sequential LNP-Cre treatments (two days apart) at 2 mg/kg total RNA (20:1, total lipid to RNA weight ratio).
  • the lungs were collected at 2 days, 7 days, 21 days, 42 days, 60 days, 120 days, 180 days, 270 days, and 360 days after the last injection.
  • Flow cytometry gating strategy for lung endothelial progenitor cells (FIG. 5A). Single cells prepared from Ail 4 mouse lungs were gated. Single cells prepared from Ail 4 mouse lungs were gated.
  • Viable (Ghost Red negative) lung endothelial progenitor cells (CD45 negative, CD31 positive, CD157 positive) expressing tdTomato fluorescence (tdTomato positive) were analyzed by flow cytometry.
  • Gene editing in mouse lung hematopoietic lung endothelial progenitor cells were obtained from three mice per each time point (FIG. 5B). Saline treated mice were served as negative control.
  • FIG. 6A-6C depicts gene editing in mouse lung hematopoietic progenitor cells with LNP-Cre.
  • Ai14 mice were dosed with two sequential LNP-Cre treatments (two days apart) at 2 mg/kg total RNA (20:1, total lipid to RNA weight ratio).
  • the lungs were collected at 2 days, 7 days, 21 days, 42 days, 60 days, 120 days, 180 days, 270 days, and 360 days after the last injection.
  • Flow cytometry gating strategy for lung hematopoietic progenitor cells (FIG. 6A). Single cells prepared from Ai14 mouse lungs were gated.
  • Viable (Ghost Red negative) lung multipotent progenitor cells lineage negative, CD45 positive, Seal negative, c-kit positive
  • lung hematopoietic stem cells lineage negative, CD45 positive, Seal positive, c-kit positive
  • tdTomato positive tdTomato positive
  • FIG. 7A-7E depicts lung SORT LNP-mediated efficient delivery into mature lung epithelial cells where vitronectin receptor expressing lung cell types exhibit enhanced editing efficiency.
  • PBS serves as a negative control.
  • FIG. 7A Quantification analysis of LNP-Cre-mediated editing in mature lung epithelium base on IHC images. Results were obtained from five to six random airway per whole slide IHC images and are presented as mean ⁇ SEM (FIG. 7B). Representative native whole slide immunofluorescence images of lung sections from a PBS treated mouse (FIG. 7C) and a LNP-Cre treated mouse (FIG. 7D), DAPI shown as blue and tdTom shown as Red. Representative native tissuecyte image of a single lung left lobe whole section from a LNP-Cre treated mouse (FIG. 7E).
  • FIG. 8A-8B depicts quantitative TissueCyte analysis of mTmG mice lung following LNP-Cre treatment.
  • Schematic representation of LNP-Cre mediated eGFP fluorescence protein expression replacing the red fluorescence in lung cells after systemic administrations (FIG. 8A).
  • a mouse was injected intravenously with a single LNP-Cre treatment at 2 mg/kg total RNA (20:1, total lipid to RNA weight ratio). The mouse lung was collected two days after the injection.
  • PBS-treated mTmG mouse was used as negative control.
  • Quantitative analysis of GFP positive (GFP + ) area % in ENP-Cre treated mTmG mouse lung left lobe by TissueCyte 3D imaging and analysis (FIG. 8B).
  • FIG. 9A-9B depicts the protein corona composition adsorbed onto Fung SORT ENP surface as determined by unbiased mass spectrometry proteomics. The most abundant proteins were ranked and plotted as a heat map (FIG. 9A) and classified into physiological classes of the identified proteins (FIG. 9B).
  • FIG. 10A-10E depicts vitronectin receptor-expressing lung cell types exhibit enhanced editing efficiency. Quantification of tdTom positivity in vitronectin receptor (CD51 + CD61 + ) expressing cells and the vitronectin receptor population in lung endothelial cell (FIG. 10A), immune cells (FIG. 10B), epithelial cells (FIG. IOC), Ngfr + cells (FIG. 1OD) and
  • FIG. 11A-11E depicts flow cytometry gating strategy for vitronectin receptor.
  • FIG. 12A-12B depicts the efficient adenine base editing in 16HBEge R553X cells.
  • mediated high level base editing efficiency >95%) in 16HBEge R553X cells at the target T7 position (FIG. 12A).
  • the A «T to G*C conversion on T7 position was analyzed using EditR analysis with Sanger sequencing data.
  • FIG. 13A-13F depicts efficient adenine base editing was achieved in lung basal cells across CF models.
  • Workflow for differentiation of HBE from a healthy donor and a CF patient with CFTR R553x/F508del i n t 0 airway epithelium and base editing strategy to correct CF R553X mutation (FIG. 13A).
  • Untreated CF HBE cells were used as negative control and HBE from healthy donor with wild-type CFTR gene was used for comparison.
  • LNP-ABE-mediated 60% of allelic base editing in both undifferentiated P2 and fully differentiated P3 culture (FIG. 13B). The frequency of desired product (the box highlighted in blue) and bystander editing was evaluated using NGS sequencing (FIG.
  • FIG. 14A-14B depicts raw data of gel images of CFTR (FIG. 14A) and Vinculin as internal standard (FIG. 14B) from JESS western blotting.
  • FIG. 15A-15B depicts the ability of LNP-ABE to restore HBE culture function.
  • FIG. 16A-16G depicts in vivo stem cell editing in CF mouse lungs.
  • Intestinal stem cells were isolated from R553X homozygous mice to generate intestinal organoids as an ex vivo model to evaluate CFTR function restoration following LNP-ABE treatment.
  • Forskolin-induced CFTR activation can facilitate ion/water transportation leading to organoid swelling. No swelling was observed from untreated group (FIG. 16B), while LNP-ABE treated group showed over 80% of intestinal organoid swelling (FIG. 16C, FIG.
  • FIG. 16D Scale bar: 500 pm.
  • Approximately 50% of base editing was confirmed using DNA sequencing; Data are mean ⁇ SD ( « 8 independent replicates); Unpaired t-test (FIG. 16E).
  • FIG. 16F Workflow of assessing LNP-ABE-mediated base editing in mouse lung basal cells after a single administration (FIG. 16F).
  • FIG. 17A-17M shows that direct in vivo gene editing was achieved in mouse lungs that persisted for one year.
  • Schematic representation of LNP-mediated gene editor delivery into lung cells after systemic administration (FIG. 17A).
  • Mice were injected with LNP-Cre at 2 mg/kg total RNA (20:1, total lipid to RNA weight ratio) with two sequential doses, 48 hours apart. Mice treated with PBS were used as negative control (FIG. 17B).
  • tdTom positive tdTomato fluorescence
  • FIG. 18A-18H depicts durable in vivo gene editing in mouse lung with LNP-Cas9.
  • Lung SORT LNPs were used to co-deliver Cas9 mRNA and sgTOMl (LNP-Cas9).
  • FIG. 19A-19E depicts the minimal toxicity observed after LNP-Cas9 treatments.
  • PBS-treated mice were used as a negative control. Data are presented as individual data points or mean ⁇ SEM. Histopathology evaluation performed by H&E staining of heart and spleen tissues 7, 60, and 120 days after LNP-Cas9 treatment (FIG. 19E). PBS- treated mice were used as a negative control. Scale bar: 100 pm.
  • the trachea harbored more immune cells (55.8%) and fewer epithelial (13.7%) and endothelial cells (8.4%) compared to the bronchus (35.4% immune cells, 28.7% epithelial cells, and 23.5% endothelial cells).
  • FIG. 21A-21B depicts gene editing in endothelial beds of various organs with LNP- Cre.
  • LNP-Cre was intravenously administered to Ai14 mice in two successive doses, 2 mg/kg total RNA, 48 hours apart.
  • tdTom expression (tdTom + ) across CD31 + endothelial beds of various organs (FIG. 21A) and total cells of various organs (FIG. 21B) were quantified by flow cytometry.
  • Tested organs include heart, lung, liver, spleen, kidney, pancreas, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum.
  • FIG. 22A-22B depicts lung SORT LNP-mediated editing in lung immune cells.
  • LNP-Cre were intravenously administered to Ai14 mice in two successive doses, each being 2 mg/kg total RNA, 48 hours apart.
  • Flow cytometry gating strategy for various lung immune cells FIG. 22A). Single cells prepared from Ai14 mouse lungs were gated.
  • FIG. 23A-23D depicts lung SORT LNP-mediated editing in P. aeruginosa infected mouse lungs.
  • Ail 4 mice were randomly allocated to P. aeruginosa infection group or non- infection group. Mice in infection group were inoculated intranasally with 50 pl of P. aeruginosa at 3.5 x 105 CFU to generate acute infection model (FIG. 23A).
  • LNP-Cre were intravenously administered to Ai14 mice, 2 mg/kg total RNA, 9 h post infection. Neutrophil invasion was observed in infected lungs 9 h after the intranasal infection compared to non- infected lungs (FIG. 23B). VtnR abundance (FIG.
  • FIG. 24A-24B depicts gene editing in mouse lung endothelial progenitor cells with LNP-Cre.
  • Ai14 mice were dosed with two sequential LNP-Cre treatments (two days apart) at 2 mg/kg total RNA (20:1, total lipid to RNA weight ratio).
  • the lungs were collected at 2, 7, 21, 42, 60, 120, 180, 270, 360, and 660 days after the last injection.
  • Flow cytometry gating strategy for lung endothelial progenitor cells (FIG. 24A). Single cells prepared from Ail 4 mouse lungs were gated.
  • Single cells prepared from Ai14 mouse lungs were gated.
  • FIG. 25A-25C depicts gene editing in mouse lung hematopoietic progenitor cells with LNP-Cre.
  • Ai14 mice were dosed with two sequential LNP-Cre treatments (two days apart) at 2 mg/kg total RNA (20:1, total lipid to RNA weight ratio).
  • the lungs inclusive of tracheas were collected at 2, 7, 21, 42, 60, 120, 180, 270, 360, and 660 days after the last injection.
  • Flow cytometry gating strategy for lung hematopoietic progenitor cells (FIG. 25 A). Single cells prepared from Ai14 mouse lungs were gated.
  • Viable (Ghost Red negative) lung multipotent progenitor cells lineage negative, CD45 positive, Seal negative, c-kit positive
  • lung hematopoietic stem cells lineage negative, CD45 positive, Seal positive, c-kit positive
  • tdTomato positive tdTomato positive
  • FIG. 26 depicts a representative whole slide images showing five to six random segments selection for quantification analysis.
  • FIG. 27A-27K depicts lung SORT LNP-mediated efficient delivery into diverse lung cell types with enhanced delivery to VtnR-expressing cells.
  • FIG. 27F quantification of tdTom positivity in VtnR+ (CD51+CD61+) cells and VtnR- (CD51-CD61-) fraction in lung endothelial cells (FIG. 27G), immune cells (FIG. 27H), epithelial cells (FIG. 271), NGFR+ cells (FIG. 27J), and KRT5+ stem cells (FIG. 27K).
  • FIG. 28A-28B depicts gene editing and persistence of gene editing in mouse lung ionocytes following systemic LNP-Cre administration.
  • Ai14 mice were dosed with two sequential LNP-Cre treatments (two days apart) at 2 mg/kg total RNA (20:1, total lipid to RNA weight ratio). The lungs were collected 2 days and 660 days after the last injection.
  • Flow cytometry gating strategy for lung ionocytes FOXI1 positive lung ionocytes were gated and analyzed for the expression of tdtomato fluorescence (FIG. 28A). Editing levels, correlated with tdtomato expression in mouse lung ionocytes, were measured from three mice at each time point (FIG. 28B).
  • FIG. 29A-29B depicts quantitative TissueCyte analysis of mTmG mice lung following LNP-Cre treatment.
  • a mouse was injected intravenously with a single LNP-Cre treatment at 2 mg/kg total RNA (20:1, total lipid to RNA weight ratio).
  • the mouse lung was collected two days after the injection.
  • PBS-treated mTmG mouse was used as negative control (FIG. 29A).
  • FIG. 30A-30B depicts the protein corona composition adsorbed onto Lung SORT LNP surface determined by unbiased mass spectrometry proteomics. The most abundant proteins were ranked and plotted as a heat map (FIG. 30A) and classified into physiological classes of the identified proteins (FIG. 30B).
  • FIG. 31A-31D depicts how vitronectin regulates SORT and non-SORT LNP efficacy in vitronectin receptor (VtnR) expressing cells in vitro.
  • 5-component 5A2-SC8 SORT LNPs, 5- component DLin-MC3-DMA (MC3) SORT LNPs, and 4-component non-SORT LNPs (standard MC3 LNPs) were preincubated with 0.25 g Vtn/g lipid prior to treating Vtn-negative liver cancer cells (Huh-7), Vtn-expressing kidney cancer cells (A-498), and Vtn-expressing human lung epithelial (16HBE14o-) cells to measure functional mRNA delivery (bioluminescence) (FIG. 31A).
  • FIG. 32 depicts the flow cytometry gating strategy for vitronectin receptor.
  • FIG. 33A-33B depicts LNP delivery in VtnR-expressing lung endothelial cells, lung multipotent progenitor cells, and lung hematopoietic stem cells.
  • VtnR CD51 + CD61 +
  • FIG. 33A depicts LNP delivery in VtnR-expressing lung endothelial cells, lung multipotent progenitor cells, and lung hematopoietic stem cells.
  • VtnR CD51 + CD61 +
  • VtnR CD5TCD61
  • Ai14 mice were dosed with two sequential LNP-Cre treatments (two days apart) at 2 mg/kg total RNA (20:1, total lipid to RNA weight ratio). The lungs were collected 2 days after the last injection. Single cells prepared from Ai14 mouse lungs were gated.
  • FIG. 34A-34C depicts profiling of VtnR expression in mouse tissues and LNP delivery in VtnR-expressing lung endothelial and non-endothelial cells.
  • VtnR CD51 + CD61 +
  • FIG. 34A shows abundance
  • VtnR CD51 + CD61 +
  • VtnR VtnR"
  • Organs were collected 2 days after the last injection. Single cells prepared from Ai14 mouse organs were gated. Viable (Ghost Red negative) total cells, vitronectin receptor expressing (CD51 positive CD61 positive) endothelial cells (CD31 positive) or non-endothelial cells (CD31 negative) with tdTomato fluorescence (tdTom positive) were analyzed by flow cytometry. Comparing to heart (3.0%), liver (2.5%), spleen (3.7%), kidney (1.1%), pancreas (1.4%), duodenum (2.2%), ovaries/testes (4.3%), lung exhibit highest level of VtnR expression (24.4%).
  • Endothelial cells of all tested organs displayed no preference for tdTom in VtnR + or VtnR- fraction.
  • tdTom expression was enriched within the VtnR + fraction compared to the VtnR" fraction in heart (24.8% vs 2.9%), lung (23.3% vs 2.9%), liver (88.8% vs 52.3%), spleen (10.4% vs 4.6%) and kidney (24.6% vs 2.7%).
  • FIG. 35A-35B depicts efficient adenine base editing in 16HBEge R553X cells.
  • high level base editing efficiency >95%) in 16HBEge R553X cells at the target T? position (FIG. 35A).
  • the A «T to G*C conversion on T? position was analyzed using EditR analysis with Sanger sequencing data.
  • FIG. 36A-36O depicts efficient adenine base editing was achieved in lung basal cells in patient-derived HBE cells and CF mouse model.
  • Workflow for differentiation of HBE from a healthy donor and a CF patient with CFTR R553x/F508del into airway epithelium and base editing strategy to correct CF R553X mutation (FIG. 36A).
  • Untreated CF HBE cells were used as negative control and HBEs from a healthy donor with wild-type CFTR gene was used for comparison.
  • FIG. 37A-37B depicts raw data of gel images of CFTR (FIG. 37A) and Vinculin as internal standard (FIG. 37B) from JessTM capillary western blotting.
  • FIG. 38A-38B depicts apical and basolateral delivery of LNP-tdTom in P3 differentiated HBE R553X/F508del cultures.
  • Cells were then treated with 12 pg LNP-tdTom (12 pg tdTomato mRNA per well) either to the apical side in liquid bolus or to the basolateral side. Untreated HBE cells were used as control. Single cells prepared from inserts were gated (FIG. 38A).
  • Cystic fibrosis also known as mucoviscidosis, is an autosomal recessive genetic disorder that affects most critically the lungs, and also the pancreas, liver, and intestine (Gibson et al., Am J Respir Crit Care Med. (2003) 168(8):918-951 ; Ratjen et al., Lancet Lond Engl. (2003) 361(9358):681-689; O’Sullivan et al., Lancet Lond Engl. (2009) 373(9678):1891-1904). Cystic fibrosis is caused by mutations in the gene encoding for the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein.
  • CFTR Cystic Fibrosis Transmembrane conductance Regulator
  • This protein functions as a channel that transports chloride ions across the membrane of cells and is required to regulate the components of mucus, sweat, saliva, tears, and digestive enzymes.
  • Disease-causing mutations in the CFTR protein cause dysfunction of its channel activity resulting in abnormal transport of chloride and sodium ions across the epithelium, leading to the thick, viscous secretions in the lung, pancreas and other organs characteristic of cycstic fibrosis disease (O’Sulliven et al., Lancet Lond Engl. (2009) 373(9678):1891-1904; Rowe et al., N Engl J Med. (2005) 352(19): 1992-2001).
  • Cystic fibrosis is the most frequent lethal genetic disease in the white population.
  • Cystic fibrosis is an autosomal recessive disorder with an estimated 89,000 individuals affected worldwide. Patients with CF have mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, mainly affecting the lungs, liver, pancreas, and intestine.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • CFTR is a membrane protein that functions as a chloride channel in the apical membrane of epithelial cells. Individuals with CF have dysregulated chloride and bicarbonate transport across the apical surface of secretory epithelia.
  • CFTR protein The decrease or loss of function of CFTR protein leads to multiorgan dysfunction and a shortened life expectancy.
  • the abnormally viscous secretions in the lung airways cause obstructions that lead to inflammation, tissue damage, frequent respiratory infections, and organ failure.
  • Other organ systems that contain epithelia are also affected, including sweat glands and the male reproductive tract.
  • obstructive lung disease is the primary cause of morbidity.
  • CFTR modulators comprised of a group of small molecules that aim to modulate and restore mutant CFTR function.
  • the compounds elexacaftor-tezacaftor-ivacaftor enhance the activity of mutant CFTR, and patients with the F508del variant taking this combination have improved lung function from 0.2% in the placebo group to 13.6%.
  • Lipid nanoparticles are the most clinically advanced non-viral delivery platform. More than a billion doses of mRNA LNP COVID-19 vaccines have been administered intramuscularly worldwide, demonstrating high safety, efficacy, and repeat dose ability (F. P. Polack et al., Safety and efficacy of the BNT162b2 mRNA Covid- 19 vaccine. New. Engl. J. Med. 383, 2603-2615 (2020); L. R. Baden et al., Efficacy and safety of the mRNA-1273 SARS- CoV-2 vaccine. New. Engl. J. Med. 384, 403-416 (2020)).
  • Lung Selective Organ Targeting (SORT) LNPs Q. Cheng et al., Selective ORgan Targeting (SORT) nanoparticles for tissue specific mRNA delivery and CRISPR/Cas gene editing. Nat. Nanotechnol. 15, 313-320 (2020)
  • SORT Selective ORgan Targeting
  • Nat. Nanotechnol. 15, 313-320 (2020) could access tissue-resident stem cells. It was shown that optimized lung-targeting LNPs can deliver CRISPR-Cas9 (M. Jinek et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012)) and adenine base editors (ABE) (N. M.
  • the methods comprise administering to a subject a lipid nanoparticle (LNP) that comprises a gene editing system.
  • the gene editing system comprises an adenine base editor (ABE), wherein the ABE comprises a tRNA adenosine deaminase (TadA) protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • the gene editing system is used to treat a subject with cystic fibrosis.
  • the adenine base editor deaminates an adenosine leading to a point mutation from adenine (A) to guanine (G).
  • the adenosine can be converted to an inosine residue.
  • inosine pairs most stably with C and therefore is read or replicated by the cell's replication machinery as a guanine (G).
  • Such base editors are useful for targeted editing of nucleic acid sequences.
  • Such base editors can be used for targeted editing of DNA in vitro, e.g., for the generation of mutant cells or animals.
  • Such base editors can be used for the introduction of targeted mutations in the cell of a living mammal. Such base editors may also be used for the introduction of targeted mutations for the correction of genetic defects in cells ex vivo, e.g., in cells obtained from a subject that are subsequently reintroduced into the same or another subject, or for multiplexed editing of a genome. These base editors may be used for the introduction of targeted mutations in vivo, e.g., the correction of genetic defects or the introduction of deactivating mutations in disease- associated genes in a subject, or for multiplexed editing of a genome.
  • the ABEs described herein are utilized for the targeted editing of G to A mutations (e.g., targeted genome editing). Reference is made to WO2021/158921 published August 12, 2021, which is incorporated herein in its entirety.
  • the methods provided herein comprise the treatment of a lung disease or lung disorder in a subject. In some embodiments, the methods provided herein comprise the treatment of cystic fibrosis. In some embodiments, the methods provided herein for the treatment of a lung disease or lung disorder in a subject comprise administering the described LNPs comprising a gene editing system or administering compositions comprising the described LNPs comprising a gene editing system to the subject. In some embodiments, the methods provided herein for the treatment of cyctic fibrosis in a subject comprise administering the described LNPs comprising a gene editing system or administering compositions comprising the described LNPs comprising a gene editing system to the subject.
  • the method comprises the treatment of cystic fibrosis in a subject, wherein the method comprises administering to a subject a LNP composition comprising a gene editing system to the subject.
  • LNPs as described in this section are described further in Section II below.
  • Gene editing systems as described in this section are described further in Section III below.
  • the methods provided herein comprise the delivery of a composition to a subject.
  • Compositions as described in this section are described further in Section IV below.
  • the methods provided herein comprise the delivery of a composition comprising a gene editing system to a lung cell type in a subject.
  • the composition comprises a LNP and a gene editing system.
  • the composition comprises a LNP, wherein the LNP comprises a gene editing system.
  • the composition comprises a LNP, wherein the LNP comprises a gene editing system, wherein the gene editing system comprises a first nucleic acid and a second nucleic acid.
  • the first nucleic acid encodes an endonuclease.
  • the first nucleic acid encodes a base editor.
  • the second nucleic acid encodes a guide RNA (gRNA).
  • the gene editing system is delivered to a lung cell type in a subject.
  • a composition comprising a LNP comprising a gene editing system, wherein the gene editing system comprises a first nucleic acid encoding a base editor and a second nucleic acid encoding a gRNA, is delivered to a lung cell type in a subject.
  • the composition is delivered to a lung cell type in a subject with cystic fibrosis.
  • the cell is a lung cell.
  • the lung cell comprises lung cell types that include, but are not limited to, an endothelial cell or an epithelial cell.
  • the lung cell type is an immune cell.
  • the lung cell type is a stem cell.
  • the epithelial cell is a ciliated cell, non-ciliated cell, a goblet cell, a brush cell (alveolar macrophage), an airway basal cell, a small granule cell, a bronchial epithelial cell, a small airway epithelial cell, and/or a tracheal epithelial cell.
  • the lung cell is a secretory cell and or ionocyte.
  • the methods provided herein comprise the modification of the nucleic acid sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a lung cell type in a subject.
  • the CFTR gene in a lung type in a subject comprises a point mutation.
  • the CFTR gene in a lung type in a subject comprises a deletion.
  • the CFTR gene in a lung type in a subject comprises a F508del deletion.
  • the CFTR gene in a lung type in a subject comprises a R553X stop codon mutation.
  • the methods provided herein comprise the modification of the nucleic acid sequence of the CFTR gene wherein the CFTR gene comprises a R553X stop codon mutation.
  • the modification of the nucleic acid comprises the contacting of the lung cell type with a composition comprising a LNP.
  • the composition comprises a LNP and a gene editing system.
  • the composition comprises a LNP, wherein the LNP comprises a first nucleic acid and a second nucleic acid.
  • the composition comprises an LNP comprising a first nucleic acid, wherein the first nucleic acid encodes a base editor, and a second nucleic acid, wherein the second nucleic acid encodes a gRNA.
  • the modification of the nucleic acid comprises the determination of the nucleic acid sequence of the CFTR gene in the lung cell type. In some embodiments, the modification of the nucleic acid of the CFTR gene comprises the removal of the R553X stop codon mutation.
  • the modification of the nucleic acid sequence of the CFTR gene in a lung cell type in a subject, wherein the CFTR gene comprises a R553X stop codon mutation comprises the contacting of the lung cell type with a composition comprising a LNP, wherein the LNP comprises a first nucleic acid encoding a base editor and a second nucleic acid encoding a gRNA, wherein the nucleic acid sequence of the CFTR gene in the lung cell type is modified to remove the R553X stop codon mutation.
  • the modification of the nucleic acid of the CFTR gene in a lung cell type occurs in a subject with cystic fibrosis.
  • the cell is a lung cell.
  • the lung cell comprises lung cell types that include, but are not limited to, an endothelial cell or an epithelial cell.
  • the lung cell type is an immune cell.
  • the lung cell type is a stem cell.
  • the epithelial cell is a ciliated cell, non-ciliated cell, a goblet cell, a brush cell (alveolar macrophage), an airway basal cell, a small granule cell, a bronchial epithelial cell, a small airway epithelial cell, and/or a tracheal epithelial cell.
  • the lung cell is a secretory cell and or ionocyte.
  • the methods provided herein comprise the increase of the expression of a full-length cystic fibrosis transmembrane conductance regulator (CFTR) protein in a lung cell type in a subject.
  • the lung cell type in a subject comprises a CFTR gene comprising a R553X stop codon mutation.
  • the CFTR gene in a lung type in a subject comprises a point mutation.
  • the CFTR gene in a lung type in a subject comprises a deletion.
  • the CFTR gene in a lung type in a subject comprises a F508del deletion.
  • the methods provided herein comprise the increase of the expression of a full length CFTR protein in a lung cell type in a subject, wherein the CFTR gene in the lung cell type comprises a R553X mutation, wherein the lung cell type is contacted with a composition comprising a LNP.
  • the composition comprises a LNP and a gene editing system.
  • the composition comprises a LNP, wherein the LNP comprises a first nucleic acid and a second nucleic acid.
  • the composition comprises an LNP comprising a first nucleic acid, wherein the first nucleic acid encodes a base editor, and a second nucleic acid, wherein the second nucleic acid encodes a gRNA.
  • the increase of the expression of a full-length CFTR protein comprises the determination of the expression of full- length CFTR protein in the lung cell type. In some embodiments, the increase of the expression of a full-length CFTR protein in a lung cell type in a subject, wherein the CFTR gene comprises a R553X stop codon mutation, comprises the contacting of the lung cell type with a composition comprising a LNP, wherein the LNP comprises a first nucleic acid encoding a base editor and a second nucleic acid encoding a gRNA, wherein the expression of full-length CFTR protein is increased in the lung cell type as compared to a lung cell type that is not contacted with the composition.
  • the increase of the expression of a full-length CFTR protein in a lung cell type occurs in a subject with cystic fibrosis.
  • the cell is a lung cell.
  • the lung cell comprises lung cell types that include, but are not limited to, an endothelial cell or an epithelial cell.
  • the lung cell type is an immune cell.
  • the lung cell type is a stem cell.
  • the epithelial cell is a ciliated cell, non-ciliated cell, a goblet cell, a brush cell (alveolar macrophage), an airway basal cell, a small granule cell, a bronchial epithelial cell, a small airway epithelial cell, and/or a tracheal epithelial cell.
  • the lung cell is a secretory cell and or ionocyte.
  • the methods provided herein comprise the modulation of the activity of a cystic fibrosis transmembrane conductance regulator (CFTR) protein in a lung cell type in a subject.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the lung cell type in a subject comprises a CFTR gene comprising a R553X stop codon mutation.
  • the methods provided herein comprise the modulation of the activity of a CFTR protein in a lung cell type in a subject, wherein the CFTR gene in the lung cell type comprises a R553X mutation, wherein the lung cell type is contacted with a composition comprising a LNP.
  • the composition comprises an LNP and a gene editing system.
  • the composition comprises a LNP, wherein the LNP comprises a first nucleic acid and a second nucleic acid.
  • the composition comprises a LNP comprising a first nucleic acid, wherein the first nucleic acid encodes a base editor, and a second nucleic acid, wherein the second nucleic acid encodes a gRNA.
  • the modulation of the activity of the CFTR protein comprises the determination of the activity of the CFTR protein in the lung cell type.
  • the modulation of the activity of the CFTR protein in a lung cell type in a subject comprises the contacting of the lung cell type with a composition comprising a LNP, wherein the LNP comprises a first nucleic acid encoding a base editor and a second nucleic acid encoding a gRNA, wherein the activity of the CFTR protein is modulated in the lung cell type as compared to a lung cell type that is not contacted with the composition.
  • the modulation of the activity of the CFTR protein in a lung cell type occurs in a subject with cystic fibrosis.
  • the cell is a lung cell.
  • the lung cell comprises lung cell types that include, but are not limited to, an endothelial cell or an epithelial cell.
  • the lung cell type is an immune cell.
  • the lung cell type is a stem cell.
  • the epithelial cell is a ciliated cell, non-ciliated cell, a goblet cell, a brush cell (alveolar macrophage), an airway basal cell, a small granule cell, a bronchial epithelial cell, a small airway epithelial cell, and/or a tracheal epithelial cell.
  • the lung cell is a secretory cell and or ionocyte.
  • the methods provided herein comprise the restoration of the function of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a subject. In some embodiments, the methods provided herein comprise the restoration of the function of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a subject with cystic fibrosis. In some embodiments, the CFTR gene in a lung type in a subject comprises a point mutation. In some embodiments, the CFTR gene in a lung type in a subject comprises a deletion. In some embodiments, the CFTR gene in a lung type in a subject comprises a F508del deletion.
  • the CFTR gene in a subject comprises a R553X stop codon mutation.
  • the CFTR gene in a lung type in a subject comprises a point mutation.
  • the CFTR gene in a lung type in a subject comprises a deletion.
  • the CFTR gene in a lung type in a subject comprises a F508del deletion.
  • the restoration of the function of the CFTR gene comprises the administration of a composition to a subject with cystic fibrosis.
  • the composition comprises a LNP and a gene editing system.
  • the composition comprises a LNP, wherein the LNP comprises a first nucleic acid and a second nucleic acid. In some embodiments, the composition comprises a LNP comprising a first nucleic acid, wherein the first nucleic acid encodes a base editor, and a second nucleic acid, wherein the second nucleic acid encodes a gRNA. In some embodiments, the restoration of the function of the CFTR gene comprises the determination of the function of the CFTR gene in the subject.
  • the restoration of the function of the CFTR gene comprises the administration of a composition to a subject, wherein the composition comprises a LNP, wherein the LNP comprises a first nucleic acid encoding a base editor and a second nucleic acid encoding a gRNA, wherein the function of the CFTR gene is restored in the subject.
  • the restorationof the function of the CFTR gene occurs in a subject with cystic fibrosis.
  • a patient in need of treatment is a male or female of 2 years or older, of 3 years or older, of 6 years or older, of 7 years or older, of 12 years or older, of 13 years or older, of 18 years or older, of 19 years or older, of 25 years or older, of 25 years or older, of 30 years or older, of 35 years or older, of 40 years or older, of 45 years or older, or of 50 years or older.
  • a patient in need of treatment is less than 50 years old, less than 45 years old, less than 40 years old, less than 35 years old, less than 30 years old, less than 25 years old, less than 20 years old, less than 19 years old, less than 18 years old, less than 13 years old, less than 12 years old, less than 7 years old, less than 6 years old, less than 3 years old, or less than 2 years old.
  • a patient in need of treatment is a male or female from 2 to 18 years old, from 2 to 12 years old, from 2 to 6 years old, from 6 to 12 years old, from 6 to 18 years old, from 12 to 16 years old, from 2 to 50 years old, from 6 to 50 years old, from 12 to 50 years old, or from 18 to 50 years old.
  • a patient in need of treatment is a female who is pregnant or who may become pregnant.
  • a patient in need of treatment has a sweat chloride value of >60 mmol/L, >65 mmol/L, >70 mmol/L, >75 mmol/L, >80 mmol/L, >85 mmol/L, >90 mmol/L, >95 mmol/L, >100 mmol/L, >110 mmol/L, >120 mmol/L, >130 mmol/L, >140 mmol/L or >150 mmol/L by quantitative pilocarpine iontophoresis (documented in the subject's medical record).
  • a patient in need of treatment has chronic sinopulmonary disease and/or gastrointestinal/nutritional abnormalities consistent with cystic fibrosis.
  • forced expiratory volume in 1 second is an established marker of cystic fibrosis disease progression that is used to capture clinical course and evaluate therapeutic efficacy.
  • a patient in need of treatment has FEVl>50% and ⁇ 90% (e.g., ⁇ 85%, ⁇ 80%, ⁇ 75%, ⁇ 70%, ⁇ 65%, ⁇ 60%, or ⁇ 55%) of the predicted normal (i.e., the average FEV of non-cycstic fibrosis patients) based on the patient's age, gender, and height.
  • a patient in need of treatment has resting oxygen saturation >92% on room air (pulse oximetry).
  • a patient in need of treatment has a body mass index >17.5 kg/m2 and weight >40 kg.
  • the method results in the production of CFTR protein in the subject. In some embodiments, the method results in an increase of CFTR protein in the subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline.
  • the increase in CFTR protein is detectable within about 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, or 48 hours of administration of the pharmaceutical composition. In some embodiments, the increase in the CFTR protein is detectable by qPCR on RNA purified from tissue samples.
  • a patient in need of treatment has received or is concurrently receiving other lung disease medications.
  • a patient in need of treatment may be receiving lumacaftor/ivacaftor combination drug (ORKAMBI®) or may have been on this treatment for at least 28 days prior to commencement of the treatment according to the present disclosure.
  • cystic fibrosis medications may include, but are not limited to, routine inhaled therapies directed at airway clearance and management of respiratory infections, such as bronchodilators, rhDNase (PULMOZYME (Dornase alfa)), hypertonic saline, antibiotics, and steroids; and other routine CF-related therapies such as systemic antibiotics, pancreatic enzymes, multivitamins, and diabetes and liver medications.
  • routine inhaled therapies directed at airway clearance and management of respiratory infections, such as bronchodilators, rhDNase (PULMOZYME (Dornase alfa)), hypertonic saline, antibiotics, and steroids
  • routine CF-related therapies such as systemic antibiotics, pancreatic enzymes, multivitamins, and diabetes and liver medications.
  • the method of treatment comprises (1) providing: a) a nebulizer, and b) a container including the LNP and base editor formulation in a pharmaceutically acceptable carrier, and (2) administering the formulation using the nebulizer.
  • the volume of the LNP and base editor formulation in the container has a volume of 10 inL. 9 inL. 8 inL. 7 niL. 6 inL. 5 inL. 4 ml. 3 inL. 2 niL. or 1 niL
  • formulations and compositions generally include a pharmaceutically acceptable carrier.
  • the carrier is preferably a liquid carrier.
  • the carrier preferably includes water and may include other components.
  • the composition including the LNP and base editor formulation is stored in an ampule, a vial, or a single-use vial prior to administrating. In some embodiments, the composition is stored in a single-use vial prior to administering.
  • the administration of the pharmaceutical composition results in the expression of a protein in a lung of the subject.
  • administration of the LNP and base editor composition results in detection of a protein in a lung of the subject between 6 and 12 hours after delivery to the subject.
  • detection of the protein in the lung is at 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.
  • the protein is detected using known techniques in the art, including, but not limited to, Western blot analysis.
  • a polypeptide delivered according to the described method results in increased protein level or activity an upper airway, a central airway, or peripheral airway of a lung of the subject by, e.g., at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or 1500-fold as compared to a control (e.g., endogenous level of protein or activity without or before the treatment according to the disclosure, or a historical reference level).
  • a control e.g., endogenous level of protein or activity without or before the treatment according to the disclosure, or a historical reference level.
  • a CFTR gRNA delivered according to the described method results in increased CFTR protein level or activity an upper airway, a central airway, or peripheral airway of a lung of the subject by, e.g., at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40- fold, 50-fold, 100-fold, 500-fold, 1000-fold, or 1500-fold as compared to a control (e.g., endogenous level of protein or activity without or before the treatment according to the disclosure, or a historical reference level).
  • a control e.g., endogenous level of protein or activity without or before the treatment according to the disclosure, or a historical reference level.
  • mRNA expression may be detected or quantified by qPCR on RNA purified from tissue samples.
  • the protein or expression may be determined by measuring immune responses to the protein.
  • Qualitative assessment of the protein may also be performed by Western blot analysis.
  • the protein activity may be measured by an appropriate activity assay.
  • Various other methods are known in the art and may be used to determine the protein expression or activity.
  • the CFTR mRNA expression may be detected or quantified by qPCR on RNA purified from tissue samples.
  • the CFTR protein or expression may be determined by measuring immune responses to CFTR protein.
  • IgG antibody to CFTR protein is measured by an enzyme-linked immunosorbent assay in collected serum samples.
  • CFTR-specific T cell responses are assessed using collected peripheral blood mononuclear cells.
  • T cell responses to CFTR are measured by a human interferon-y enzyme-linked immunospot assay as described by Calcedo et al. (Calcedo et al., Hum Gene Ther Clin Dev. (2013) 24:108-15).
  • CFTR protein activity may be measured by CFTR chloride channel activity in appropriate tissue cells. A stable potential with the mean value of a 10 second scoring interval after perfusion of solution is recorded. CFTR activity is estimated by the change in potential difference following perfusion with chloride-free isoproterenol.
  • Various other methods are known in the art and may be used to determine the CFTR mRNA and CFTR protein expression or activity.
  • the administration of the LNP comprising a gene editing system in accord with the provided methods effectively treats a subject with a mutation in the CFTR gene.
  • the mutation is a R553X CFTR nonsense mutation.
  • the expression level of the CFTR gene in a subject administered the LNP comprising a gene editing system is increased 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 at least 100% as compared to the expression level in the subject prior to administration of the LNP.
  • the expression level of the CFTR gene in a subject administered the LNP comprising a gene editing system is increased between 0 to 10%, between 5% to 15%, between 20% to 30%, between 25% to 35%, between 30% to 40%, between 35% to 45%, between 40% to 50%, between 45% to 55%, between 50% to 60%, between 55% to 65%, between 60% to 70%, between 70% to 80%, between 75% to 85%, between 80% to 90%, between 85% to 95%, between 90% to 100% as compared to the expression level in the subject prior to administration of the LNP.
  • administration of the LNP comprising a gene editing system enhances the expression or activity of CFTR protein by way of increasing the amount of a functional CFTR gene, transcript or protein in the cell (e.g., by at least about 1.1-fold) relative to a corresponding control.
  • the method yields a therapeutically effective amount of a functional of CFTR gene, transcript or protein in the cell.
  • the administration of the LNP comprising a gene editing system yields at least about 5%, 10%, 15%, 20%, 25%A 30%, 35%, 40%, 45%, or 50% by mole or by weight, increase in detectable CFTR gene, transcript or protein in the cell as compared to the cell prior to administration of the LNP composition.
  • the method for enhancing the expression or activity of CFTR protein comprises the enhancement (e.g., chloride) ion transport in cell(s) (e.g., by at least about 1.1 -fold) relative to a cell prior to administration of the LNP composition.
  • the method reduces defective export from or import to cell(s) of chloride, such as chloride anion or in the form of a chloride salt or other chloride-containing compound.
  • the method enhances or stimulates ion (e.g., chloride) transport in cell(s).
  • the enhanced or stimulated ion (e.g., chloride) transport results in secretion or absorption of (e.g., chloride) ions.
  • enhanced (e.g., chloride) ion transport is determined by evaluating CFTR-mediated currents across cell(s) by employing standard Ussing chamber (see Ussing and Zehrahn, Acta. Physiol. Scand. 23:110-127, 1951) or nasal potential difference measurements (see Knowles et al., Hum. Gene Therapy 6:445-455,
  • the enhanced chloride transport is be determined by the leq (equivalent current) assay using the TECC-24 system as described in Vu et al., J. Med. Chem. 2017, 60, 458-473, which is hereby incorporated by reference in its entirety.
  • the enhanced (e.g., chloride) ion transport is determined by CFTR-dependent whole-cell current measurement(s), as described in International Patent Application No. PCT/US2017/032967, published as W02017201091, which is hereby incorporated by reference in its entirety.
  • the method further comprises deriving (e.g., by cell culturing) a cell composition (e.g., a lung cell composition) from the cell.
  • response assessment may be performed at baseline (e.g. prior to any of the methods provided herein), 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and/or 24 months following administration of the LNP. In some aspects, response assessment is be performed at baseline, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and 24 months following administration of the LNP.
  • the method described herein comprises delivery of one or more polynucleotides to a cell.
  • the method comprises contacting a cell with a LNP comprising a gene editing system.
  • the method comprises expressing a protein or an RNA in a cell.
  • the method comprises a method of increasing chloride flux in a cell.
  • the method comprises contacting the cell with a LNP and base editor composition, wherein optionally the cell comprises homozygous inactivating mutations in the CFTR gene.
  • the method maintains transpeithelial electrical resistance (TEER) or reduces TEER by at most 10%, at most 20%, or at most 30%.
  • the cell is a lung cell.
  • the lung cell comprises lung cell types that include, but are not limited to, an endothelial cell or an epithelial cell.
  • the lung cell type is an immune cell.
  • the lung cell type is a stem cell.
  • the epithelial cell is a ciliated cell, non-ciliated cell, a goblet cell, a brush cell (alveolar macrophage), an airway basal cell, a small granule cell, a bronchial epithelial cell, a small airway epithelial cell, and/or a tracheal epithelial cell.
  • the lung cell is a secretory cell and or ionocyte.
  • the method comprises the delivery of the one or more polynucleotides to the lung of a subject, wherein the method comprises administering to the subject a composition comprising a LNP comprising a gene editing system. In some embodiments, the method comprises the treating or preventing lung disease in a subject, wherein the method comprises administering to the subject a composition comprising a LNP comprising a gene editing system.
  • the methods provided herein comprise a method for lung cell editing.
  • the methods comprise a method for genetic correction of cystic fibrosis transmembrane conductance regulator (CFTR) in a lung (e.g., basal) cell, comprising: contacting the lung (e.g., basal) cell with a composition that comprises a nucleic acid editing system assembled with a lipid composition, thereby delivering the nucleic acid editing system to the lung (e.g., basal) cell.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the methods provided herein comprise a method for genetic correction of cystic fibrosis transmembrane conductance regulator (CFTR) in a cell composition, comprising: contacting the cell composition comprising a plurality of lung (e.g., basal) cells with a composition that comprises a nucleic acid editing system assembled with a lipid composition, thereby delivering the nucleic acid editing system, e.g., to at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, or 70% of the plurality of lung (e.g., basal) cells.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the methods provided herein comprise a method for genetic correction of cystic fibrosis transmembrane conductance regulator (CFTR) in a cell composition, comprising: contacting the cell composition with a composition that comprises a nucleic acid editing system assembled with a lipid composition, which cell composition comprise a lung (e.g., basal) cell and a lung non-basal cell, thereby delivering the nucleic acid editing system to the lung (e.g., basal) cell in a greater amount than that delivered to the lung non-basal cell.
  • the non-basal cell may be an ionocyte (e.g., exhibiting or determined to exhibit to FOXI1), a ciliated cell, or a secretory cell (such as goblet cell and club cell).
  • the methods provided herein comprise methods for genetic correction of CFTR.
  • the lung (e.g., basal) cell or the plurality of lung (e.g., basal) cells is/are determined to exhibit a mutation in CFTR gene.
  • the lung (e.g., basal) cell or the plurality of lung (e.g., basal) cells exhibit(s) a mutation in CFTR gene.
  • the methods provided herein comprise methods for genetic correction of CFTR wherein the contacting is ex vivo.
  • the contacting is in vitro.
  • the contacting is in vivo.
  • a cell or pluralty of cells is isolated from the subject.
  • the compositions as described elsewhere here may be contacted with the cell outside of the subject. Upon administration of the composition or therapeutic, the cell may be re-injected or otherwise re-introduced into the subject.
  • the cell is a cell line.
  • the cell is a lung cell.
  • the lung cell is a lung airway cell. Examples of lung airway cells that can be targeted by the delivery of the present application includes but is not limited to basal cell, secretory cell such as goblet cell and club cell, ciliated cell and any combination thereof.
  • the method comprising nebulizing the composition prior to the administering step.
  • the composition is administered, as an aerosolized composition, by inhalation.
  • the method delivers to the lung an effective amount of the composition.
  • the composition comprises a LNP comprising a gene editing system.
  • the method delivers to the lung an amount effective of the composition to treat the lung disease.
  • the method is more effective than contacting the cell with and/or adminsitering to the subject elexacaftor, tezacaftor, lumacaftor, ivacaftor, or a combination thereof.
  • the method is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% more effective.
  • the method is 10%-70%, 20%-70%, 30%-70%, 40%-70%, 50%-70%, or 60%- 70% more effective.
  • LNP lipid nanoparticle
  • the LNP comprises a nucleic acid encoding a base editor that is administered to a subject.
  • the method comprises the administration of a LNP comprising a gene editing system to the subject.
  • the LNP comprises a nucleic acid encoding a recombinase.
  • the method comprises administration of an LNP comprising a first nucleic acid and a second nucleic acid.
  • the methods comprise the administration of a LNP comprising a gene editing system to a subject.
  • the LNP comprises an ionizable lipid, a phospholipid, a polyethylene glycol (PEG) lipid, a sterol, and one or more DNA molecules or an RNA molecules.
  • the methods comprise the delivery of the nucleic acids enveloped by the LNP to a target organ and/or target cell.
  • the LNP comprises an ionizable lipid.
  • the ionizable lipid is an ionizable cationic lipid.
  • the cationic ionizable lipids contain one or more groups which is protonated at physiological pH but may deprotonated and has no charge at a pH above 8, 9, 10, 11, or 12.
  • the ionizable cationic group comprises one or more protonatable amines which are able to form a cationic group at physiological pH.
  • the cationic ionizable lipid compound may also further comprise one or more lipid components such as two or more fatty acids with C6-C24 alkyl or alkenyl carbon groups.
  • these lipid groups may be attached through an ester linkage or may be further added through a Michael addition to a sulfur atom.
  • these compounds may be a dendrimer, a dendron, a polymer, or a combination thereof.
  • the LNP comprises one or more ionizable (e.g., ionizable amino) lipids (e.g., lipids that may have a positive or partial positive charge at physiological pH).
  • Ionizable lipids may be selected from the group comprising, but not limited to, 3- (didodecylamino)-N 1 ,N 1 ,4-tridodecy 1-1 -piperazineethanamine (KL 10) , N 1 - [2- (didodecylamino)ethyl] Nl,N4,N4-tridodecyl-l,4- piperazinediethanamine (KL22), 14,25- ditridecy 1-15,18,21 ,24-tetraaza-octatriacontane (KL25) , 1 ,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilino
  • ionizable lipids can also be the compounds disclosed in International Publication No. WO 2017/075531 Al, hereby incorporated by reference in its entirety. In some embodiments, ionizable lipids can also be the compounds disclosed in International Publication No. WO 2015/199952 Al, hereby incorporated by reference in its entirety. In some embodiments, the ionizable lipid may be selected from, but not limited to, an ionizable lipid described in International Publication Nos.
  • a cationic lipid may be selected from (20Z,23Z)-N,N- dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,Ndimemylhexacosa-17,20-dien-9-amine, (lZ,19Z)-N5N-dimethylpentacosa-l 6, 19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16- dien-5-amine, (12Z,15Z)-N,N dimethylhenicosa-12,15- dien-4-amine, (14Z,17Z)-N,N- dimethyltricosa-14,1 7-di en-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,Ndimethylheptacosa-18,21-dien-10-amine
  • the ionizable cationic lipids refer to lipid and lipid-like molecules with nitrogen atoms that can acquire charge (pKa). These molecules with amino groups typically have between 2 and 6 hydrophobic chains, often alkyl or alkenyl such as C 6 -C 24 alkyl or alkenyl groups, but may have at least 1 or more that 6 tails.
  • these cationic ionizable lipids are dendrimers, which are a polymer exhibiting regular dendritic branching, formed by the sequential or generational addition of branched layers to or from a core and are characterized by a core, at least one interior branched layer, and a surface branched layer. (See Petar R.
  • the term “dendrimer” as used herein is intended to include, but is not limited to, a molecular architecture with an interior core, interior layers (or “generations”) of repeating units regularly attached to this initiator core, and an exterior surface of terminal groups attached to the outermost generation.
  • a “dendron” is a species of dendrimer having branches emanating from a focal point which is or can be joined to a core, either directly or through a linking moiety to form a larger dendrimer.
  • the dendrimer structures have radiating repeating groups from a central core which doubles with each repeating unit for each branch.
  • the dendrimers described herein may be described as a small molecule, medium-sized molecules, lipids, or lipid-like material. These terms may be used to describe compounds described herein which have a dendron like appearance (e.g. molecules which radiate from a single focal point).
  • dendrimers are polymers, dendrimers may be preferable to traditional polymers because they have a controllable structure, a single molecular weight, numerous and controllable surface functionalities, and traditionally adopt a globular conformation after reaching a specific generation.
  • Dendrimers can be prepared by sequentially reactions of each repeating unit to produce monodisperse, tree-like and/or generational structure polymeric structures. Individual dendrimers consist of a central core molecule, with a dendritic wedge attached to one or more functional sites on that central core.
  • the dendrimeric surface layer can have a variety of functional groups disposed thereon including anionic, cationic, hydrophilic, or lipophilic groups, according to the assembly monomers used during the preparation.
  • Modifying the functional groups and/or the chemical properties of the core, repeating units, and the surface or terminating groups, their physical properties can be modulated. Some properties which can be varied include, but are not limited to, solubility, toxicity, immunogenicity and bioattachment capability. Dendrimers are often described by their generation or number of repeating units in the branches. A dendrimer consisting of only the core molecule is referred to as Generation 0, while each consecutive repeating unit along all branches is Generation 1, Generation 2, and so on until the terminating or surface group. In some embodiments, half generations are possible resulting from only the first condensation reaction with the amine and not the second condensation reaction with the thiol.
  • Dendrimer synthesis can be of the convergent or divergent type. During divergent dendrimer synthesis, the molecule is assembled from the core to the periphery in a stepwise process involving attaching one generation to the previous and then changing functional groups for the next stage of reaction. Functional group transformation is necessary to prevent uncontrolled polymerization. Such polymerization would lead to a highly branched molecule that is not monodisperse and is otherwise known as a hyperbranched polymer.
  • the dendrimers of G1-G10 generation are specifically contemplated.
  • the dendrimers comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range derivable therein.
  • the dendrimers used herein are GO, Gl, G2, or G3. However, the number of possible generations (such as 11, 12, 13, 14, 15, 20, or 25) may be increased by reducing the spacing units in the branching polymer.
  • dendrimers have two major chemical environments: the environment created by the specific surface groups on the termination generation and the interior of the dendritic structure which due to the higher order structure can be shielded from the bulk media and the surface groups. Because of these different chemical environments, dendrimers have found numerous different potential uses including in therapeutic applications.
  • the dendrimers are assembled using the differential reactivity of the acrylate and methacrylate groups with amines and thiols.
  • the dendrimers may include secondary or tertiary amines and thioethers formed by the reaction of an acrylate group with a primary or secondary amine and a methacrylate with a mercapto group.
  • the repeating units of the dendrimers may contain groups which are degradable under physiological conditions. In some embodiments, these repeating units may contain one or more germinal diethers, esters, amides, or disulfides groups.
  • the core molecule is a monoamine which allows dendritic polymerization in only one direction.
  • the core molecule is a polyamine with multiple different dendritic branches which each may comprise one or more repeating units.
  • the dendrimer may be formed by removing one or more hydrogen atoms from this core. In some embodiments, these hydrogen atoms are on a heteroatom such as a nitrogen atom.
  • the terminating group is a lipophilic groups such as a long chain alkyl or alkenyl group. In other embodiments, the terminating group is a long chain haloalkyl or haloalkenyl group. In other embodiments, the terminating group is an aliphatic or aromatic group containing an ionizable group such as an amine (-NH2) or a carboxylic acid (-CO2H). In still other embodiments, the terminating group is an aliphatic or aromatic group containing one or more hydrogen bond donors such as a hydroxide group, an amide group, or an ester.
  • the cationic ionizable lipids contain one or more asymmetrically-substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form.
  • Cationic ionizable lipids may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained.
  • the chiral centers of the cationic ionizable lipids of the present application can have the .S- or the R configuration.
  • one or more of the cationic ionizable lipids may be present as constitutional isomers.
  • the compounds have the same formula but different connectivity to the nitrogen atoms of the core.
  • the constitutional isomers may present the fully reacted primary amines and then a mixture of reacted secondary amines.
  • Chemical formulas used to represent cationic ionizable lipids of the present application will typically only show one of possibly several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given formula, and regardless of which one is most prevalent, all tautomers of a given chemical formula are intended.
  • the cationic ionizable lipids have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g., higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.
  • a better pharmacokinetic profile e.g., higher oral bioavailability and/or lower clearance
  • atoms making up the cationic ionizable lipids include all isotopic forms of such atoms.
  • Isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium
  • isotopes of carbon include 13 C and 14 C.
  • the ionizable clipid is a dendrimer or dendron.
  • the ionizable lipid comprises an ammonium group which is positively charged at physiological pH and contains at least two hydrophobic groups. In some embodiments, the ammonium group is positively charged at a pH from about 6 to about 8. In some embodiments, the ionizable lipid is a dendrimer or dendron. In some embodiments, the ionizable lipid comprises at least two C 6 -C 24 alkyl or alkenyl groups.
  • Modifying the functional groups and/or the chemical properties of the core, repeating units, and the surface or terminating groups, their physical properties can be modulated. Some properties which can be varied include, but are not limited to, solubility, toxicity, immunogenicity and bioattachment capability. Dendrimers are often described by their generation or number of repeating units in the branches. A dendrimer consisting of only the core molecule is referred to as Generation 0, while each consecutive repeating unit along all branches is Generation 1, Generation 2, and so on until the terminating or surface group. In some embodiments, half generations are possible resulting from only the first condensation reaction with the amine and not the second condensation reaction with the thiol.
  • Dendrimer synthesis can be of the convergent or divergent type. During divergent dendrimer synthesis, the molecule is assembled from the core to the periphery in a stepwise process involving attaching one generation to the previous and then changing functional groups for the next stage of reaction. Functional group transformation is necessary to prevent uncontrolled polymerization. Such polymerization would lead to a highly branched molecule that is not monodisperse and is otherwise known as a hyperbranched polymer.
  • the dendrimers of G1-G10 generation are specifically contemplated.
  • the dendrimers comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range derivable therein.
  • the dendrimers used herein are GO, Gl, G2, or G3. However, the number of possible generations (such as 11, 12, 13, 14, 15, 20, or 25) may be increased by reducing the spacing units in the branching polymer.
  • dendrimers have two major chemical environments: the environment created by the specific surface groups on the termination generation and the interior of the dendritic structure which due to the higher order structure can be shielded from the bulk media and the surface groups. Because of these different chemical environments, dendrimers have found numerous different potential uses including in therapeutic applications.
  • the dendrimers are assembled using the differential reactivity of the acrylate and methacrylate groups with amines and thiols.
  • the dendrimers include secondary or tertiary amines and thioethers formed by the reaction of an acrylate group with a primary or secondary amine and a methacrylate with a mercapto group.
  • the repeating units of the dendrimers contain groups which are degradable under physiological conditions. In some embodiments, these repeating units may contain one or more germinal diethers, esters, amides, or disulfides groups.
  • the core molecule is a monoamine which allows dendritic polymerization in only one direction.
  • the core molecule is a polyamine with multiple different dendritic branches which each may comprise one or more repeating units.
  • the dendrimer may be formed by removing one or more hydrogen atoms from this core. In some embodiments, these hydrogen atoms are on a heteroatom such as a nitrogen atom.
  • the terminating group is a lipophilic groups such as a long chain alkyl or alkenyl group. In other embodiments, the terminating group is a long chain haloalkyl or haloalkenyl group.
  • the terminating group is an aliphatic or aromatic group containing an ionizable group such as an amine (-NH2) or a carboxylic acid (-CO2H).
  • the terminating group is an aliphatic or aromatic group containing one or more hydrogen bond donors such as a hydroxide group, an amide group, or an ester.
  • the cationic ionizable lipids contain one or more asymmetrically-substituted carbon or nitrogen atoms, and can be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a chemical formula are intended, unless the specific stereochemistry or isomeric form is specifically indicated.
  • cationic ionizable lipids occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained.
  • the chiral centers of the cationic ionizable lipids of the present application can have the S or the R configuration.
  • one or more of the cationic ionizable lipids may be present as constitutional isomers.
  • the compounds have the same formula but different connectivity to the nitrogen atoms of the core.
  • such cationic ionizable lipids exist because the starting monomers react first with the primary amines and then statistically with any secondary amines present.
  • the constitutional isomers may present the fully reacted primary amines and then a mixture of reacted secondary amines.
  • Chemical formulas used to represent cationic ionizable lipids of the present application will typically only show one of possibly several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given formula, and regardless of which one is most prevalent, all tautomers of a given chemical formula are intended.
  • the cationic ionizable lipids of the present application may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g., higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.
  • a better pharmacokinetic profile e.g., higher oral bioavailability and/or lower clearance
  • atoms making up the cationic ionizable lipids of the present application are intended to include all isotopic forms of such atoms.
  • Isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium
  • isotopes of carbon include 13 C and 14 C.
  • the ionizable cationic lipid is a dendrimer or dendron.
  • the ionizable lipid comprises an ammonium group which is positively charged at physiological pH and contains at least two hydrophobic groups. In some embodiments, the ammonium group is positively charged at a pH from about 6 to about 8. In some embodiments, the ionizable lipid is a dendrimer or dendron. In some embodiments, the ionizable lipid comprises at least two C 6 -C 24 alkyl or alkenyl groups.
  • the ionizable lipid comprises at least two C 8 -C 24 alkyl groups. In some embodiments, the ionizable lipid is a dendrimer further defined by the formula:
  • Core- (Repeating Unit) n -Terminating Group (D-I) wherein one or more hydrogen atoms of the core are replaced with a repeating unit and wherein: the core has the formula: wherein:
  • Xi is amino or C 1 -C 12 alkylamino, C 1 -C 12 dialkylamino, C 3 -C 12 heterocycloalkyl, C 5 -C 12 heteroaryl, or a substituted version thereof;
  • R 1 is amino, hydroxy, mercapto, C 1 -C 12 alkylamino, or C 1 -C 12 dialkylamino, or a substituted version of either of these groups; and a is 1, 2, 3, 4, 5, or 6; or the core has the formula:
  • X 2 is N(R 5 ) y ;
  • R 5 is hydrogen, C 1 -C 18 alkyl, or substituted C 1 -C 18 alkyl; and y is 0, 1, or 2, provided that the sum of y and z is 3;
  • R 2 is amino, hydroxy, mercapto, C 1 -C 12 alkylamino, or C 1 -C 12 dialkylamino, or a substituted version of either of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, or 3; provided that the sum of z and y is 3; or the core has the formula: wherein:
  • X 3 is -NR 6 -, wherein R 6 is hydrogen, C 1 -C 8 alkyl, or C 1 -C 8 substituted alkyl, -O-, or C 1 -C 8 alkylaminodiyl, C 1 -C 8 alkoxydiyl, C 6 -C 8 arenediyl, C 8 -C 8 heteroarenediyl, C 3 -C 8 heterocycloalkanediyl, or a substituted version of any of these groups;
  • R 3 and R4 are each independently amino, hydroxy, mercapto, C 1 -C 12 alkylamino, or C 1 - C 12 dialkylamino, or a substituted version of either of these groups; or a group of the formula: wherein: e and f are each independently 1, 2, or 3; provided that the sum of e and f is 3;
  • R c , R d , and R f are each independently hydrogen, C 1 -C 6 alkyl, or substituted C 1 -C 6 alkyl; c and d are each independently 1, 2, 3, 4, 5, or 6; or the core is C 1 -C 18 alkylamine, C 1 -C 36 dialkylamine, C 3 -C 12 heterocycloalkane, or a substituted version of any of these groups; wherein the repeating unit comprises a degradable diacyl or a degradable diacyl and a linker; the degradable diacyl group has the formula: wherein:
  • a 1 and A 2 are each independently -O- , -S-, or -NR a -, wherein:
  • R a is hydrogen, C 1 -C 6 alkyl, or substituted C 1 -C 6 alkyl
  • Y 3 is C 1 -C 12 alkanediyl, C 1 -C 12 alkenediyl, C 6 -C 12 arenediyl, or a substituted version of any of these groups; or a group of the formula: wherein:
  • X 3 and X4 are C 1 -C 12 alkanediyl, C 2 -C 12 alkenediyl, C 6 -C 12 arenediyl, or a substituted version of any of these groups;
  • Y 5 is a covalent bond, C 1 -C 12 alkanediyl, C 1 -C 12 alkenediyl, C 6 -C 12 arenediyl, or a substituted version of any of these groups;
  • R 9 is C 1 -C 8 alkyl or substituted C 1 -C 8 alkyl; the linker group has the formula: wherein:
  • Y 1 is C 1 -C 12 alkanediyl, C 1 -C 12 alkenediyl, C 6 -C 12 arenediyl, or a substituted version of any of these groups; and wherein each independently denotes a point of attachment to another repeating unit or a terminating group; and the terminating group has the formula: wherein:
  • Y 4 is alkanediyl or an Ci -C 18 alkanediyl wherein one or more of the hydrogen atoms on the C 1 -C 18 alkanediyl has been replaced with -OH, -F, -Cl, -Br, -I, -SH, -OCH 3 , -OCH 2 CH 3 , -SCH 3 , or -OC(O)CH 3 ;
  • R 10 is hydrogen, carboxy, hydroxy, C 6 -C 12 aryl, C 1 -C 12 alkylamino, C 1 -C 12 dialkylamino, C 3 -C 12 N-heterocycloalkyl, -C(O)N(Rn)- C 1 -C 6 alkanediyl- C 3 -C 12 heterocycloalkyl, -C(O)- C 1 -C 12 alkylamino, — C(O)— C 1 -C 12 dialkylamino, or -C(O)- C 3 -C 12 N-heterocycloalkyl, wherein:
  • R11 is hydrogen, C 1 -C 6 alkyl, or substituted C 1 -C 6 alkyl; wherein the final degradable diacyl in the chain is attached to a terminating group; n is 0, 1, 2, 3, 4, 5, or 6; or a pharmaceutically acceptable salt thereof.
  • the terminating group is further defined by the formula: wherein:
  • Y4 is Cl -Cl 8 alkanediyl
  • R 10 is hydrogen.
  • Ai and A2 are each independently -O- or
  • the terminating group is a structure selected from the structures in Table 2.
  • the core is further defined by the formula:
  • X 2 is N(R 5 ) y ;
  • R 5 is hydrogen or C 1 -C 8 alkyl, or substituted C 1 -C 18 alkyl; and y is 0, 1, or 2, provided that the sum of y and z is 3;
  • R 2 is amino, hydroxy, or mercapto, or C 1 -C 12 alkylamino, C 1 -C 12 dialkylamino, or a substituted version of either of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, 3; provided that the sum of z and y is 3.
  • the core is further defined by the formula: wherein:
  • X 3 is -NR 6 -, wherein R 6 is hydrogen, C 1 -C 8 alkyl, or substituted C 1 -C 8 alkyl, -O-, or C 1 -C 8 alkylaminodiyl, C 1 -C 8 alkoxydiyl, C 1 -C 8 arenediyl, C 1 -C 8 heteroarenediyl, C 1 -C 8 heterocycloalkanediyl, or a substituted version of any of these groups;
  • R 3 and R4 are each independently amino, hydroxy, or mercapto, or C 1 -C 12 alkylamino, dialkylamino, or a substituted version of either of these groups; or a group of the formula: wherein: e and f are each independently 1, 2, or 3; provided that the sum of e and f is 3;
  • R c , Rd, and Rf are each independently hydrogen, C 1 -C 6 alkyl, or substituted C 1 -C 6 alkyl; c and d are each independently 1, 2, 3, 4, 5, or 6.
  • the terminating group is represented by the formula: wherein:
  • Y4 is alkanediyl(c ⁇ 18).
  • R 10 is hydrogen
  • a core of the structure of formula (D-IV) is:
  • the core comprises a structural formula set forth in Table 2 and pharmaceutically acceptable salts thereof, wherein * indicates a point of attachment of the core to a repeating unit (i.e., where a hydrogen of the core is relaced with a repeating unit).
  • the degradable diacyl is further defined as:
  • the linker is further defined as wherein Y 1 is C 1 -C 8 alkanediyl or substituted C 1 -C 12 alkanediyl.
  • R 6 is H. In some embodiments, in the core of formula (D-IV), R 6 is C 1 -C 8 alkyl. In some embodiments, in the core of formula (D-IV), R 6 is substituted alkyl (e.g., alkyl substituted with -NH2, alkyl substituted with -NHCH 3 , or alkyl substituted with -NHCH 2 CH 3 ).
  • one or two hydrogen atoms of the core are replaced with a repeating unit. In some embodiments three or four hydrogen atoms of the core is replaced with a repeating unit. In some embodiments five hydrogen atoms of the core is replaced with a repeating unit. In some embodiments six hydrogen atoms of the core is replaced with a repeating unit.
  • the dendrimer is selected from the group consisting of:
  • the ionizable lipid is a dendrimer of the formu . In some embodiments, the ionizable lipid is a dendrimer of the formula [0173] In some embodiments of the lipid composition, the ionizable lipid is a dendrimer of a generation (g) having a structural formula: or a pharmaceutically acceptable salt thereof, wherein:
  • the core comprises a structural formula (X core ): wherein:
  • Q is independently at each occurrence a covalent bond, -O-, -S-, -NR 2 -, or -CR 3a R 3b -;
  • R 2 is independently at each occurrence R lg or -L 2 -NR le R lf ;
  • R 3a and R 3b are each independently at each occurrence hydrogen or an optionally substituted (e.g., C 1 -C 6 , such as C 1 -C 3 ) alkyl;
  • R la , R lb , R lc , R ld , R le , R lf , and R lg are each independently at each occurrence a point of connection to a branch, hydrogen, or an optionally substituted (e.g., C 1 -C 12 ) alkyl;
  • L°, L 1 , and L 2 are each independently at each occurrence selected from a covalent bond, alkylene, heteroalkylene, [alkylene] -[heterocycloalkyl] -[alkylene], [alkylene] -(ary lene)- [alkylene], heterocycloalkyl, and arylene; or, alternatively, part of L 1 form a (e.g., C 4 -C 6 ) heterocycloalkyl (e.g., containing one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur) with one of R lc and R ld ; and x 1 is 0, 1, 2, 3, 4, 5, or 6; and
  • each branch of the plurality (N) of branches independently comprises a structural formula (X B ranch): wherein:
  • each diacyl group independently comprises a structural formula , wherein:
  • ** indicates a point of attachment of the diacyl group at the distal end thereof
  • Y 3 is independently at each occurrence an optionally substituted (e.g., C 1 -C 12 ); alkylene, an optionally substituted (e.g., C 1 -C 12 ) alkenylene, or an optionally substituted (e.g., C 1 -C 12 ) arenylene;
  • a 1 and A 2 are each independently at each occurrence -O-, -S-, or -NR 4 -, wherein:
  • R 4 is hydrogen or optionally substituted (e.g., C 1 -C 6 ) alkyl; m 1 and m 2 are each independently at each occurrence 1, 2, or 3; and
  • R 3C , R 3d , R 3e , and R 3f are each independently at each occurrence hydrogen or an optionally substituted (e.g., C 1 -C 8 ) alkyl;
  • each linker group independently comprises a structural formula wherein:
  • ** indicates a point of attachment of the linker to a proximal diacyl group
  • Y 1 is independently at each occurrence an optionally substituted (e.g., C 1 -C 12 ) alkylene, an optionally substituted (e.g., C 1 -C 12 ) alkenylene, or an optionally substituted (e.g., C 1 -C 12 ) arenylene; and
  • each terminating group is independently selected from optionally substituted
  • C 1 -C 18 such as C 4 -C 18 alkylthiol
  • optionally substituted e.g., C 1 -C 18 , such as C 4 - C 18 alkenylthiol.
  • R la , R lb , R lc , R ld , R le , R lf , and R lg are each independently at each occurrence a point of connection to a branch, hydrogen, or an optionally substituted alkyl.
  • R la , R lb , R lc , R ld , R le , R lf , and R lg are each independently at each occurrence a point of connection to a branch, hydrogen.
  • L°, L 1 , and L 2 are each independently at each occurrence selected from a covalent bond, C 1 -C 6 alkylene (e.g., C 1 -C 3 alkylene), C 2 -C 12 (e.g., C 2 -C 8 ) alkyleneoxide (e.g., oligo(ethyleneoxide), such as -(CH 2 CH 2 O)I-4-(CH 2 CH 2 )-), [(C 1 -C 4 ) alkylene] -[(C 4 -C 6 ) heterocycloalkyl] -[(C 1 -C 4 ) alkylene] (e.g. , ), and [(C 1 -C 4 ) alkylene] -phenylene- [(C 1 -C 4 ) alkylene] (e.g., ).
  • C 1 -C 6 alkylene e.g., C 1 -C 3 alkylene
  • C 2 -C 12 e.g., C
  • L°, L 1 , and L 2 are each independently at each occurrence selected from [(C 1 -C 4 ) alkylene]- [(C 4 -C 6 ) heterocycloalkyl] -[(C 1 -C 4 ) alkylene] (e.g., -(C 1 -C 3 alkylene)-phenylene-(C 1 -C 3 alkylene)-) and [(C 1 -C 4 ) alkylene] -[(C 4 -C 6 ) heterocycloalkyl] -[(C 1 -C 4 ) alkylene] (e.g., -(C 1 -C 3 alky lene)-piperaziny 1- (C 1 -C 3 alkylene) -) .
  • the core comprises a structural formula set forth in
  • the plurality (N) of branches comprises at least 3 branches, at least 4 branches, at least 5 branches. In some embodiments, the plurality (N) of branches comprises at least 3 branches. In some embodiments, the plurality (N) of branches comprises at least 4 branches. In some embodiments, the plurality (N) of branches comprises at least 5 branches.
  • g is 1, 2, 3, or 4. In some embodiments of X Branch , g is 1. In some embodiments of X Branch , g is 2. In some embodiments of X Branch , g is 3. In some embodiments of X Branch , g is 4.
  • each branch of the plurality of branches comprises a structural formula
  • each branch of the plurality of branches comprises a structural formula
  • each branch of the plurality of branches comprises a structural formula
  • each branch of the plurality of branches comprises a structural formula
  • the diacyl group independently comprises a structural formula * indicates a point of attachment of the diacyl group at the proximal end thereof, and ** indicates a point of attachment of the diacyl group at the distal end thereof.
  • Y 3 is independently at each occurrence an optionally substituted; alkylene, an optionally substituted alkenylene, or an optionally substituted arenylene. In some embodiments of the diacyl group of X Branch , Y 3 is independently at each occurrence an optionally substituted alkylene (e.g., C 1 -C 12 ). In some embodiments of the diacyl group of X Branch , Y 3 is independently at each occurrence an optionally substituted alkenylene (e.g., C 1 -C 12 ). In some embodiments of the diacyl group of X Branch , Y 3 is independently at each occurrence an optionally substituted arenylene (e.g., C 1 -C 12 ).
  • a 1 and A 2 are each independently at each occurrence -O-, -S-, or -NR 4 -. In some embodiments of the diacyl group of X Branch , A 1 and A 2 are each independently at each occurrence -O-. In some embodiments of the diacyl group of X Branch , A 1 and A 2 are each independently at each occurrence -S-. In some embodiments of the diacyl group of X Branch , A 1 and A 2 are each independently at each occurrence -NR 4 - and R 4 is hydrogen or optionally substituted alkyl (e.g., C 1 -C 6 ).
  • m 1 and m 2 are each independently at each occurrence 1, 2, or 3. In some embodiments of the diacyl group of X Branch , m 1 and m 2 are each independently at each occurrence 1. In some embodiments of the diacyl group of X Branch , m 1 and m 2 are each independently at each occurrence 2. In some embodiments of the diacyl group of X Branch , m 1 and m 2 are each independently at each occurrence 3. In some embodiments of the diacyl group of X Branch , R 3C , R 3d , R 3e , and R 3f are each independently at each occurrence hydrogen or an optionally substituted alkyl.
  • diacyl group of X Branch , R 3C , R 3d , R 3e , and R 3f are each independently at each occurrence hydrogen. In some embodiments of the diacyl group of X Branch , R 3c , R 3d , R 3e , and R 3f are each independently at each occurrence an optionally substituted (e.g., C 1 -C 8 ) alkyl.
  • a 1 is -O- or -NH-. In some embodiments of the diacyl group, A 1 is -O-. In some embodiments of the diacyl group, A 2 is -O- or -NH-.
  • a 2 is -O-.
  • Y 3 is C 1 -C 12 (e.g., C 1 -C 6 , such as C 1 -C 3 ) alkylene.
  • the diacyl group independently at each occurrence comprises a structural formula optionally R 3c , R 3d ,
  • R 3e , and R 3f are each independently at each occurrence hydrogen or C 1 -C 3 alkyl.
  • linker group independently comprises a structural formula
  • Y 1 is independently at each occurrence an optionally substituted alkylene, an optionally substituted alkenylene, or an optionally substituted arenylene. In some embodiments of the linker group of X Branch if present, Y 1 is independently at each occurrence an optionally substituted alkylene (e.g., C 1 -C 12 ). In some embodiments of the linker group of X Branch if present, Y 1 is independently at each occurrence an optionally substituted alkenylene (e.g., C 1 -C 12 ). In some embodiments of the linker group of Xuranch if present, Y i is independently at each occurrence an optionally substituted arenylene (e.g., C 1 -C 12 ).
  • each terminating group is independently selected from optionally substituted alkylthiol and optionally substituted alkenylthiol.
  • each terminating group is an optionally substituted alkylthiol (e.g., C 1 -C 18 , such as C 4 -C 18 ).
  • each terminating group is optionally substituted alkenylthiol (e.g., C 1 -C 18 , such as C 4 -C 18 ).
  • each terminating group is independently C 1 -C 18 alkenylthiol or C 1 -C 18 alkylthiol, and the alkyl or alkenyl moiety is optionally substituted with one or more substituents each independently selected from halogen, C 6 -C 12 aryl, C 1 -C 12 alkylamino, C 4 -C 6 AMieterocycloalkyl , -OH, -C(O)OH, -C(O)N(CI-C 3 alkyl)-(C 1 -C 6 alkylene)-(C 1 -C 12 alkylamino), -C(O)N(CI-C 3 alkyl)-(C 1 -C 6 alkylene)-(C 4 -C 6 JV-heterocycloalkyl), -C(O)-(C 1 -C 12 alkylamino), and -C(O)-(C 4
  • each terminating group is independently C 1 -C 18 (e.g., C 4 -C 18 ) alkenylthiol or C 1 -C 18 (e.g., C 4 -C 18 ) alkylthiol, wherein the alkyl or alkenyl moiety is optionally substituted with one or more substituents each independently selected from halogen, C 6 -C 12 aryl (e.g., phenyl), C 1 -C 12 (e.g., C 1 -C 8 ) alkylamino (e.g., C 1 -C 6 mono-alkylamino (such as -NHCH 2 CH 2 CH 2 CH 3 ) or C 1 -C 8 di-alkylamino (such as )), C 4 -C 6 N-heterocycloalkyl (e.g..).
  • C 6 -C 12 aryl e.g., phenyl
  • C 1 -C 12 e.g., C 1
  • each terminating group is independently C 1 -C 18 (e.g., C 4 -C 18 ) alkylthiol, wherein the alkyl moiety is optionally substituted with one substituent -OH.
  • each terminating group is independently C 1 -C 18 (e.g., C 4 -C 18 ) alkylthiol, wherein the alkyl moiety is optionally substituted with one substituent selected from C 1 -C 12 (e.g., C 1 -C 8 ) alkylamino (e.g., C 1 -C 6 mono-alkylamino (such as -NHCH 2 CH 2 CH 2 CH 3 ) or C 1 -C 8 di- alkylamino (such as , )) and C 4 -C 6 N-heterocycloalkyl
  • each terminating group is independently C 1 - C 18 (e.g., C 4 -C 18 ) alkenylthiol or C 1 -C 18 (e.g., C 4 -C 18 ) alkylthiol.
  • each terminating group is independently C 1 -C 18 (e.g., C 4 -C 18 ) alkylthiol.
  • the core comprises a structural formula selected from
  • each terminating group is independently a structure selected from the structurea in Table 3.
  • the dendrimers described herein can comprise a terminating group or pharmaceutically acceptable salt, or thereof selected in Table 3.
  • the dendrimer of Formula (X) is selected from those set forth in Table 4 and pharmaceutically acceptable salts thereof.
  • a is 1. In some embodiments of the cationic lipid of formula (D-I’), b is 2. In some embodiments of the cationic lipid of formula (D-I’), m is 1. In some embodiments of the cationic lipid of formula (D-I’), n is 1. In some embodiments of the cationic lipid of formula (D-I’), R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or -CH 2 CH(OH)R 7 . In some embodiments of the cationic lipid of formula
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or .
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or .
  • R is C 3 -C 18 alkyl (e.g., C 6 -C 12 alkyl).
  • the cationic lipid of formula (D-I’) is 13,16,20-tris(2- hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-l l,25-diol: [0207] In some embodiments, the cationic lipid of formula (D-I’) is (117?,257?)-13,16,20- tris((7?)-2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-l l,25-diol:
  • Additional cationic lipids that can be used in the compositions and methods of the present application include those cationic lipids as described in J. McClellan, M. C. King, Cell 2010, 141, 210-217, and International Patent Publication WO 2010/144740, WO 2013/149140, WO 2016/118725, WO 2016/118724, WO 2013/063468, WO 2016/205691, WO 2015/184256, WO 2016/004202, WO 2015/199952, WO 2017/004143, WO 2017/075531, WO 2017/117528, WO 2017/049245, WO 2017/173054 and WO 2015/095340, which are incorporated herein by reference for all purposes.
  • Examples of those ionizable cationic lipids include but are not limited to those as shown in Table 5.
  • the ionizable lipid is present in an amount of from about from about 20 mol% to about 23 mol%. In some embodiments, the ionizable lipid is present in an amount of about 20 mol%, about 20.5 mol%, about 21 mol%, about 21.5 mol%, about 22 mol%, about 22.5 mol%, or about 23 mol%. In other embodiments, the ionizable lipid is present in an amount of from about 7.5 mol% to about 20 mol%.
  • the ionizable lipid is present in an amount of about 7.5 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, or about 20 mol%.
  • the lipid composition comprises the ionizable lipid in an amount of from about 20mol% to about 30mol%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the ionizable lipid in an amount of of at least (about) 5 mol%, at least (about) 10 mol%, at least (about) 15 mol%, at least (about) 20 mol%, at least (about) 25 mol%, or at least (about) 30 mol%.
  • a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond.
  • an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide.
  • Such reactions may be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as targeting or imaging moiety (e.g., a dye).
  • the LNP comprises about 5% to about 30% weight of phoshpolipid. In some embodiments, the LNP comprises about 5% weight, or 10%, or 12%, or 15%, or 18%, or 20%, or 25%, or 30% weight of phospholipid.
  • the LNP comprises the phospholipid at a molar percentage from about 8% to about 23%. In some embodiments, the LNP comprises the phospholipid at a molar percentage from about 10% to about 20%. In some embodiments, the LNP comprises the phospholipid at a molar percentage from about 15% to about 20%. In some embodiments, the LNP comprises the phospholipid at a molar percentage from about 8% to about 15%. In some embodiments, the LNP comprises the phospholipid at a molar percentage from about 10% to about 15%. In some embodiments, the LNP comprises the phospholipid at a molar percentage from about 12% to about 18%.
  • the molecular weight of the PEG modification is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000,
  • lipids that may be used in the present application are taught by U.S. Patent 5,820,873, WO 2010/141069, or U.S. Patent 8,450,298, which is incorporated herein by reference.
  • the PEG-lipid has a structural formula:
  • R 12 and R 13 are each independently alkyl(c ⁇ 24), alkenyl(c ⁇ 24), or a substituted version of either of these groups;
  • R e is hydrogen, alkyl(c ⁇ 8), or substituted alkyl(c ⁇ 8>; and x is 1-250.
  • R e is alkyl(c ⁇ 8) such as methyl.
  • R12 and R13 are each independently alkyl(c ⁇ 4-20).
  • x is 5-250.
  • x is 5-125 or x is 100-250.
  • the PEG-lipid is 1,2- dimyristoyl-sn-glycerol, methoxypolyethylene glycol.
  • the PEG-lipid has a structural formula: , wherein: m is an integer between 1 and 100 and n2 and m are each independently selected from an integer between 1 and 29. In some embodiments, m is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
  • compositions may further comprise a molar percentage of the PEG-lipid to the total lipid composition from about 4.0 to about 4.6.
  • the molar percentage is from about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, to about 4.6 or any range derivable therein. In other embodiments, the molar percentage is from about 1.5 to about 4.0. In some embodiments, the molar percentage is from about 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, to about 4.0 or any range derivable therein.
  • the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 0.5% to about 10%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 1% to about 8%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 2% to about 7%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 3% to about 5%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 5% to about 10%.
  • the lipid composition comprises the polymer-conjugated lipid at a molar percentage of at least (about) 0.5%, at least (about) 1%, at least (about) 1.5%, at least (about) 2%, at least (about) 2.5%, at least (about) 3%, at least (about) 3.5%, at least (about) 4%, at least (about) 4.5%, at least (about) 5%, at least (about) 5.5%, at least (about) 6%, at least (about) 6.5%, at least (about) 7%, at least (about) 7.5%, at least (about) 8%, at least (about) 8.5%, at least (about) 9%, at least (about) 9.5%, or at least (about) 10%.
  • the lipid composition comprises the polymer-conjugated lipid at a molar percentage of at most (about) 0.5%, at most (about) 1%, at most (about) 1.5%, at most (about) 2%, at most (about) 2.5%, at most (about) 3%, at most (about) 3.5%, at most (about) 4%, at most (about) 4.5%, at most (about) 5%, at most (about) 5.5%, at most (about) 6%, at most (about) 6.5%, at most (about) 7%, at most (about) 7.5%, at most (about) 8%, at most (about) 8.5%, at most (about) 9%, at most (about) 9.5%, or at most (about) 10%.
  • the LNP comprises a steroid or steroid derivative.
  • the term “steroid” is a class of compounds with a four ring 17 carbon cyclic structure which can further comprises one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or a double bond between two or more carbon atoms.
  • the ring structure of a steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring as shown in the formula:
  • a steroid derivative comprises the ring structure above with one or more non-alkyl substitutions.
  • the steroid or steroid derivative is a sterol wherein the formula is further defined as: .
  • the steroid or steroid derivative is a cholestane or cholestane derivative.
  • the ring structure is further defined by the formula: .
  • a cholestane derivative includes one or more non-alkyl substitution of the above ring system.
  • the cholestane or cholestane derivative is a cholestene or cholestene derivative or a sterol or a sterol derivative.
  • the cholestane or cholestane derivative is both a cholestere and a sterol or a derivative thereof.
  • the compositions may further comprise a molar percentage of the steroid to the total lipid composition from about 40 to about 46.
  • the molar percentage is from about 40, 41, 42, 43, 44, 45, to about 46 or any range derivable therein.
  • the molar percentage of the steroid relative to the total lipid composition is from about 15 to about 40. In some embodiments, the molar percentage is 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or any range derivable therein.
  • the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 15% to about 46%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 20% to about 40%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 25% to about 35%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 30% to about 40%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 20% to about 30%.
  • the lipid composition comprises the steroid or steroid derivative at a molar percentage of at least (about) 15%, of at least (about) 20%, of at least (about) 25%, of at least (about) 30%, of at least (about) 35%, of at least (about) 40%, of at least (about) 45%, or of at least (about) 46%.
  • the lipid composition comprises the steroid or steroid derivative at a molar percentage of at most (about) 15%, of at most (about) 20%, of at most (about) 25%, of at most (about) 30%, of at most (about) 35%, of at most (about) 40%, of at most (about) 45%, or of at most (about) 46%.
  • the cationic agent is a cationic lipid which is a sterol amine.
  • a sterol amine has, for its hydrophobic portion, a sterol, and for its hydrophilic portion, an amine group.
  • the sterol group is selected from, but not limited to, cholesterol, sitosterol, campesterol, stigmasterol or derivatives thereof.
  • the amine group can comprise one to five primary, secondary, tertiary amines, or mixtures thereof. At least one of the amines has a pKa of 8 or greater and is charged at physiological pH.
  • the amine can be contained in a three to eight membered heteroalkyl or heteroaryl ring.
  • the method comprises the administration of a LNP comprising a gene editing system, wherein the LNP comprises one or more selective organ targeting (SORT) molecules.
  • SORT selective organ targeting
  • LNPs comprising SORT molecules include a supplemental SORT molecule, wherein the chemical structure of the SORT molecule determines the tissue- specific activity of the LNP.
  • the method comprises administration of the LNP comprising a SORT molecule, wherein LNP is delivered to organs and cells other than the liver.
  • the LNP comprising a gene editing system comprising a SORT molecule is preferentially delivered to a target organ.
  • the target organ is a lung, a lung tissue or a lung cell.
  • the lung cell type is an endothelial cell or an epithelial cell.
  • the lung cell type is an immune cell.
  • the lung cell is a stem cell.
  • the epithelial cell is a ciliated cell, non-ciliated cell, a goblet cell, a brush cell (alveolar macrophage), an airway basal cell, a small granule cell, a bronchial epithelial cell, a small airway epithelial cell, and/or a tracheal epithelial cell.
  • the lung cell is a secretory cell and or ionocyte.
  • the term “preferentially delivered” is used to refer to a composition, upon being delivered, which is delivered to the target organ (e.g., lung), tissue, or cell in at least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%) of the amount administered.
  • the LNP comprises one or more SORT molecules.
  • the SORT molecule comprises a cationic lipid or an anionic lipid.
  • the SORT molecule comprises a permanently cationic lipid or a permanently anionic lipid.
  • the SORT molecule comprises a cationic lipid or a permanently cationic lipid.
  • the SORT molecule comprises permanently positively charged moiety.
  • the permanently positively charged moiety may be positively charged at a physiological pH such that the SORT molecule comprises a positive charge upon delivery of a polynucleotide to a cell.
  • the positively charged moiety is quaternary amine or quaternary ammonium ion.
  • the SORT molecule comprises, or is otherwise complexed to or interacting with, a counterion.
  • the SORT molecule comprises two or more alkyl or alkenyl chains of C 6 -C24.
  • the one or more SORT molecule is a permanently cationic lipid (i.e., comprising one or more hydrophobic components and a permanently cationic group).
  • the permanently cationic lipid may contain a group which has a positive charge regardless of the pH.
  • One permanently cationic group that may be used in the permanently cationic lipid is a quaternary ammonium group.
  • the one or more SORT molecule is ionizable cationic lipid (i.e., comprising one or more hydrophobic components and an ionizable cationic group).
  • the ionizable positively charged moiety may be positively charged at a physiological pH.
  • One ionizable cationic group that may be used in the ionizable cationic lipid is a tertiary ammine group.
  • the one or more SORT molecule comprises 18:1 DOTMA; DORI, DC-6-14; 12:0 EPC (Chloride Salt); 14:0 EPC (Chloride Salt); 16:0 EPC (Chloride Salt); 18:0 EPC (Chloride Salt); 18:1 EPC (Chloride Salt); 16:0-18:1 EPC (Chloride Salt); 14:1 EPC (Triflate Salt); 18:0 DDAB (Dimethyldioctadecylammonium (Bromide Salt)); 14:0 TAP; 16:0 TAP; 18:0 TAP; 18:1 TAP (DOTAP); 18:1 TAP (DOTAP, MS Salt); 18:1 DODAP, or 18:1 PA (l,2-dioleoyl-sn-glycero-3-phosphate (sodium salt)).
  • the SORT molecule comprises DOTAP (l,2-dioleoyl-3-trimethylammonium propane). In some embodiments, the SORT molecule comprises 18:1 PA. In some embodiments, the SORT molecule comprises DODAP.
  • the SORT molecule comprises between 5% to 70% molar percentage of the LNP. In some embodiments, the SORT molecule comprises up to about 5%, up to about 10%, up to about 15%, up to about 20%, up to about 25%, up to about 30%, up to about 35%, up to about 40%, up to about 45%, up to about 50%, up to about 55%, up to about 60%, up to about 65%, or up to about 70% molar percentage of the LNP. In some embodiments, the SORT molecule comprises about 20% molar percentage of the LNP. In some embodiments, the SORT molecule comprises about 25% molar percentage of the LNP. In some embodiments, the SORT molecule comprises about 30% molar percentage of the LNP.
  • the SORT molecule comprises about 35% molar percentage of the LNP. In some embodiments, the SORT molecule comprises about 40% molar percentage of the LNP. In some embodiments, the SORT molecule comprises about 45% molar percentage of the LNP.
  • the SORT molecule comprises from about 5% to about 70% molar percentage of the LNP, from about 10% to about 70% molar percentage of the LNP, from about 15% to about 70% molar percentage of the LNP, from about 20% to about 70% molar percentage of the LNP, from about 25% to about 70% molar percentage of the LNP, from about 30% to about 70% molar percentage of the LNP, from about 35% to about 70% molar percentage of the LNP, from about 40% to about 70% molar percentage of the LNP, from about 45% to about 70% molar percentage of the LNP, from about 50% to about 70% molar percentage of the LNP, from about 55% to about 70% molar percentage of the LNP, from about 60% to about 70% molar percentage of the LNP, from about 65% to about 70% molar percentage of the LNP, from about 5% to about 65% molar percentage of the LNP, from about 10% to about 65% molar percentage of the LNP, from about 15% to about 65%
  • the SORT molecule comprises from about 20% to about 40% molar percentage of the LNP. In some embodiments, the SORT molecule comprises from about 35% to about 40% molar percentage of the LNP. In some embodiments, the SORT molecule comprises from about 40% to about 45% molar percentage of the LNP. In some embodiments, the SORT molecule comprises from about 35% to about 45% molar percentage of the LNP.
  • the SORT molecule facilitates the preferential delivery of the one or more polynucleotides comprising one or more nucleic acids encapsulated in the LNP to a target organ and/or a target cell.
  • an LNP comprising a SORT molecule delivers a greater percentage of the enveloped nucleic acid than a reference LNP without a SORT molecule.
  • the LNP comprising a SORT molecule delivers at least 5% more, at least 10% more, at least 15% more, at least 20% more, at least 25% more, at least 30% more, at least 35% more, at least 40% more, at least 45% more, at least 50% more, at least 55% more, at least 60% more, at least 65% more, at least 70% more, at least 75% more, at least 80% more, at least 85% more, at least 90% more, at least 95% more, or at least 99% more encapsulated nucleic acids to the target organ and/or the target cell than a reference LNP not comprising a SORT molecule.
  • the target organ is the lung.
  • target cells may comprise, but are not limited to, basal cells, secretory cells such as goblet cells and club cells, ciliated cells, and any combination thereof.
  • the LNP comprising a SORT molecule achieves a greater therapeutic effect compare to a reference LNP that does not comprise a SORT molecule. In some embodiments of the method, the LNP comprising a SORT molecule achieves about 1.1 - fold to about 20-fold therapeutic effect compared to that achieved with a reference LNP. In some embodiments of the method, the LNP comprising a SORT molecule achieves about 1.1 - fold to about 10-fold therapeutic effect compared to that achieved with a reference LNP. In some embodiments of the method, the LNP comprising a SORT molecule achieves about 1.1 - fold to about 5-fold therapeutic effect compared to that achieved with a reference LNP.
  • the LNP comprising a SORT molecule achieves about 5-fold to about 10-fold therapeutic effect compared to that achieved with a reference LNP. In some embodiments of the method, the LNP comprising a SORT molecule achieves about 10-fold to about 20-fold therapeutic effect compared to that achieved with a reference LNP.
  • the LNP comprising a SORT molecule achieves at least about 1.1 - fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold therapeutic effect compared to that achieved with a reference LNP.
  • the additional lipid is a permanently cationic lipid (z.e., comprising one or more hydrophobic components and a permanently cationic group).
  • the permanently cationic lipid may contain a group which has a positive charge regardless of the pH.
  • One permanently cationic group that may be used in the permanently cationic lipid is a quaternary ammonium group.
  • the permanently cationic lipid may comprise a structural formul (S-I), wherein: Y 1 , Y 2 , or Y 3 are each independently XiC(O)R 1 or X2N + R 3 R4R 5 ; provided at least one of Y 1 , Y 2 , and Y 3 is X2N + R 3 R4R 5 ;
  • R 1 is C 1 -C 24 alkyl, C 1 -C 24 substituted alkyl, C 1 -C 24 alkenyl, C 1 -C 24 substituted alkenyl;
  • X 1 is O or NR a , wherein R a is hydrogen, C 1 -C 4 alkyl, or C 1 -C 4 substituted alkyl; X2 is C 1 -C 6 alkanediyl or C 1 -C 6 substituted alkanediyl;
  • R 3 , R4, and R5 are each independently C 1 -C 24 alkyl, C 1 -C 24 substituted alkyl, C 1 -C 24 alkenyl, C 1 -C 24 substituted alkenyl; and
  • R 1 and R 2 are each independently C 8 -C 24 alkyl, C 8 -C 24 alkenyl, or a substituted version of either group;
  • R 3 , R 3 ', and R 3 " are each independently C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl;
  • R 1 and R 2 are each independently C 8 -C 24 alkyl, C 8 -C 24 alkenyl, or a substituted version of either group;
  • R 3 , R 3 ', and R 3 " are each independently C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl;
  • X- is a monovalent anion
  • the additonal lipid e.g., additonal lipid (e.g., SORT lipid)
  • the additonal lipid is an ethylphosphocholine.
  • the ethylphosphocholine may be, by way of example, without being limited to, l,2-dimyristoleoyl-sn-glycero-3- ethylphosphocholine, 1 ,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1 ,2-distearoyl-sn-glycero- 3-ethylphosphocholine, 1 ,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1 ,2-dimyristoyl-sn- glycero-3-ethylphosphocholine, l,2-dilauroyl-sn-glycero-3-ethylphosphocholine, l-palmitoyl-2- oleoy 1- sn-gly cero- 3 -ethy Ipho sphocholine .
  • the lipid has a structural formula: wherein:
  • R 1 and R 2 are each independently C 8 -C 24 alkyl, C 8 -C 24 alkenyl, or a substituted version of either group;
  • R 3 , R 3 ', and R 3 " are each independently C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl;
  • X- is a monovalent anion
  • a additonal lipid e.g., additonal lipid (e.g., SORT lipid)
  • a structural formula of the immediately preceding paragraph is l,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., chloride salt).
  • DOTAP l,2-dioleoyl-3-trimethylammonium-propane
  • the additonal lipid e.g., additonal lipid (e.g., SORT lipid)
  • R4 and R4' are each independently alkyl (C6-c24) , alkenyl (C6-c24) , or a substituted version of either group;
  • R4" is alkyl(c ⁇ 24), alkenyl(c ⁇ 24), or a substituted version of either group;
  • R4"' is alkyl(ci-cs), alkenyl(C2-cs), or a substituted version of either group
  • X2 is a monovalent anion.
  • a additonal lipid e.g., additonal lipid (e.g., SORT lipid) of the structural formula of the immediately preceding paragraph is dimethyldioctadecylammonium (DDAB).
  • DDAB dimethyldioctadecylammonium
  • the additional lipid is selected from the lipids set forth in Table 6.
  • Example additonal lipid e.g., SORT lipids
  • X- is a counterion (e.g., O-, Br ⁇ etc.)
  • the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage from about 20% to about 65%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage from about 25% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage from about 30% to about 55%.
  • the additonal lipid e.g., SORT lipid
  • the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage from about 20% to about 50%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage from about 30% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage from about 25% to about 60%.
  • the additonal lipid e.g., SORT lipid
  • the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage of at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, at least (about) 45%, at least (about) 50%, at least (about) 55%, at least (about) 60%, or at least (about) 65%.
  • the additonal lipid e.g., SORT lipid
  • the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage of at most (about) 25%, at most (about) 30%, at most (about) 35%, at most (about) 40%, at least (about) 45%, at most (about) 50%, at most (about) 55%, at most (about) 60%, or at most (about) 65%.
  • the additonal lipid e.g., SORT lipid
  • the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%, or of a range between (inclusive) any two of the foregoing values.
  • Additonal lipid e.g., SORT lipid
  • Illustrative LNP compositions are presented below in Table 7.
  • Table 7 Illustrative LNP compositions.
  • the method disclosed herein comprises the administration of a LNP, wherein the LNP comprises one or more polynucleotides.
  • delivery of the one or more polynucleotides to a target cell can be used for therapeutic potential. For instance, it can allow delivery of nucleic acids (e.g. mRNA) encoding a protein that stimulates the immune system near the target cell or leads the target cells to express the protein in such a way that immune cells kill the target cell. In some aspects, such delivery is beneficial in the context of treating or preventing tumors or cancer, for prophylactic uses or in the context of vaccination.
  • the LNP comprises a one or more polynucleotides.
  • the polynucleotide comprises a gRNA. In some embodiments, the polynucleotide comprises the mRNA of a base editor. In some embodiments, the polynucleotide comprises a gRNA and the mRNA of a base editor.
  • the LNP is used to deliver a polynucleotide to a target cell.
  • the polynucleotide is a DNA.
  • the polynucleotide comprises a ribonucleic acid (RNA).
  • the RNA is a circRNA, an mRNA, a siRNA, an RNAi, and/or a microRNA.
  • the RNA enhances the anti-tumor effect or the therapeutic effect of the nanoparticle.
  • the polynucleotide comprises a deoxyribonucleic acid (DNA).
  • the RNA and/or enhances the therapeutic effect of the LNP.
  • siRNA or small interfering RNA or silencing RNA are RNA molecules that are double stranded, non-coding RNA molecules. These RNA molecules are typically 20-24 base pairs in length and produced through the Dicer enzyme catalyzing production from long double- stranded RNA molecules.
  • siRNA can bind to mRNA with a complementary sequence and lead to mRNA degradation. Delivery of siRNA to a cell can be used to reduce specific mRNA transcripts, leading to decreased protein production from the mRNA. This is a form of RNA interference or RNAi.
  • Another form of RNAi includes microRNA, which are similar to siRNAs but may contain post-transcriptional modifications. MicroRNAs also work to deplete target mRNAs and thereby decrease proteins produced by the mRNAs. Delivery of microRNAs to target cells offer therapeutic potentials by decreasing production of negative proteins.
  • mRNA is RNA that can be translated into a protein and its delivery into target cells can lead to localized production of protein within the target cell. mRNA is rapidly degraded and so needs to be efficiently delivered to the target cell.
  • the nanoparticle is used to deliver mRNA to a target cell.
  • the RNA is a linear RNA molecule.
  • the linear RNA is at least 600 nucleotides in length, at least 1000 nucleotides in length, or at least 1200 nucleotides in length. In some embodiments, the linear RNA is less than 2000 nucleotides in length.
  • the linear RNA is at least 600 nucleotides in length but less than 2000 nucleotides in length, at least 1000 nucleotides in length but less than 2000 nucleotides in length, at least 1200 nucleotides in length but less than 2000 nucleotides in length, at least 1400 nucleotides in length but less than 2000 nucleotides in length, at least 600 nucleotides in length but less than 1400 nucleotides in length, or at least 600 nucleotides in length but less than 2000 nucleotides in length.
  • the RNA comprises a cleaved linear RNA comprising a hydroxyl group at the 5' terminus, and a 2', 3 '-cyclic phosphate at the 3' terminus.
  • the RNA is a messenger RNA (mRNA) molecule.
  • the nucleic acid is a capped mRNA.
  • the mRNA molecule is greater than 2000 nucleotides, greater than 2500 nucleotides, greater than 3000 nucleotides, greater than 3500 nucleotides, greater than 4000 nucleotides, greater than 4500 nucleotides, or greater than 5000 nucleotides in length.
  • the mRNA molecule is about 2000 nucleotides in length. In some embodiments, the mRNA molecule is about 2500 nucleotides in length.
  • the mRNA molecule is about 3000 nucleotides in length. In some embodiments, the mRNA molecule is about 3500 nucleotides in length. In some embodiments, the mRNA molecule is about 4000 nucleotides in length. In some embodiments, the mRNA molecule is about 4500 nucleotides in length. In some embodiments, the mRNA molecule is about 5000 nucleotides in length.
  • CircRNA is a covalently closed continuous loop of single- stranded RNA. CircRNA can be divided into four categories including exonic circRNA (ecircRNA), circular intronic RNA (ciRNA), exon-intron circRNA (ElciRNA), and intergenic circRNA.
  • ecircRNA exonic circRNA
  • ciRNA circular intronic RNA
  • ElciRNA exon-intron circRNA
  • intergenic circRNA intergenic circRNA
  • circRNA results in several advantages compared to linear mRNA, including resistance to exonuclease-mediated degradation, increased stability, extended half-life, increased protein expression, and reduced immunogenicity (Chen, RNA Biol, 12(4):381-388 (2015); Wesselhoeft et al., Nat Commun, 9(1):2629 (2016)).
  • circRNA generally has a longer half-life compared to their linear mRNA counterparts (Wesselhoeft et al., Nat Commun, 9(1):2629 (2018)). Accordingly, circRNA improves protein expression (e.g., of the encoded gene product) over its lifetime compared to linear mRNAs.
  • CircRNA can be synthesized in vitro by chemical, enzymatic, and ribozymatic approaches.
  • PIE splicing systems based on Group I introns that are naturally found in the rRNA, tRNA, and mRNA genes of bacteria and non-metazoan eukaryotes can produce circRNA by self-splicing.
  • the circular RNA is at least 600 nucleotides in length, at least 1000 nucleotides in length, or at least 1200 nucleotides in length. In some embodiments, the circular RNA is less than 2000 nucleotides in length.
  • the circular RNA is at least 600 nucleotides in length but less than 2000 nucleotides in length, at least 1000 nucleotides in length but less than 2000 nucleotides in length, at least 1200 nucleotides in length but less than 2000 nucleotides in length, at least 1400 nucleotides in length but less than 2000 nucleotides in length, at least 600 nucleotides in length but less than 1400 nucleotides in length, or at least 600 nucleotides in length but less than 2000 nucleotides in length.
  • the circular RNA molecule is greater than 2000 nucleotides, greater than 2500 nucleotides, greater than 3000 nucleotides, greater than 3500 nucleotides, greater than 4000 nucleotides, greater than 4500 nucleotides, or greater than 5000 nucleotides in length. In some embodiments, the circular RNA molecule is about 2000 nucleotides in length. In some embodiments, the circular RNA molecule is about 2500 nucleotides in length. In some embodiments, the circular RNA molecule is about 3000 nucleotides in length. In some embodiments, the circular RNA molecule is about 3500 nucleotides in length.
  • the circular RNA molecule is about 4000 nucleotides in length. In some embodiments, the circular RNA molecule is about 4500 nucleotides in length. In some embodiments, the circular RNA molecule is about 5000 nucleotides in length.
  • the polynucleotide comprises a DNA molecule.
  • the DNA is a naked DNA molecule.
  • the DNA is a double- stranded DNA molecule.
  • the DNA is a single- stranded DNA molecule.
  • the DNA is a modified DNA molecule.
  • the DNA is modified to enhance its stability.
  • the DNA is a closed-ended DNA molecule.
  • the DNA is a naked closed-ended DNA molecule.
  • the DNA is at least 600 nucleotides, at least 1000 nucleotides, or at least 1200 nucleotides in length. In some embodiments, the DNA is less than 2000 nucleotides in length. In some embodiments, the DNA is at least 600 nucleotides but less than 2000 nucleotides in length, at least 1000 nucleotides but less than 2000 nucleotides in length, at least 1200 nucleotides but less than 2000 nucleotides in length, at least 1400 nucleotides but less than 2000 nucleotides in length, at least 600 nucleotides but less than 1400 nucleotides in length, or at least 600 nucleotides but less than 2000 nucleotides in length.
  • the polynucleotide is from 2000 nucleotides to 5000 nucleotides in length. In some embodiments, the polynucleotide is from 2500 to 5000 nucleotides in length. In some embodiments, the polynucleotide is from 3000 to 5000 nucleotides in length. In some embodiments, the polynucleotide is from 3500 to 5000 nucleotides in length. In some embodiments, the polynucleotide is from 4000 to 5000 nucleotides in length. In some embodiments, the polynucleotide is from 4500 to 5000 nucleotides in length.
  • the polynucleotide has a concentration of 0.5-3.0 mg/mL, of 1.0-3.0 mg/mL, of 2.0-3.0 mg/mL of 1.0 mg/mL. In some embodiments, the polynucleotide has a concentration of 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL, 1.4 mg/mL, or 1.5 mg/mL. In some embodiments, the polynucleotide has a concentration of 1.0 mg/mL.
  • the polynucleotide is from 2000 to 5000 nucleotides in length and at a concentration of 1.0 mg/mL. In some embodiments, the polynucleotide is from 2500 to 5000 nucleotides in length and at a concentration of 1.0 mg/mL. In some embodiments, the polynucleotide is from 3000 to 5000 nucleotides in length and at a concentration of 1.0 mg/mL. In some embodiments, the polynucleotide is from 3500 to 5000 nucleotides in length and at a concentration of 1.0 mg/mL.
  • the polynucleotide is from 4000 to 5000 nucleotides in length and at a concentration of 1.0 mg/mL. In some embodiments, the polynucleotide is from 4500 to 5000 nucleotides in length and at a concentration of 1.0 mg/mL.
  • the polynucleotide is from 2000 to 5000 nucleotides in length and at a concentration of 0.9 mg/mL. In some embodiments, the polynucleotide is from 2500 to 5000 nucleotides in length and at a concentration of 0.9 mg/mL. In some embodiments, the polynucleotide is from 3000 to 5000 nucleotides in length and at a concentration of 0.9 mg/mL. In some embodiments, the polynucleotide is from 3500 to 5000 nucleotides in length and at a concentration of 0.9 mg/mL.
  • the polynucleotide is from 4000 to 5000 nucleotides in length and at a concentration of 0.9 mg/mL. In some embodiments, the polynucleotide is from 4500 to 5000 nucleotides in length and at a concentration of 0.9 mg/mL.
  • the polynucleotide is from 2000 to 5000 nucleotides in length and at a concentration of 0.8 mg/mL. In some embodiments, the polynucleotide is from 2500 to 5000 nucleotides in length and at a concentration of 0.8 mg/mL. In some embodiments, the polynucleotide is from 3000 to 5000 nucleotides in length and at a concentration of 0.8 mg/mL. In some embodiments, the polynucleotide is from 3500 to 5000 nucleotides in length and at a concentration of 0.8 mg/mL.
  • the polynucleotide is from 4000 to 5000 nucleotides in length and at a concentration of 0.8 mg/mL. In some embodiments, the polynucleotide is from 4500 to 5000 nucleotides in length and at a concentration of 0.8 mg/mL.
  • the polynucleotide is from 2000 to 5000 nucleotides in length and at a concentration of 0.7 mg/mL. In some embodiments, the polynucleotide is from 2500 to 5000 nucleotides in length and at a concentration of 0.7 mg/mL. In some embodiments, the polynucleotide is from 3000 to 5000 nucleotides in length and at a concentration of 0.7 mg/mL. In some embodiments, the polynucleotide is from 3500 to 5000 nucleotides in length and at a concentration of 0.7 mg/mL.
  • the polynucleotide is from 4000 to 5000 nucleotides in length and at a concentration of 0.7 mg/mL. In some embodiments, the polynucleotide is from 4500 to 5000 nucleotides in length and at a concentration of 0.7 mg/mL.
  • the polynucleotide is from 2000 to 5000 nucleotides in length and at a concentration of 0.6 mg/mL. In some embodiments, the polynucleotide is from 2500 to 5000 nucleotides in length and at a concentration of 0.6 mg/mL. In some embodiments, the polynucleotide is from 3000 to 5000 nucleotides in length and at a concentration of 0.6 mg/mL. In some embodiments, the polynucleotide is from 3500 to 5000 nucleotides in length and at a concentration of 0.6 mg/mL.
  • the polynucleotide is from 4000 to 5000 nucleotides in length and at a concentration of 0.6 mg/mL. In some embodiments, the polynucleotide is from 4500 to 5000 nucleotides in length and at a concentration of 0.6 mg/mL.
  • the polynucleotide has an average molecular weight of up to 20,000,000 Da. In some embodiments, the polynucleotide can have an average molecular weight of up to 2,000,000 Da. In some embodiments, the polynucleotide may have an average molecular weight of up to 150,000 Da. In some embodiments, the polynucleotide has an average molecular weight of up to 15,000 Da, 5,000 Da or 1,000 Da.
  • the method described herein comprises the LNP composition, comprising an LNP comprising a gene editing system, wherein the LNP comprises 1,2-dioleoyl- 3 -dimethylammonium propane (DODAP) at a molar percentage less then 25% or less then 20%; cholesterol at a molar percentage greater than 40%; and/or messenger RNA (mRNA) at a lipid:mRNA ratio less than 40:1.
  • DODAP 1,2-dioleoyl- 3 -dimethylammonium propane
  • mRNA messenger RNA
  • the method described herein comprises a LNP composition, comprising an LNP comprising a gene editing system, wherein the LNP specifically transduces lung cells; and/or the LNP delivers mRNA to lung cells or in an amount effective to increase expression and/or function of a polypeptide or polynucleotide encoded by the mRNA.
  • the lung cells comprise lung cell types that include, but are not limited to, endothelial cells or epithelial cells.
  • the lung cell type is an immune cell.
  • the lung cell type is a stem cell.
  • the epithelial cell is a ciliated cell, non-ciliated cell, a goblet cell, a brush cell (alveolar macrophage), an airway basal cell, a small granule cell, a bronchial epithelial cell, a small airway epithelial cell, and/or a tracheal epithelial cell.
  • the lung cell type is a secretory cell and or ionocyte.
  • the LNP specifically transduces secretory cells and/or ionocytes; and/or wherein the LNP delivers mRNA to lung cells in an amount effective to increase expression and/or function of a polypeptide or polynucleotide encoded by the mRNA.
  • the LNP comprises an ionizable cationic lipid; a neutral phospholipid; a polyethylene-glycol (PEG)-lipid; and/or a/the cholesterol.
  • the LNP comprises a second ionizable cationic lipid.
  • the LNP comprises an anionic lipid.
  • the LNP comprises a permanently cationic lipid.
  • the LNP comprises DODAP at a molar percentage less than 25% or less then 20%. In some embodiments, the LNP comprises a DODAP at a molar percentage of less than 5%, less than 10%, less than 15%, less than 16%, than 17%, than 18%, than 19%, than 20%, than 21%, than 22%, than 22%, than 23%, than 24% or of less than 25%.
  • the LNP comprises DODAP at a molar percentage between 5% and 25%, between 7.5% and 25%, between 10% and 25%, between 15% and 25%, between 20% and 25%, between 5% and 20%, between 7.5% and 20%, between 10% and 20%, between 15% and 20%, between 5% and 15%, between 7.5% and 15%, between 10% and 15%, between 5% and 10%, or between 7.5% and 10%.
  • the LNP comprises DODAP at a molar percentage between 17.5% and 20%, between 17.5% and 22.5%, between 17.5% and 25%, between 5% and 17.5%, between 7.5% and 17.5%, between 10% and 17.5%, between 12.5% and 17.5% or between 15% and 17.5%.
  • the LNP comprises DODAP at a molar percentage of 16%.
  • the LNP comprises cholesterol at a molar percentage greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99%.
  • the LNP comprises cholesterol at a molar percentage between 40% and 60%, between 45% and 60%, between 50% and 60%, between 55% and 60%, between 40% and 55%, between 40% and 50%, between 40% and 45%, between 45% and 55%, between 45% and 50% or between 50% and 55%.
  • the LNP comprises cholesterol at a molar percentage of 50%.
  • Exemplary LNP formulations are presented below in Table 8. Table 8: Exemplary fomulations
  • the LNP comprises messenger RNA (mRNA). In some embodiments, the LNP comprises mRNA at a lipid:mRNA ratio less than 40:1. In some embodiments, the lipid:mRNA ratio is between 20:1 and 40:1, between 25:1 and 40:1, between
  • the lipid:mRNA ratio is 36:1. In some embodiments, the lipid:mRNA ratio is 25:1. In some embodiments, the ionizable cationic lipid is 5A2-SC 8 or 4A3-SC7; the neutral phospholipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and/or the polyethylene-glycol (PEG)- lipid is DMG-PEG, optionally DMG-PEG2000. In some embodiments, the ionizable cationic lipid is 4A3-SC7; the neutral phospholipid is DOPE; and the polyethylene-glycol (PEG)-lipid is DMG-PEG.
  • DOPE dioleoyl-sn-glycero-3-phosphoethanolamine
  • DSPC l,2-distearoyl-sn-glycero-3-
  • the LNP comprises a second cationic lipid and the second cationic lipid is DODAP.
  • the LNP comprises a second cationic lipid and the second cationic lipid is l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA).
  • DOTMA l,2-di-O-octadecenyl-3-trimethylammonium propane
  • the ionizable cationic lipid is 4A3-SC7 and the LNP comprises 4A3-SC7 at a molar percentage between 13% and 15%, between 13.5% and 15%, between 14% and 15%, between 14.5 and 15%, between 13% and 14.5%, between 13.5% and 14.5%, between 14% and 14.5%, between 13% and 14%, between 13.5% and 14% or between 13% and 13.5%.
  • the LNP comprises PEG-lipid at a molar percentage between 2% and 8%, between 4% and 8%, between 6% and 8%, between 2% and 6%, between 4% and 6%, between 2% and 4%, between 2% and 3%, between 3% and 4%, between 2.5% and 3.5%, between 2.5% and 3% or between 3% and 3.5%.
  • the PEG-lipid at a molar percentage of (about) 3%.
  • the LNP comprises a neutral phospholipid and the neutral phospholipid is DOPE.
  • the LNP comprises DOPE at a molar percentage between 10% and 25%, between 10% and 20%, between 10% and 15%, between 10% and 12.5%, between 15% and 25, between 15% and 20% or between 20% and 25%. In some embodiments, the LNP comprises DOPE at a molar percentage of 11% or 22%.
  • the LNP comprises a poly nucleotide.
  • the polynucleotide is a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the mRNA comprises between 100 bases and 8 kilobases (kb).
  • the mRNA comprises between 1 Ikb and 8 kb, between 2 kb and 8 kb, between 3 kb and 8 kb, between 4 kb and 8 kb, between 5 kb and 8 kb, between 6 kb and 8 kb, between 7 kb and 8 kb, between 1 kb and 7 kb, between 2 kb and 7 kb, between 3 kb and 7 kb, between 4 kb and 7 kb, between 5 kb and 7 kb, or between 6 kb and 7 kb, between 1 kb and 6 kb, between 2 kb and 6 kb, between 3 kb and 6 kb, between 4 kb and 6 kb, or between 5 kb and 6 kb.
  • the mRNA comprises (about) 2 kb. In some embodiments, the mRNA comprises (about) 4.6 kb. In some embodiments, the mRNA encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In some embodiments, the nucleic acid sequence of the mRNA is set forth in SEQ ID NOG. In some embodiments, the mRNA encodes a Cre recombinase. In some embodiments, the mRNA encodes a CRISPR-Cas protein. In some embodiments, the CRISPR-Cas protein is Cas9, or a variant thereof.
  • CRISPR-Cas protein is Cas9, or a variant thereof.
  • the CRISPR-Cas protein is Casl2, or a variant thereof.
  • the mRNA encodes a base editor.
  • the mRNA encodes an adenine base editor (ABE).
  • the mRNA encodes a cytosine base editor (CBE).
  • the composition is a pharmaceutical composition. In some embodiments, the composition is an aerosolized composition. In some embodiments, the LNP composition has an encapsulation efficiency of between 50% and 99%, between 60% and 99%, between 70% and 99%, between 80% and 99%, between 90% and 99%, between 95% and 99%, between 50% and 95%, between 60% and 95%, between 70% and 95%, between 80% and 95%, between 85% and 95%, or between 90% and 95%. [0301] In some embodiments, the LNP composition is substantively free of any anionic lipid, of any permanently cationic lipid, or of any anionic lipid and any permanently cationic lipid. In some embodiments, the LNP composition is substantively free of any ionizable cationic lipids.
  • the methods disclosed herein comprise the administration of a LNP, wherein the LNP encapsulates a gene editing system.
  • the LNP comprises a nucleic acid encoding a base editor.
  • the LNP comprises a nucleic acid encoding a nuclease.
  • the LNP comprises a nucleic acid encoding a CRIS PR-associated (Cas) polypeptide or a variant thereof.
  • the LNP comprises a guide RNA (gRNA).
  • CRISPR-Cas systems The discovery and engineering of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems for genome editing has substantially expanded and improved the editing capabilities in eukaryotic cells. Initially identified in bacteria and archaeal organisms, CRISPR-Cas systems are classified into two main groups based on the number of effector proteins involved in the cleavage of nucleic acids: class 1, which cleaves nucleic acids with multiprotein complexes and class 2, which uses single-protein effectors for cleavage (Koonin EV, Makarova KS, Zhang F. Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol.
  • Class 2 is further subdivided by the type of Cas protein, including the DNA-targeting type-II Cas9 and type-V Casl2, and the RNA-targeting type- VI Casl3; these systems are more widely used due to the technical advantages of using a single-protein effector domain.
  • Other important characteristics to consider when selecting an editing strategy include cell type, cellular environment, expression method of the CRISPR-Cas system, and method of delivery, as they can affect editing efficiency and frequency of undesired genomic editing events.
  • CRISPR-Cas class 2 systems are principally comprised of an endonuclease protein encoded by a set of CRIS PR-associated (cas) genes and a short RNA sequence called guide RNA (gRNA) that guides the protein.
  • gRNA guide RNA
  • CRISPR RNAs crRNAs
  • tracrRNAs trans-activating crRNAs
  • Engineered approaches can utilize a single gRNA (sgRNA).
  • sgRNA is a single RNA molecule that contains both the crRNA sequence fused to the tracrRNA sequence.
  • sgRNA can be synthetically generated or made in vitro or in vivo from a DNA template.
  • the DNA-targeting systems also require a protospacer-adjacent motif (PAM), a short-required sequence, to occur near the target DNA site.
  • PAM protospacer-adjacent motif
  • Type-II CRISPR- Cas9 derived from Streptococcus pyogenes is one of the most commonly used types, and its main components are RNA-guided Cas9 endonuclease and a sgRNA.
  • the PAM sequence is located 3’ of the protospacer on the DNA strand not complementary to the guide RNA (Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E.
  • sgRNA and Cas9 nuclease form a Cas9 ribonucleoprotein that can search, bind, and cleave the specific target sequence.
  • the endonuclease Once the target site is located, the endonuclease generates a double- stranded break that is followed by two self-repair mechanisms: the error-prone non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway. Editing of the nucleic acids occurs after the nuclease treatment.
  • NHEJ error-prone non-homologous end joining
  • HDR homology-directed repair
  • NHEJ can introduce insertions or deletions (indels), generating frameshift mutations or premature stop codons that inactivate the target gene.
  • the HDR pathway can introduce precise genomic modifications but requires a homologous DNA repair template and is typically less efficient than NHEJ.
  • CRISPR-Cas tools In addition to nucleases that cleave target sequences, there are three other classes of CRISPR-Cas tools: base editors, transposases, and prime editors. These four classes can mediate different types of genomic edits, including conversion, deletion, or insertion of nucleic acids. These tools can use a nuclease dead Cas or deactivated Cas (dCas) system. The nuclease domains are mutated to abolish cleavage activity and obtain a dCas, such as for Cas9, where two point mutations are introduced to attain dCas9 (Xu, Y., & Li, Z. (2020).
  • CRISPR-Cas systems Overview, innovations and applications in human disease research and gene therapy. Computational and Structural Biotechnology Journal., 18, 2401- -15, which is incorporated by reference herein in its entirety). Importantly, the DNA binding activity of dCas9 is not affected. Fusing dCas systems with other effector domains can further extend CRISPR-Cas applications as catalytically inactive Cas nucleases are useful programmable proteins that localize the fused proteins to the target regions.
  • CRISPR-dCas9 system fused to transcriptional activators (CRISPRa) or repressors (CRISPRi) can be used to activate or inhibit the transcription of target genes, respectively (Xu, Y., & Li, Z, (2020).
  • CRISPR-Cas systems Overview, innovations and applications in human disease research and gene therapy. Computational and Structural Biotechnology Journal., 18, 2401—15, which is incorporated by reference herein in its entirety).
  • the development of the prime editing system a versatile fusion between Cas9, a reverse transcriptase, and a prime editing gRNA (pegRNA), can mediate insertions, deletions, and all 12 types of base substitutions without double-strand breaks or donor templates (Anzalone, A.V., Randolph, P.B., Davis, J.R. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157 (2019), which is incorporated by reference herein in its entirety).
  • the methods provided herein comprises the administration of an LNP, wherein the LNP comprises a nucleic acid encoding a base editor.
  • Base editors comprise fusions between impaired Cas enzymes which are unable to create double stranded breaks (DSBs), and a base-modification enzyme that modifies single-stranded nucleic acids only.
  • Base editing can precisely convert one nucleic acid or base pair into another in genomic DNA or cellular RNA without double-strand breaks, DNA repair templates, or relying on repair mechanisms.
  • CBEs cytosine base editors
  • ABEs adenine base editors
  • CBGEs cytosine to guanine base editors
  • the base editor is a cytosine base editor.
  • the first generation of CBEs were made up of a cytidine deaminase enzyme, such as an Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 1 (APOBEC1), fused to the amino terminus of a catalytically impaired Cas, which can only edit single- stranded DNA.
  • the Cas protein can either be a catalytically inactive dCas, or a partially inactive Cas nickase (nCas), which includes mutations that only allow the enzyme to nick the non-edited strand (Porto, E.M., Komor, A.C., et al.
  • Improved versions also include uracil glycosylase inhibitor (UGI) in the CBE fusion complex to improve editing efficiency.
  • UGI inhibits uracil DNA glycosylase (UNG), an enzyme which eliminates uracil bases through the base-excision repair (BER) pathway.
  • the CBE base editing process begins with sgRNA directing the Cas protein to the target locus.
  • Cas binding to the target denatures the DNA duplex to generate a ssDNA R-loop formation that exposes a region of DNA with target cytosines that the cytidine deaminase enzyme can deaminate.
  • CBEs convert a C-G base pair to a T-A base pair by deaminating the target cytosine to generate uracil, which will be read as a thymine by polymerases (Porto, E.M., Komor, A.C., et al. Base editing: advances and therapeutic opportunities.
  • ABEs are highly relevant in the context of correcting disease-causing mutations.
  • ABEs contain a catalytically impaired Cas protein, either a dCas, with no endonuclease activity, or nCas, which yield single- stranded breaks, fused to a DNA modifying enzyme, Escherichia coli tRNA adenosine deaminase (ecTadA).
  • TadA As ssDNA adenosine deaminase enzymes are not naturally occurring, TadA required extensive engineering and development through the directed mutagenesis to produce the first generation of ABE. Similar to CBEs, sgRNA guides the Cas domain to the intended target locus, which exposes a stretch of ssDNA in an R-loop for editing. TadA deaminates an adenine’s exocyclic amine to yield inosine, which is read as guanine by polymerases, converting A-T base pairs to G-C base pairs (Porto, E.M., Komor, A.C., et al. Base editing: advances and therapeutic opportunities. Nat Rev Drug Discov 19, 839 -859 (2020), which is incorporated by reference herein in its entirety).
  • ABEs are more restricted. Optimization and protein engineering of ABEs have been performed to improve editing efficiency and expand the targeting range.
  • ABE7.10 a previous version of ABE, ABE7.10, was compatible with limited Cas9 enzymes and exhibited lower DNA editing efficiency than CBEs.
  • the adenosine deaminase enzyme of ABE7.10, TadA-7.10 was evolved to include 8 additional mutations, yielding TadA-8e, which allowed for greater compatibility with more Cas9 and Cas 12a homologs, higher deamination rates, and overall improved DNA editing efficiency.
  • the base editor variant ABE8e contains an ecTadA-8e fused to a Streptococcus pyogenes Cas9 nickase (SpCas9n).
  • ABE8e has a broader base-editing window than ABE7.10, resulting in an editing window that is on par with that of CBEs (Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol. 2020 Jul;38(7):883-891, which is incorporated by reference herein in its entirety).
  • CRISPR-Cas systems can be used in a number of disease-relevant contexts.
  • the generation of animal and cell models of human disease has been facilitated by CRISPR-Cas systems as it allows for the generation of knockout, knock-in, and mutagenesis models.
  • the high sensitivity and single-base specificity also allow CRISPR-Cas systems to be used in the molecular diagnosis of disease by screening for susceptibility genes and pathogenic genes.
  • CRISPR-mediated genome-editing therapies are increasingly relevant in treating specific conditions caused by mutated or defective genes, including monogenic diseases caused by mutations of a single allele or a pair of alleles on homologous chromosomes.
  • the present disclosure provides a method comprising administering a lipid nanoparticle (LNP) that comprises a gene editing system.
  • LNP lipid nanoparticle
  • the term “gene editing system” refers to a DNA or RNA editing system that comprises an enzyme element that can bind to DNA or RNA.
  • the enzyme element can comprise an enzyme with nuclease activity, including but not limited to endonuclease activity, or a nucleic acid encoding such an enzyme.
  • the enzyme element can comprise an enzyme with recombinase activity, or a nucleic acid encoding such an enzyme.
  • the gene editing system can further comprise a guide RNA (gRNA) element that comprises a RNA molecule comprising a nucleotide sequence substantially complementary to a nucleotide sequence at one or more target genomic regions.
  • gRNA guide RNA
  • the enzyme element can comprise an enzyme that is guided or brought to a target genomic region(s) by a guide RNA element, or a nucleic acid encoding such an enzyme.
  • the enzyme element can be naturally occurring.
  • the enzyme element can comprise a fusion protein.
  • the gene editing system comprises a CRISPR-Cas enzyme, or a variant thereof, and a guide RNA (gRNA).
  • the gene editing system comprises a fusion enzyme.
  • the fusion enzyme is a fusion of a Cas enzyme, or a variant thereof, with another enzyme, such as a recombinase, a polymerase, a deaminase, a reverse transcriptase, or another enzyme that binds to nucleic acids.
  • the fusion enzyme is a fusion between a catalytically impaired Cas enzyme capable of binding to a specific nucleotide sequence and a base-modifying enzyme.
  • the base-modifying enzyme is an adenosine deaminase. In another embodiment, the base-modifying enzyme is a cytidine deaminase.
  • a base editor comprises a base-modifying enzyme, such as an adenosine deaminase or a cytidine deaminase, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • the gene editing system comprises a base editor and a guide RNA (gRNA).
  • the base editor is an adenine base editor (ABE).
  • the base editor is a cytosine base editor (CBE).
  • An ABE comprises a tRNA adenosine deaminase (TadA) protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • a CBE comprises a cytidine deaminase protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • the catalytically impaired Cas protein is a dead or deactivated Cas9 (dCas), In some embodiments, the catalytically impaired Cas is a Cas9 nickase (nCas). In some embodiments, a dCas has no endonuclease activity. In other embodiments, a nCas creates single-stranded breaks in a nucleic acid.
  • dCas dead or deactivated Cas9
  • nCas Cas9 nickase
  • a dCas has no endonuclease activity.
  • a nCas creates single-stranded breaks in a nucleic acid.
  • the methods described herein include a gene editing system, a composition comprising a gene editing system, or a gene editing system assembled with the lipid composition.
  • the lipid composition comprises one or more polypeptides.
  • Some polypeptides may include endonucleases such as any one of the nuclease enzymes described herein.
  • the nuclease enzyme may include from CRIS PR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFN); transcription activator- like effector nucleases (TALEN); meganucleases; RNA-binding proteins (RBP); CRISPR- associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute (
  • the gene editing system comprises a zinc finger nuclease (ZFN).
  • ZFN zinc finger nuclease
  • a zine-finger nuclease (ZFN) comprises a zinc-finger DNA binding domain fused to a DNA cleavage domain.
  • fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.
  • the cleavage domain is from the Type IIS restriction endonuclease FokI, which generally catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other.
  • FokI Type IIS restriction endonuclease FokI
  • Some gene-specific engineered zinc fingers are available commercially.
  • a platform called CompoZr for zinc-finger construction is available that provides specifically targeted zinc fingers for thousands of targets. See, e.g,, Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405. In some cases, commercially available zinc fingers are used or are custom designed.
  • the gene editing system comprises a Transcription Activator like Effector (TALE).
  • TALE proteins are from the bacterial species Xanthomonas and comprise a plurality of repeated sequences, each repeat comprising di-residues in position 12 and 13 (RVD) that are specific to each nucleotide base of the nucleic acid targeted sequence. Binding domains with similar modular base-per-base nucleic acid binding properties (MBBBD) can also be derived from different bacterial species.
  • a “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains/units.
  • the repeat domains are involved in binding of the TALE to its cognate target DNA sequence.
  • a single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein.
  • TALE proteins may be designed to bind to a target site using canonical or non-canonical RVDs within the repeat units. See, e.g., U.S. Pat. Nos. 8,586,526 and 9,458,205.
  • the gene editing system comprises a TAL-effector nuclease (TALEN).
  • TALEN TAL-effector nuclease
  • TALEN is a fusion protein comprising a nucleic acid binding domain typically derived from a Transcription Activator Like Effector (TALE) and a nuclease catalytic domain that cleaves a nucleic acid target sequence.
  • the catalytic domain comprises a nuclease domain or a domain having endonuclease activity, like for instance I-TevI, ColE7, NucA and Fok-I.
  • the TALE domain can be fused to a meganuclease like for instance I-Crel and I-Onul or functional variant thereof.
  • the TALEN is a monomeric TALEN.
  • a monomeric TALEN is a TALEN that does not require dimerization for specific recognition and cleavage, such as the fusions of engineered TAL repeats with the catalytic domain of I-TevI described in WO2012138927.
  • TALENs have been described and used for gene targeting and gene modifications (see, e.g., Boch et al.
  • Cas protein or “CRISPR-Cas protein” refers to a full- length Cas protein obtained from nature, a recombinant Cas protein having a sequence that differs from a naturally occurring Cas protein, or any fragment of a Cas protein that nevertheless retains all or a significant amount of the requisite basic functions needed for the disclosed methods, i.e., (i) possession of nucleic-acid binding of the Cas protein to a target DNA, (ii) ability to create double-strand breaks in the target DNA sequence; and/or (iii) ability to nick the target DNA sequence on one strand.
  • the Cas proteins contemplated herein comprise CRISPR Cas9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9 nickase (nCas9) or nuclease inactive Cas9 (dCas9) homologs, orthologs, or paralogs, whether naturally occurring or non- naturally occurring (e.g., engineered or recombinant), and may include a Cas9 equivalent from any type of CRISPR system (e.g., type II, V, VI), including Cpfl (a type-V CRISPR-Cas systems), C2cl (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type V CRISPR-Cas system).
  • CRISPR Cas9 proteins as well as Cas9 equivalents, variants (e.g., Cas9 nickase (nCas9) or nuclease inactive Cas9 (d
  • C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector
  • Cas9 or “Cas9 domain” comprises any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally-occurring or engineered.
  • Cas9 is not meant to be particularly limiting and may be referred to as a “Cas9 or equivalent.” Additional Cas9 sequences and structures are well known to those of skill in the art (see, e.g., "Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al., J.J., McShan W.M., Ajdic D .J., Savic DJ., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q readily Zhu H respect Song L prefer White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc.
  • Examples of Cas9 and Cas9 equivalents are provided as follows; however, these specific examples are not meant to be limiting.
  • the base editors of the present disclosure may use any suitable CRISPR-Cas domain, including any suitable Cas9 or Cas9 equivalent.
  • the disclosure provides base editors comprising one or more adenosine deaminase variants disclosed herein and a CRISPR-Cas protein.
  • the CRISPR-Cas protein comprises a Cas homolog.
  • the CRISPR-Cas protein may be selected from any CRISPR associated protein, including but not limited to a Cas9, a Cas9n, a dCas9, a CasX, a CasY, a C2cl, a C2c2, a C2c3, a GeoCas9, a CjCas9, a Cas 12a, a Casl2b, a Casl2g, a Casl2h, a Casl2i, a Casl3b, a Casl3c, a Casl3d, a Cas14, a C 8 n2, an xCas9, an SpCas9-NG, an SpCas9-NG-CP1041, an SpCas9-NG-VRQR, an LbCasl2a, an AsCasl2a, a Cas9-KKH, a circularly permuted Cas9, an
  • the CRISPR-Cas protein comprises or is a Cas9 protein or a Cas 12a protein derived from .S-. pyogenes or .S-. aureus.
  • the CRISPR-Cas protein comprises a nuclease dead Cas9 (dCas9) protein, a Cas9 nickase (nCas9) protein, or a nuclease active Cas9 protein.
  • the Cas protein may be complexed with a guide polynucleotide.
  • Exemplary CRISPR-Cas proteins include but are not limited to .S-, pyogenes Cas9 nickase (SpCas9n) and .S-. aureus Cas9 nickase (SaCas9n). Additional exemplary CRISPR-Cas proteins include .S-.
  • the CRISPR-Cas protein comprises a Cas9 nickase (nCas9) protein. In some embodiments, the CRISPR-Cas protein comprises an SpCas9n protein.
  • the CRISPR-Cas protein of any of the disclosed base editors is a SaCas9n. In certain embodiments, the CRISPR-Cas protein of any of the disclosed base editors is an SpCas9-NRCH. In certain embodiments, the CRISPR-Cas protein of any of the disclosed base editors is an LbCasl2a, e.g., a catalytically inactive or "dead" LbCasl2a. In certain embodiments, the CRISPR-Cas protein of any of the disclosed base editors is an AsCasl2a, e.g., an enAsCasl2a.
  • the nuclease in the compositions described herein may be Cas9 (e.g., from .S-. pyogenes or .S-. pneumonia).
  • the CRISPR-Cas protein can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence of any one of the genes described herein.
  • the CRISPR enzyme may be directed and cleaved a genomic locus of CFTR.
  • the CRISPR-Cas protein may be mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR-Cas protein lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • an aspartate-to-alanine substitution (D10A) in the RuvC catalytic domain of Cas9 from .S-, pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
  • a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target.
  • guide sequence(s) e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target.
  • This combination allows both strands to be nicked and used to induce non-homologous end-joining (NHEJ) or homology directed repair (HDR).
  • NHEJ non-homologous end-joining
  • HDR homology directed repair
  • Adenine base editors can deaminate an adenosine that leads to a point mutation from adenine (A) to guanine (G).
  • the adenine base editors can comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein including any naturally occurring variant, mutant, or otherwise engineered version of Cas9 that is known or which can be made or evolved through a directed evolutionary or otherwise mutagenesis process.
  • the CRISPR-Cas protein has nickase activity, i.e., can only cleave one strand of the target DNA sequence.
  • the CRISPR-Cas protein has an inactive nuclease, e.g., are “dead” or deactivated proteins.
  • Other variant Cas9 proteins that may be used are those having a smaller molecular weight than the canonical SpCas9 (e.g., for easier delivery) or having modified or rearranged primary amino acid sequence (e.g., the circular permutant forms).
  • the adenine base editors described herein can also comprise Cas9 equivalents, including Cas 12a/Cpfl and Cas 12b proteins.
  • the CRISPR-Cas proteins used herein can also contain various modifications that alter/enhance their PAM specificities.
  • the disclosure contemplates any Cas9, Cas9 variant, or Cas9 equivalent which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%', at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as a reference SpCas9 canonical sequence , a reference SaCas9 canonical sequence or a reference Cas9 equivalent (e.g., Casl2a/Cpfl).
  • a reference Cas9 sequence such as a reference SpCas9 canonical sequence , a reference SaCas9 canonical sequence or a reference Cas9 equivalent (e.g., Casl2a/Cpfl).
  • the adenine base editors contemplated herein can include a Cas9 protein that is of smaller molecular weight than the canonical SpCas9 sequence.
  • the smaller-sized Cas9 variants may facilitate delivery to cells, e.g., by an expression vector, nanoparticle, or other means of delivery.
  • the canonical SpCas9 protein is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons.
  • Smaller- sized Cas9 variants can be at least 1300 amino acids, or at least less than 1290 amino acids, or than less than 1280 amino acids, or less than 1270 amino acid, or less than 1260 amino acid, or less than 1250 amino acids, or less than 1240 amino acids, or less than 1230 amino acids, or less than 1220 amino acids, or less than 1210 amino acids, or less than 1200 amino acids, or less than 1190 amino acid, or less than 1180 amino acids, or less than 1170 amino acids, or less than 1160 amino acids, or less than 1150 amino acids, or less than 1140 amino acids, or less than 1130 amino acids, or less than 1120 amino acids, or less than 1110 amino acids, or less than 1100 amino acids, or less than 1050 amino acids, or less than 1000 amino acids, or less than 950 amino acids, or less than 900 amino acids, or less than 850 amino acids, or less than 800 amino acids, or less than 750 amino acids, or less than 700 amino acids, or less than 650 amino acids, or less than
  • the base editors may comprise the “canonical SpCas9” nuclease from .S-. pyogenes, which has been widely used as a tool for genome engineering.
  • This Cas9 protein is a large, multi-domain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish one or both nuclease activities, resulting in a nickase Cas9 (nCas9) or dead Cas9 (dCas9), respectively, that still retains its ability to bind DNA in a sgRNA programmed manner.
  • Cas9 or variant thereof can target that protein to virtually any DNA sequence simply by co-expression with an appropriate sgRNA.
  • the canonical SpCas9 protein refers to the wild type protein from .S-. pyogenes having the following amino acid sequence.
  • the base editors described herein may include canonical SpCas9, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with a wild type Cas9 sequence provided above.
  • the adenine base editors described herein may include any of the above SpCas9 sequences, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
  • the Cas9 protein can be a wild type Cas9 ortholog from another bacterial species.
  • the Cas9 protein is an ortholog comprising a sequence of at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the below orthologs.
  • the adenine base editor may include any of the above Cas9 ortholog sequences, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
  • the CRISPR-Cas domain may include any suitable homologs and/or orthologs or naturally occurring enzymes, such as Cas9.
  • Cas9 homologs and/or orthologs have been described in various species, including, but not limited to, .S-. pyogenes and .S-, thermophilus.
  • the Cas moiety is configured (e.g., mutagenized, recombinantly engineered, or otherwise obtained from nature) as a nickase.
  • Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.
  • a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase.
  • the disclosed base editors may comprise a catalytically inactive, deactivated, or “dead,” CRISPR-Cas domain.
  • Exemplary catalytically inactive domains in the disclosed adenine base editors are dead .S-. pyogenes Cas9 (dSpCas9), dead .S-. aureus Cas9 (dSaCas9) and dead Lachnospiraceae bacterium Casl2a (dLbCasl2a).
  • the base editors described herein may include a dead Cas9, e.g.. dead SpCas9, which has no nuclease activity due to one or more mutations that inactivate both nuclease domains of SpCas9, namely the RuvC domain (which cleaves the nonprotospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand).
  • the nuclease inactivation may be due to one or mutations that result in one or more substitutions and/or deletions in the amino acid sequence of the encoded protein, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
  • the D10A and N580A mutations in the wild-type .S-. aureus Cas9 amino acid sequence may be used to form a dSaCas9.
  • the CRISPR-Cas domain of the base editors provided herein comprises a dSaCas9 that has D10A and N580A mutations relative to the wild-type SaCas9 sequence.
  • dCas9 refers to a nuclease-inactive Cas9 or nuclease-dead Cas9.
  • the term dCas9 is not meant to be particularly limiting and may be referred to as a “dCas9 or equivalent.” Any suitable mutation which inactivates both Cas9 endonucleases may be used to form the dCas9.
  • dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity.
  • Cas9 variants having mutations may result in the full or partial inactivation of the endogenous Cas9 nuclease activity (e.g., dCas9 or nCas9, respectively).
  • variants or homologues of Cas9 are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the sequence of wild type Cas9 from Streptococcus pyogenes.
  • the CRISPR-Cas protein of any of the disclosed base editors comprises a dead .S-. pyogenes Cas9 (dSpCas9).
  • the CRISPR-Cas protein of any of the disclosed base editors comprises a dead Lachnospiraceae bacterium Casl2a (dLbCasl2a).
  • the disclosed base editors may comprise a CRISPR-Cas protein that comprises a nickase.
  • the base editors described herein comprise a Cas9 nickase.
  • Cas9 nickase refers to a variant of Cas9 which is capable of introducing a single-strand break in a double strand DNA molecule target.
  • the Cas9 nickase comprises only a single functioning nuclease domain.
  • the wild type Cas9 e.g., the canonical SpCas9
  • the wild type Cas9 comprises two separate nuclease domains, namely, the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand).
  • the Cas9 nickase comprises a mutation in the RuvC domain which inactivates the RuvC nuclease activity.
  • the catalytically impaired Cas9 protein can be, but is not limited to NRRH, NRTH, NRCH, xCas9, SpCas9-NG, SpCas9, SpG, SpRY, SauriCas9, SaCas9, Nme2Cas9, VRER- SpCas9, and VQRSpCas9.
  • the catalytically impaired Cas9 protein is SpCas9-NG.
  • the CRISPR-Cas protein of any of the disclosed base editors comprises an 5. pyogenes Cas9 nickase (SpCas9n). In some embodiments, the CRISPR-Cas protein of any of the disclosed base editors comprises an 5. aureus Cas9 nickase (SaCas9n).
  • the CRISPR-Cas proteins used in the base editors described herein may also include other Cas9 variants that area at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art.
  • any reference Cas9 protein including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art.
  • the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9.
  • a reference Cas9 e.g., a gRNA binding domain or a DNA-cleavage domain
  • the fragment is 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% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.
  • the disclosure also may utilize Cas9 fragments which retain their functionality and which are fragments of any herein disclosed Cas9 protein.
  • the Cas9 fragment is at least 100 amino acids in length.
  • the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.
  • the base editors disclosed herein may comprise one of the Cas9 variants described as follows, or a Cas9 variant thereof having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 variants.
  • the base editors described herein can include any Cas9 equivalent.
  • Cas9 equivalent is a broad term that encompasses any CRISPR-Cas protein that serves the same function as Cas9 in the present adenine base editors despite that its amino acid primary sequence and/or its three-dimensional structure may be different and/or unrelated from an evolutionary standpoint.
  • Cas9 equivalents include any Cas9 ortholog, homolog, mutant, or variant described or embraced herein that are evolutionarily related
  • the Cas9 equivalents also embrace proteins that may have evolved through convergent evolution processes to have the same or similar function as Cas9, but which do not necessarily have any similarity with regard to amino acid sequence and/or three dimensional structure.
  • the adenine base editors described here embrace any Cas9 equivalent that would provide the same or similar function as Cas9 despite that the Cas9 equivalent may be based on a protein that arose through convergent evolution.
  • Cas9 equivalents contemplated herein may also be obtained from archaea, which constitute a domain and kingdom of single-celled prokaryotic microbes different from bacteria.
  • the gene editing system comprises a base editor and a guide RNA (gRNA ).
  • the base editor is an adenine base editor (ABE).
  • the base editors is a cytosine base editor (CBE).
  • An ABE comprises a tRNA adenosine deaminase (TadA) protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • a CBE comprises a cytidine deaminase protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • the present disclosure provides base editors having adenosine deaminase domains that are mutated (e.g. evolved to have mutations) that enable the deaminase domain to have improved activity when used with Cas homologs (e.g., homologs other than SpCas9). Accordingly, the present disclosure provides variants of adenosine deaminases (e.g., variants of TadA-7.10).
  • TadA-8e which contains eight additional mutations relative to the TadA- 7.10 deaminase domain (where TadA-7.10 contains the mutations W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N in the ecTadA sequence).
  • TadA-8e is broadly compatible with diverse Cas9 or Casl2 homologs, and exhibits improved editing efficiencies when paired with previously incompatible Cas9 or Cas 12 homologs.
  • adenosine deaminase variants such as TadA-8e exhibit higher editing efficiencies when paired in a base editor with certain Cas9 variants, such as circularly permuted variants CP1041 and CP1028, than exhibited by the TadA-7.10 deaminase.
  • the adenosine deaminase is TadA-7.10.
  • the adenosine deaminase is TadA-8e.
  • the base editor is ABE 0.1, ABE 0.2, ABE 1.1, ABE 1.2, ABE
  • the base editor is an ABE8 variant. In some embodiments, the base editor is ABE8e.
  • Exemplary ABEs include, but are not limited to, ABE7.10 (or ABEmax), ABE8e, SaKKH-ABE8e, NG-ABE8e, ABE-xCas9, SaKKH-ABE7.10, NG-ABE7.10, ABE7.10-VRQR, ABE8e-NRTH, ABE8e-NRRH, ABE8e-NRCH, NG-CP1041-ABE8e, ABE8eCP1041, ABE8e- CP 1028, and ABE8e-VRQR.
  • the ABE used in the disclosed methods is an ABE8e or an ABE7.10.
  • ABE8e may be referred to herein as “ABE8” or “ABE8.0.”
  • the ABE8e base editor and variants thereof may comprise an adenosine deaminase domain containing a TadA-8e adenosine deaminase monomer (monomer form) or a TadA-8e adenosine deaminase homodimer or heterodimer (dimer form).
  • ABE8e is further described in Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR.
  • any of the disclosed base editors are capable of deaminating adenosine in a nucleic acid sequence (e.g., DNA or RNA).
  • the adenosine deaminases of the base editors hydrolytically deaminate a targeted adenosine in a nucleic acid of interest to an inosine, which is read as a guanosine (G) by DNA polymerase enzymes.
  • the base editor is an ABE.
  • the base editor is ABE8e.
  • the base editor is a CBE.
  • the gene editing system comprises a ABE and a guide RNA (gRNA).
  • the gene editing system comprises a CBE and a guide RNA (gRNA).
  • any of the adenosine deaminases provided herein are capable of deaminating adenine, e.g., deaminating adenine in a deoxyadenosine residue of DNA.
  • the adenosine deaminase may be derived from any suitable organism (e.g., E. coli).
  • the adenosine deaminase is a naturally -occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA).
  • the adenosine deaminase is derived from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the disclosed adenosine deaminases are variants of a TadA derived from a species other than Escherichia coli, such as Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Elaemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli.
  • the base editor includes mutations that confer reduced off- target effects, such as reduced RNA editing activity and off-target DNA editing activity, on the adenine base editor.
  • the disclosure provides an ABE that has one or more amino acid variations introduced into the amino acid sequence of the adenosine deaminase domain relative to the amino acid sequence of the reference adenosine deaminase domain.
  • the ABE may include variants in one or more components or domains of the base editor (e.g., variations introduced into the adenosine deaminase domain, or variations introduced into both the adenine deaminase domain and the CRISPR-Cas domain).
  • the disclosed adenosine deaminase variants may be at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a reference adenosine deaminase domain.
  • the adenosine deaminase domain of any of the disclosed base editors comprises a single adenosine deaminase, or a monomer. In some embodiments, the adenosine deaminase domain comprises 2, 3, 4 or 5 adenosine deaminases. In some embodiments, the adenosine deaminase domain comprises two adenosine deaminases, or a dimer. In some embodiments, the deaminase domain comprises a dimer of an engineered (or evolved) deaminase and a wild-type deaminase, such as a wild-type E.
  • Patent Publication No. 2015/0166980 published June 18, 2015; U.S. Patent No. 9,840,699, issued December 12, 2017; and U.S. Patent No. 10,077,453, issued September 18, 2018, and International Patent Application No. PCT/US2020/28568, filed April 16, 2020: all of which are incorporated herein by reference in their entireties.
  • Exemplary ABEs of this disclosure comprise the monomer and dimer versions of the following editors: ABE8e, SaABE8e, SaKKH-ABE8e, NG-ABE8e, ABE-xCas9, ABE8e- NRTH, ABE8e-NRRH, ABE8e-NRCH, ABE8e-NG-CP1041, ABE8e-VRQR-CP1041, ABE8e- CP1041, ABE8e-CP1028, ABE8e-VRQR.
  • ABE8e-LbCasl2a (LbABE8e), ABE8eAsCasl2a (enAsABE8e), ABE8e-SpyMac, ABE8e (TadA-8e V 106W), ABE8e (K20A, R 2 1A), and ABE8e (TadA-8e V82G).
  • the monomer version refers to an editor having an adenosine deaminase domain that comprises a TadA-8e and does not comprise a second adenosine deaminase enzyme.
  • the dimer version refers to an editor having an adenosine deaminase domain that comprises a first and second adenosine deaminase, i.e., a wild-type TadA enzyme and a TadA-8e enzyme.
  • the ABE is a ABE8e.
  • any two or more of the adenosine deaminases described herein may be connected to one another (e.g., by a linker, such as a peptide linker) within an adenosine deaminase domain of the base editors provided herein.
  • the base editor comprises two adenosine deaminases (e.g., a first adenosine deaminase and a second adenosine deaminase).
  • the base editors provided herein may contain exactly two adenosine deaminases.
  • the first and second adenosine deaminases are any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminases are the same. In some embodiments, the adenosine deaminases are different. In some embodiments, the first adenosine deaminase and second adenosine deaminase are derived from the same bacterial species. In some embodiments, the first adenosine deaminase and second adenosine deaminase are derived from different bacterial species.
  • the base editor comprises a heterodimer of a first adenosine deaminase and a second adenosine deaminase.
  • the first adenosine deaminase is N-terminal to the second adenosine deaminase in the base editor.
  • the first adenosine deaminase is C-terminal to the second adenosine deaminase in the base editor.
  • the first adenosine deaminase and the second deaminase are fused directly to each other or via a linker.
  • the first adenosine deaminase is fused N-terminal to the CRISPR-Cas protein via a linker
  • the second deaminase is fused C-terminal to the CRISPR-Cas protein via a linker
  • the second adenosine deaminase is fused N-terminal to the CRISPR-Cas protein via a linker
  • the first deaminase is fused C-terminal to the CRISPR-Cas protein via a linker.
  • the base editors described herein may comprise one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the base editor components).
  • a base editor may comprise any additional protein sequence, and optionally a linker sequence between any two domains.
  • Other exemplary features that may be present are localization sequences, such as cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags.
  • linkers may be used to link any of the peptides or peptide domains or domains of the base editor (e.g., a CRISPR-Cas protein covalently linked to an adenosine deaminase domain).
  • the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100- 110, 110- 120, 120-130, 130-140, 140-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the linker is 32 amino acids in length.
  • the linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length.
  • the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like.
  • the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.).
  • the linker is a carbon-nitrogen bond of an amide linkage.
  • the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker.
  • the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3- aminopropanoic acid, 4- aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx).
  • Ahx aminohexanoic acid
  • the linker is based on a carbocyclic moiety (e.g., cyclopentane, cydohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring.
  • the linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.
  • the present disclosure provides compositions comprising the ABE as described herein and one or more guide RNAs, e.g., a single-guide RNA (“sgRNA”).
  • sgRNA single-guide RNA
  • the present disclosure provides for nucleic acid molecules encoding and/or expressing the adenine base editors as described herein, as well as expression vectors or constructs for expressing the adenine base editors described herein and a gRNA, host cells comprising said nucleic acid molecules and expression vectors, and optionally one or more gRNAs, and compositions for delivering and/or administering nucleic acid-based embodiments described herein.
  • the LNP comprises a gene editing system, wherein the gene editing system comprises a gRNA and the mRNA of the base editor.
  • the gRNA and the mRNA of the base editor are present at a molar ratio that is 1 : 1.
  • the gRNA and the mRNA of the base editor are present at a molar ratio that is not 1:1.
  • the gRNA and the mRNA of the base editor are present at a molar or weight ratio less than 1:1.
  • the gRNA and the mRNA of the base editor are present at a molar or weight ratio of at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1 :10, 1:11, 1:12, 1:13, 1 :14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1 :29, or 1:30.
  • the gRNA and the mRNA of the base editor are present at a molar or weight ratio of at least about 1:30, 1:29, 1:28, 1 :27, 1:26, 1:25, 1:24, 1 :23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1 :4, 1 :3, 1:2, or 1 :1.
  • the gRNA and the mRNA of the base editor are present at a molar or weight ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1 :27, 1:28, 1:29, or 1:30, or a range between any two of the foregoing values.
  • the mRNA of the base editor and the gRNA are present at a molar or weight ratio that is not 1:1. In some embodiments, the mRNA of the base editor and the gRNA are present at a molar or weight ratio less than 1:1.
  • the mRNA of the base editor and the gRNA are present at a molar or weight ratio of at most about 1 :1, 1 :2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 :21 , 1:22, 1:23, 1:24, 1 :25, 1 :26, 1:27, 1:28, 1 :29, or 1 :30.
  • the mRNA of the base editor and the gRNA are present at a molar or weight ratio of at least about 1:30, 1:29, 1:28, 1:27, 1 :26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1 :9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1.
  • the mRNA of the base editor and the gRNA are present at a molar or weight ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1 :6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 :21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, or 1:30, or a range between any two of the foregoing values. 3. Guide RNA
  • the one or more polynucleotides comprise a guide RNA (gRNA).
  • gRNAs are designed to recognize target sequences in a gene or genome of interest. Such gRNAs may be designed to have guide sequences (or "spacers") having complementarity to a protospacer within the target sequence.
  • the gRNA is complexed with a recombinant nuclease capable of inducing a DNA break.
  • the gRNA is a single guide RNA (sgRNA).
  • the gRNA comprises a crispr RNA (crRNA) and a tracrRNA.
  • the gRNA is modified.
  • the gRNA is modified with an end modification (e.g. (3 x 2'-O-methyl-3'-phosphorothioate (MS) on 5’ end and 3 x 2'-O-methyl-3'- phosphonoacetat (MP) on 3’end (K. A. Hajj, K. A. Whitehead, Nat. Rev. Mater. 2, 17056 (2017)).
  • the gRNA can be heavily modified (T. Wei et al., ACS Nano 14, 9243-9262 (2020).
  • gRNAs can be used with one or more of the disclosed ABEs, e.g., in the disclosed methods of editing a nucleic acid molecule.
  • Such gRNAs may be designed to have guide sequences having complementarity to a protospacer within a target sequence to be edited, and to have backbone sequences that interact specifically with the CRISPR-Cas protein of any of the disclosed base editors, such as Cas9 nickase proteins of the disclosed base editors.
  • the guide sequence becomes associated or bound to the base editor and directs its localization to a specific target sequence having complementarity to the guide sequence or a portion thereof.
  • a guide sequence will depend upon the nucleotide sequence of a genomic target sequence (i.e., the desired site to be edited) and the type of CRISPR-Cas protein (e.g., type of Cas9 protein) present in the base editor, among other factors, such as PArvl sequence locations, percent G/C content in the target sequence, the degree of microhomology regions, and/or secondary structures.
  • a genomic target sequence i.e., the desired site to be edited
  • type of CRISPR-Cas protein e.g., type of Cas9 protein
  • the polynucleotide encodes a guide polynucleotide (such as guide RNA (gRNA) or guide DNA (gDNA)) that is at least partially complementary to the genomic region of a gene, where upon binding of the guide polynucleotide to the gene the guide polynucleotide recruits the guide polynucleotide guided CRISPR-Cas protein to cleave and genetically modified the region.
  • a CFTR gene can be modified by the guide polynucleotide-guided CRISPR-Cas protein.
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR-Cas protein (e.g., a Cas9 or Cas9 variant) to the target sequence.
  • the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP, and Maq (available at maq.sourceforge.net).
  • any suitable algorithm for aligning sequences non-limiting example of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP, and Maq (available at maq.sourceforge.net).
  • a guide sequence is above or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.
  • each gRNA comprises a guide sequence of at least 10 contiguous nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) that is complementary to a target sequence (or off target site).
  • a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length.
  • the ability of a guide sequence to direct sequence- specific binding of a base editor to a target sequence may be assessed by any suitable assay.
  • the components of a base editor, including the guide sequence to be tested may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of a base editor disclosed herein, followed by an assessment of preferential cleavage within the target sequence.
  • cleavage of a target polynucleotide sequence may be evaluated in situ by providing the target sequence, components of a base editor, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • Other assays are possible, and will occur to those skilled in the art.
  • the gRNA comprises a sequence that is complementary to a target sequence. In some embodiments, the gRNA comprises a sequence that is complementary to a target sequence of a cystic fibrosis transmembrane conductance regulator (CFTR) gene or transcript. In some embodiments, the gRNA comprises a sgRNA. In some embodiments, the sequence of the gRNA is shown in Table 9.
  • the gRNA comprises the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1.
  • the gRNA comprises the nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2.
  • a guide RNA comprising a sequence that is complementary to a target sequence (e.g. CFTR gene) and is used with RNA-guided nucleases, e.g., Cas, to induce a DNA break at the target site or target position.
  • a target sequence e.g. CFTR gene
  • RNA-guided nucleases e.g., Cas
  • the gRNA is a unimolecular or chimeric gRNA comprising, from 5’ to 3’: a targeting domain which targets a target site (e.g., a locus in the CFTR gene); a first complementarity domain; a linking domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and optionally, a tail domain.
  • a targeting domain which targets a target site (e.g., a locus in the CFTR gene); a first complementarity domain; a linking domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and optionally, a tail domain.
  • the gRNA is a modular gRNA comprising first and second strands.
  • the first strand preferably includes, from 5’ to 3’: a targeting domain (which targets a target site e.g., at CFTR gene); and a first complementarity domain.
  • the second strand generally includes, from 5’ to 3’: optionally, a 5’ extension domain; a second complementarity domain; a proximal domain; and optionally, a tail domain.
  • compositions comprising lipid nanoparticles (LNPs), such as LNPs comprising a gene editing system.
  • the composition comprises LNPs in any formulation such as those described in Section IID.
  • the method comprises administration of the LNPs comprising a gene editing system and/or compositions containing said LNPs to a subject for the treatment of disease or condition.
  • the LNPs or compositions comprising said LNPs are administered to a subject, such as a subject with a disease or condition, or to prevent or reduce the severity of a disease or condition.
  • the method comprises administration of the LNPs comprising a gene editing system and/or compositions containing said LNPs to a subject for the treatment of cystic fibrosis.
  • the composition is a pharmaceutical composition.
  • the composition comprises a pharmaceutically acceptable excipient.
  • the nanoparticle is administered (e.g., in a lipoplex particle or liposome) intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intracranial, intrathoracic, or subcutaneous administration. Dosing and administration may depend in part on whether the administration is brief or chronic. Various dosing schedules include but are not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion.
  • the LNPs, or compositions comprising the same are administered intravenously.
  • the LNPs or compositions comprising the same are administered by way of aerosolized delivery.
  • the LNPs are formulated with a pharmaceutically acceptable carrier.
  • the choice of carrier is determined in part by the particular cell or agent and/or by the method of administration. Accordingly, there are a variety of suitable formulations.
  • the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A.
  • the formulations can include aqueous solutions.
  • the formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells or agents, where the respective activities do not adversely affect one another.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the pharmaceutical composition further includes other pharmaceutically active agents or drugs.
  • the pharmaceutical composition comprises the LNP comprising a gene editing system in amounts effective to treat the disease or condition, such as a therapeutically effective or prophylactic ally effective amount.
  • Therapeutic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful and can be determined.
  • the desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
  • the LNP comprising a gene editing system may be administered using standard administration techniques, formulations, and/or devices.
  • formulations and devices such as syringes and vials, for storage and administration of the compositions.
  • a therapeutic composition e.g., a pharmaceutical composition containing a LNP
  • it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
  • pharmaceutical compositions are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH.
  • Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues.
  • Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
  • Sterile injectable solutions can be prepared by incorporating the LNPs in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
  • a suitable carrier such as a suitable carrier, diluent, or excipient
  • sterile water such as physiological saline, glucose, dextrose, or the like.
  • physiological saline such as glucose, dextrose, or the like.
  • the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • compositions comprising the LNPs described herein.
  • Such compositions can be used for the treatment of a lung disease in a patient or subject.
  • the pharmaceutical compositions of the disclosure may include a pharmaceutically acceptable carrier, and a thorough discussion of such carriers is available in Chapter 30 of Remington: The Science and Practice of Pharmacy (23rd ed divide 2021).
  • aerosolized composition comprises LNPs for selective delivery to one or more of goblet cells, secretory cells, club cells, basal cells or ionocytes.
  • the aerosolized pharmaceutical composition comprises LNPs for selective delivery to one or more of ciliated cells, club cells, or basal cells.
  • the aerosolized pharmaceutical compositions include one or more of a poloxamer (e.g., Poloxamer 188) polyethylene glycol (“PEG”), sucrose, and a buffer, wherein the buffer comprises a citrate buffer, an acetate buffer, or a Tris buffer.
  • the PEG has a concentration from 1% to 4% (w/v).
  • the PEG has a concentration from 1% to 5%, or 2 to 4%.
  • the aerosolized pharmaceutical compositions includes Poloxamer 188 at a concentration of between about 0.001% w/v and 0.5% w/v.
  • the composition includes sucrose.
  • the sucrose is at a concentration from 1% to 15% w/v, 5% to 15% w/v, 1% to 10% w/v, or 5% to 10% w/v.
  • the composition includes a citrate buffer.
  • the citrate buffer is at a pH from 4 to 8.
  • the buffer is an acetate buffer and has a pH from 4 to 8.
  • the composition includes a Tris buffer, and the Tris buffer has a pH from 4 to 8.
  • the composition has a pH of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.
  • composition has pKa 4 to 7.
  • the pharmaceutical compositions comprising the LNPs described have at least one pharmaceutically acceptable excipient or carrier.
  • the pharmaceutically acceptable excipient or carrier is for nebuilzation of the composition.
  • the pharmaceutical compositions can also include excipients and/or additives.
  • these are surfactants, stabilizers, complexing agents, antioxidants, or preservatives which prolong the duration of use of the finished pharmaceutical formulation, flavorings, vitamins, or other additives known in the art.
  • Complexing agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as the disodium salt, citric acid, nitrilotriacetic acid and the salts thereof.
  • preservatives include, but are not limited to, those that protect the solution from contamination with pathogenic particles, including benzalkonium chloride or benzoic acid, or benzoates such as sodium benzoate.
  • Antioxidants include, but are not limited to, vitamins, provitamins, ascorbic acid, vitamin E, salts or esters thereof.
  • one or more tonicity agents may be added to provide the desired ionic strength.
  • Tonicity agents for use herein include those which display no or only negligible pharmacological activity after administration. Both inorganic and organic tonicity adjusting agents may be used.
  • the disclosure provides a method of making an LNP composition comprising mixing lipid components and polynucleotides in conditations effective to assemble LNPs comprising the polynucleotides.
  • the method comprises nebulizing the composition to generate an aerosolized LNP composition.
  • kits and articles of manufacture such as those containing reagents for performing the methods provided herein, e.g., reagents for producing LNPs and compositions thereof and/or reagents for introducing one or more nucleic acid molecules into a lung type cell using LNPs and/or compositions thereof.
  • the kits or articles of manufacture can contain reagents and/or nucleic acids for use in engineering or manufacturing processes to generate the LNP.
  • kits can contain reagents and/or consumables required for producing LNPs and compositions thereof. In some embodiments, the kits can contain reagents and/or consumables required for delivery of nucleic acid into the lung type cells using such LNPs and/or compositions thereof.
  • the various components of the kit may be present in separate containers or certain compatible components may be precombined into a single container. In some embodiments, the kits further contain instructions for using the components of the kit to practice the provided methods.
  • the kit includes a lipid nanoparticle composition comprising one or more of a phospholipid, an ionizable lipid, a PEG-lipid, cholesterol, and a base editor. In some embodiments, the kit comprises a LNP composition and a base editor and a nebulizer mask and/or a mesh suitable for use in a nebulizer.
  • articles of manufacture which may include a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container in some embodiments holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition.
  • the container has a sterile access port.
  • Exemplary containers include an intravenous solution bags, vials, including those with stoppers pierceable by a needle for injection, or bottles or vials for orally administered agents.
  • the label or package insert may indicate that the composition is used for treating a disease or condition.
  • the article of manufacture may further include a package insert indicating that the compositions can be used to treat a particular condition.
  • the article of manufacture may further include another or the same container comprising a pharmaceutically-acceptable buffer. It may further include other materials such as other buffers, diluents, filters, needles, and/or syringes.
  • percent (%) amino acid sequence identity and “percent identity” and “sequence identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
  • abasic editor refers any one of the abasic editors described in PCT/JP2015/080958 and US20170321210, which are incorporated herein by reference.
  • lipid composition generally refers to a composition comprising lipid compound(s), including but not limited to, a lipoplex, a liposome, a lipid particle.
  • lipid compositions include suspensions, emulsions, and vesicular compositions.
  • lipid nanoparticle refers to a vesicle formed by one or more lipid components typically used as carriers for nucleic acid, protein, or oligonucleotide delivery in the context of pharmaceutical development.
  • lipid nanoparticle compositions for delivery are composed of synthetic ionizable or cationic lipids, phospholipids, cholesterol, and a polyethylene glycol (PEG) lipid.
  • PEG polyethylene glycol
  • these compositions may also include other lipids.
  • neutral phospholipid refers to phospholipids that have little or no net charge at physiological pH.
  • neutral phospholipids are zwitterions, although other types of net neutral phospholipids are known and may be used.
  • the term “PEG-lipid” refers to a lipid modified with a polyethylene glycol unit.
  • the PEG-lipid comprises DMG.
  • the PEG-lipid comprises DSPE.
  • PEG-OH lipid refers to a PEG-lipid having one or more hydroxyl (-OH) groups on the lipid.
  • adenosine deaminase refers to a polypeptide or fragment thereof capable of catalyzing the hydrolytic deamination of adenine or adenosine.
  • the deaminase or deaminase domain is an adenosine deaminase catalyzing the hydrolytic deamination of adenosine to inosine or deoxy adenosine to deoxyinosine.
  • the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA).
  • the adenosine deaminases e.g., engineered adenosine deaminases, evolved adenosine deaminases
  • the adenosine deaminases may be from any organism, such as a bacterium.
  • ionizable lipid refers to a lipid comprising one or more charged moieties.
  • an ionizable lipid may be positively charged or negatively charged.
  • an ionizable lipid may be positively charged at lower pHs, in which case it could be referred to as “cationic lipid.”
  • an ionizable lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipids.
  • a “charged moiety” refers to a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc.
  • the charged moiety may be anionic (z.e., negatively charged) or cationic (/. ⁇ ?., positively charged).
  • positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidazolium groups.
  • the charged moieties comprise amine groups.
  • negatively- charged groups or precursors thereof include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like.
  • the charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired.
  • partial negative charge and “partial positive charge” are given its ordinary meaning in the art.
  • a “partial negative charge” may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom.
  • bonds that can become polarized in this way are given its ordinary meaning in the art.
  • treatment refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse event, effect, or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
  • adverse event refers to any new untoward medical occurrence or worsening of a pre-existing medical condition occurring in a clinical investigation participant after signing of informed consent, whether or not considered related to the study intervention.
  • An adverse event can therefore be any unfavorable and unintended sign (such as an abnormal laboratory test result), symptom, or disease temporally associated with the study intervention.
  • delay development of a disease means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or subject being treated. As sufficient or significant delay can, in effect, encompass prevention, in that the subject does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
  • Preventing includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease.
  • the provided molecules and compositions are used to delay development of a disease or to slow the progression of a disease.
  • a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition.
  • an antibody or composition or cell which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody or composition or cell.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • a “subject” or an “individual” is a mammal.
  • a “mammal” includes humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, monkeys, etc.
  • the subject is human.
  • lung disease broadly refer to diseases or disorders of the lung. Lung diseases may be characterized by symptoms including but not limited to difficulty breathing, coughing, airway discomfort and inflammation, increased mucus, and/or pulmonary fibrosis.
  • composition treats the cystic fibrosis in the subject.
  • gRNA guide RNA
  • Embodiment 3 The method of embodiment 2, wherein the administration of the composition results in an increase in the expression of the full-length cystic fibrosis transmembrane conductance regulator (CFTR) protein in the subject, as compared to a subject with cystic fibrosis and whose CFTR gene comprises a R553X stop codon mutation, and that is not administered the composition.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Embodiment 4 The method of embodiment 2, wherein the administration of the composition results in an increase in the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in the subject, as compared to a subject with cystic fibrosis and whose CFTR gene comprises a R553X stop codon mutation, and that is not administered the composition.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Embodiment 5 The method of any of embodiments 1-4, wherein the nucleic acid encoding the base editor is RNA.
  • Embodiment 6 The method of any of embodiments 1-5, wherein the base editor is an adenine base editor (ABE).
  • ABE adenine base editor
  • Embodiment 7 The method of embodiment 6, wherein the ABE comprises a tRNA adenosine deaminase (TadA) protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • TadA tRNA adenosine deaminase
  • Embodiment 8 The method of embodiment 6, wherein the ABE is ABE8e.
  • Embodiment 9 The method of any of embodiments 1-5, wherein the base editor is a cytosine base editor (CBE).
  • CBE cytosine base editor
  • Embodiment 10 The method of embodiment 9, wherein the CBE comprises a cytidine deaminase protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • Embodiment 11 The method of any of embodiments 6-10, wherein the ABE or the CBE further comprises a Cas9 enzyme that does not have any nuclease activity.
  • Embodiment 12 The method of any of embodiments 1-11, wherein the LNP comprises an ionizable cationic lipid, a zwitterionic phospholipid, a cholesterol, and a PEG lipid.
  • Embodiment 13 The method of any of embodiments 1-12, wherein the LNP comprises 5A2-SC 8 , l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, DMG-PEG, and one or more selective organ targeting (SORT) molecules.
  • DOPE l,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • SORT selective organ targeting
  • Embodiment 14 A method of delivering a gene editing system to a lung cell type in a subject, the method comprising administering to the subject a composition comprising a lipid nanoparticle (LNP) that comprises a gene editing system, wherein the gene editing system comprises (i) a first nucleic acid encoding an endonuclease or a base editor; and (ii) a second nucleic acid encoding a guide RNA (gRNA), and wherein the gene editing system is delivered to a lung cell type in a subject.
  • LNP lipid nanoparticle
  • gRNA guide RNA
  • Embodiment 15 The method of embodiment 14, wherein the lung cell type is an endothelial cell or an epithelial cell.
  • Embodiment 16 The method of embodiment 14 or 15, wherein the lung cell type is an immune cell.
  • Embodiment 17 The method of any of embodiments 14-16, wherein the lung cell type is a stem cell.
  • Embodiment 18 The method of any of embodiments 14-17, wherein the endonuclease is a Cas nuclease of the CRISPR-Cas system.
  • Embodiment 19 The method of embodiment 18, wherein the Cas nuclease is a Cas9 nuclease, a Casl2 nuclease, or a Casl3 nuclease.
  • Embodiment 20 The method of any of embodiments 14-19, wherein the nucleic acid encoding the endonuclease is DNA.
  • Embodiment 21 The method of any of embodiments 14-19, wherein the nucleic acid encoding the endonuclease is RNA.
  • Embodiment 22 The method of any of embodiments 14-17, wherein the nucleic acid encoding the base editor is DNA.
  • Embodiment 23 The method of embodiment 14-17, wherein the nucleic acid encoding the base editor is RNA.
  • Embodiment 24 The method of any of embodiments 14-17, 22, and 23, wherein the base editor is an adenine base editor (ABE).
  • ABE adenine base editor
  • Embodiment 25 The method of embodiment 24, wherein the ABE comprises a tRNA adenosine deaminase (TadA) protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • TadA tRNA adenosine deaminase
  • Embodiment 26 The method of embodiment 24, wherein the ABE is ABE8e.
  • Embodiment 27 The method of any of embodiments 14-17, 22, and 23, wherein the base editor is a cytosine base editor (CBE).
  • CBE cytosine base editor
  • Embodiment 28 The method of embodiment 27, wherein the CBE comprises a cytidine deaminase protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence.
  • Embodiment 29 The method of any of embodiments 14-17 and 22-28, wherein the ABE or the CBE further comprises a Cas9 enzyme that does not have any nuclease activity.
  • Embodiment 30 The method of any of embodiments 1-29, wherein the ratio of the first nucleic acid to the second nucleic acid is 1:1 on a molecule: molecule basis.
  • Embodiment 31 The method of any of embodiments 1-29, wherein the ratio of the first nucleic acid to the second nucleic acid is 1:1 on a weight basis.
  • Embodiment 32 The method of any of embodiments 1-29, wherein the ratio of the first nucleic acid to the second nucleic acid is 2:1 on a molecule: molecule basis.
  • Embodiment 33 The method of any of embodiments 1-29, wherein the ratio of the first nucleic acid to the second nucleic acid is 2:1 on a weight basis.
  • Embodiment 34 The method of any one of embodiments 14-33, wherein the LNP comprises an ionizable cationic lipid, a zwitterionic phospholipid, a cholesterol, and a PEG lipid.
  • Embodiment 35 The method of any one of embodiments 14-34, wherein the LNP comprises 5A2-SC 8 , l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, DMG-PEG, and one or more selective organ targeting (SORT) molecules.
  • DOPE l,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • SORT selective organ targeting
  • Embodiment 36 The method of embodiment 13 or 35, wherein the one or more SORT molecules comprises permanently positively charged moiety.
  • Embodiment 37 The method of embodiment 36, wherein the one or more SORT molecules is selected from the group consisting of 18:1 DOTMA (DOTMA); DORI, DC-6-14; 12:0 EPC (Chloride Salt); 14:0 EPC (Chloride Salt); 16:0 EPC (Chloride Salt); 18:0 EPC (Chloride Salt); 18:1 EPC (Chloride Salt); 16:0-18:1 EPC (Chloride Salt); 14:1 EPC (Triflate Salt); 18:0 DDAB (Dimethyldioctadecylammonium (Bromide Salt)); 14:0 TAP; 16:0 TAP; 18:0 TAP; 18:1 TAP (DOTAP); 18:1 TAP (DOTAP, MS Salt); 18:1 DODAP, or 18:1 PA (1,2- dioleoyl-sn-glycero-3-phosphate (sodium salt)) (18PA).
  • DOTMA DOTMA
  • MS Salt 18:1 DOD
  • Embodiment 38 The method of embodiment 13 or 35, wherein the one or more SORT molecule comprises DOTAP (l,2-dioleoyl-3-trimethylammonium propane).
  • DOTAP l,2-dioleoyl-3-trimethylammonium propane
  • Embodiment 39 The method of embodiment 36 or 37, wherein the one or more SORT molecule comprises 18PA.
  • Embodiment 40 The method of embodiment 36 or 37, wherein the one or more SORT molecule comprises DODAP.
  • Embodiment 41 The method of embodiment 40, wherein the DODAP comprises about 20% molar ratio of the total lipids.
  • Embodiment 42 The method of embodiment 38, wherein the DOTAP comprises about 50% molar ratio of the total lipids.
  • Embodiment 43 The method of embodiment 39, wherein the 18PA comprises about 10% molar ratio of the total lipids.
  • Embodiment 44 The method of embodiment 36 or 37, wherein the one or more SORT molecule comprises DOTMA.
  • Embodiment 45 The method of embodiments 35, 36 or 37, wherein the LNP comprises a ratio of DOPE:DOTMA between 3:1 and 1:3.
  • Embodiment 46 The method of embodiment 45, wherein the ratio of DOPE:DOTMA is about 3:1.
  • Embodiment 47 The method of embodiment 45, wherein the ratio of DOPE:DOTMA is about 1:1.
  • Embodiment 48 The method of any of embodiments 13, 35-47, wherein the SORT molecule comprises from about 5% to about 60% molar percentage of the LNP.
  • Embodiment 49 The method of any of embodiments 13, 35-47, wherein the SORT molecule comprises about 40% molar percentage of the LNP.
  • Embodiment 50 The method of any of embodiments 13, 35-47, wherein the SORT molecule comprises about 50% molar percentage of the LNP.
  • Embodiment 51 The method of any of embodiments 1-50, wherein the LNP binds vitronectin.
  • Embodiment 52 The method of any one of embodiments 1-51, wherein the guide RNA comprises a circular RNA.
  • Embodiment 53 The method of any one of embodiments 1-51, wherein the guide RNA comprises a linear RNA.
  • Embodiment 54 The method of any of embodiments 1-51, wherein the guide RNA is a single guide RNA (sgRNA).
  • sgRNA single guide RNA
  • Embodiment 55 The method of any of embodiments 1-54, wherein the guide RNA comprises a target sequence that is complementary with a target sequence of a cystic fibrosis transmembrane conductance regulator (CFTR) gene.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Embodiment 56 The method of any of embodiments 1-55, wherein the nucleotide sequence of the guide RNA is AAGTAAAACCTCTACAAATG (SEQ ID NO: 1) or TTGCTCATTGACCTCCACTC (SEQ ID NO: 2).
  • Embodiment 57 The method of any one of embodiments 1 to 56, wherein the composition comprises a pharmaceutically acceptable carrier.
  • Embodiment 58 The method of any one of embodiments 1-57, wherein the composition is administered intravenously.
  • Embodiment 59 The method of any one of embodiments 1-58, wherein the subject is a human.
  • Embodiment 60 The method of any one of embodiments 14-59, wherein the subject has cystic fibrosis.
  • Embodiment 61 A method of modifying the nucleic acid sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a lung cell type, wherein the CFTR gene comprises a R553X stop codon mutation, the method comprising:
  • nucleic acid sequence of the CFTR gene in the lung cell type is modified to remove the R553X stop codon mutation.
  • Embodiment 63 A method of increasing the expression of full-length cystic fibrosis transmembrane conductance regulator (CFTR) protein in a lung cell type, wherein a CFTR gene in the lung cell type comprises a R553X mutation, the method comprising:
  • a composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises (i) a first nucleic acid encoding a base editor; and (ii) a second nucleic acid encoding a guide RNA;
  • LNP lipid nanoparticle
  • Embodiment 66 The method of embodiment 65, wherein expression of the CFTR protein is determined by analysis of chloride levels in the sweat of the subject.
  • Embodiment 67 The method of embodiment 65, wherein the chloride levels in the sweat of the subject after being administered the composition are decreased as compared to the chloride levels in the sweat of the subject before being administered the composition.
  • Embodiment 70 The method of any one of embodiments 61-69 wherein the lung cell type is an endothelial cell or an epithelial cell.
  • Embodiment 79 The method of any of embodiments 75-78, wherein the ABE or the CBE further comprises a Cas9 enzyme that does not have any nuclease activity.
  • Embodiment 80 The method of any one of embodiments 61-79, wherein the ratio of the first nucleic acid to the second nucleic acid is 1:1 on a molecule: molecule basis.
  • Embodiment 81 The method of any one of embodiments 61-79, wherein the ratio of the first nucleic acid to the second nucleic acid is 1:1 on a weight basis.
  • Embodiment 82 The method of any one of embodiments 61-79, wherein the ratio of the first nucleic acid to the second nucleic acid is 2:1 molecule: molecule basis.
  • Embodiment 83 The method of any one of embodiments 61-79, wherein the ratio of the first nucleic acid to the second nucleic acid is 2:1 on a weight basis.
  • Embodiment 84 The method of any one of embodiments 61-83, wherein the LNP comprises an ionizable cationic lipid, a zwitterionic phospholipid, a cholesterol, and a PEG lipid.
  • Embodiment 85 The method of any one of embodiments 61-84, wherein the LNP comprises 5A2-SC 8 , l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, DMG-PEG, and one or more selective organ targeting (SORT) molecules.
  • DOPE l,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • SORT selective organ targeting
  • Embodiment 86 The method of embodiment 85, wherein the one or more SORT molecules comprises permanently positively charged moiety.
  • Embodiment 87 The method of embodiment 85 or 86, wherein the one or more SORT molecule is selected from the group consisting of 18:1 DOTMA (DOTMA); DORI, DC- 6-14; 12:0 EPC (Chloride Salt); 14:0 EPC (Chloride Salt); 16:0 EPC (Chloride Salt); 18:0 EPC (Chloride Salt); 18:1 EPC (Chloride Salt); 16:0-18:1 EPC (Chloride Salt); 14:1 EPC (Triflate Salt); 18:0 DDAB (Dimethyldioctadecylammonium (Bromide Salt)); 14:0 TAP; 16:0 TAP; 18:0 TAP; 18:1 TAP (DOTAP); 18:1 TAP (DOTAP, MS Salt); 18:1 DODAP, or 18:1 PA (1,2- dioleoyl-sn-glycero-3-phosphate (sodium salt)) (18PA).
  • DOTMA DOTMA
  • Embodiment 88 The method of any of embodiments 85-87, wherein the one or more SORT molecules comprises DOTAP (l,2-dioleoyl-3-trimethylammonium propane).
  • DOTAP l,2-dioleoyl-3-trimethylammonium propane
  • Embodiment 89 The method of any of embodiments 85-87, wherein the one or more SORT molecules comprises 18PA.
  • Embodiment 90 The method of any of embodiments 85-87, wherein the one or more SORT molecules comprises DODAP.
  • Embodiment 91 The method of embodiment 90, wherein the DODAP comprises about 20% molar ratio of the total lipids.
  • Embodiment 92 The method of embodiment 88, wherein the DOTAP comprises about 50% molar ratio of the total lipids.
  • Embodiment 93 The method of embodiment 89, wherein the 18PA comprises about 10% molar ratio of the total lipids.
  • Embodiment 94 The method of any of embodiments 85-87, wherein the SORT molecule comprises DOTMA.
  • Embodiment 95 The method of embodiment 85, 86 or 87, wherein the LNP comprises a ratio of DOPE:DOTMA of between 3:1 and 1:3.
  • Embodiment 96 The method of embodiment 95, wherein the ratio of DOPE:DOTMA is about 3:1.
  • Embodiment 97 The method of embodiment 95, wherein the ratio of DOPE:DOTMA is about 1:1.
  • Embodiment 98 The method of any of embodiments 85-97, wherein the SORT molecule comprises from about 5% to about 60% molar percentage of the LNP.
  • Embodiment 99 The method of any of embodiments 85-98, wherein the SORT molecule comprises about 40% molar percentage of the LNP.
  • Embodiment 100 The method of any of embodiments 85-98, wherein the SORT molecule comprises from 50% molar percentage of the LNP.
  • Embodiment 101 The method of any of embodiments 61-100, wherein the LNP binds vitronectin.
  • Embodiment 102 The method of any one of embodiments 61-101, wherein the guide RNA comprises a circular RNA.
  • Embodiment 103 The method of any one of embodiments 61-101, wherein the guide RNA comprises a linear RNA.
  • Embodiment 104 The method of any one of embodiments 61-101, wherein the guide RNA is a single guide RNA (sgRNA).
  • Embodiment 105 The method of any of embodiments 61-104, wherein the guide RNA comprises a target sequence that is complementary with a target sequence of a cystic fibrosis transmembrane conductance regulator (CFTR) gene.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Embodiment 106 The method of any of embodiments 61-105, wherein the nucleotide sequence of the guide RNA is AAGTAAAACCTCTACAAATG (SEQ ID NO: 1) or TTGCTCATTGACCTCCACTC (SEQ ID NO: 2).
  • Embodiment 107 The method of embodiment 4, wherein the function of the CFTR protein is determined by one or more bioassays comprising sweat chloride concentration assay, P-adrenergic sweat assay, and nasal potential difference assay.
  • Embodiment 110 The method of any one of embodiments 61-109, wherein the composition comprises a pharmaceutically acceptable carrier.
  • Embodiment 114 The method of embodiment 113, wherein about 5% to about 95% of the function of the CFTR gene is restored.
  • Embodiment 115 The method of embodiment 113 or 114, wherein the restoring of the function of the CFTR gene is determined by the increase of CFTR protein expression.
  • Embodiment 116 The method of embodiment 115, wherein expression of the CFTR protein is determined by one or more bioassays comprising sweat chloride concentration assay, P-adrenergic sweat assay, and nasal potential difference assay.
  • Embodiment 117 The method of embodiment 115 or 116 wherein expression of the CFTR protein is determined by analysis of chloride levels in the sweat of the subject.
  • Embodiment 118 The method of embodiment 117, wherein chloride levels in the sweat of the subject after being administered the composition are decreased as compared to levels in a subject before being administered the composition.
  • Embodiment 119 The method of embodiment 115, wherein the expression is measured using western blotting, immunoprecipitation, and anti-CFTR antibodies.
  • Embodiment 120 The method of any one of embodiments 113-119, wherein the nucleic acid encoding the base editor is DNA.
  • Embodiment 121 The method of any one of embodiments 113-119, wherein the nucleic acid encoding the base editor is RNA.
  • Embodiment 122 The method of any one of embodiments 113-121, wherein the base editor is an adenine base editor (ABE).
  • ABE adenine base editor
  • Embodiment 123 The method of embodiment 122, wherein the base editor is ABE8e.
  • Embodiment 125 The method of any one of embodiments 113-121, wherein the base editor is a cytosine base editor (CBE).
  • CBE cytosine base editor
  • Embodiment 132 The method of any one of embodiments 113-131, wherein the LNP comprises an ionizable cationic lipid, a zwitterionic phospholipid, a cholesterol, and a PEG lipid.
  • Embodiment 133 The method of any one of embodiments 113-132, wherein the LNP comprises 5A2-SC 8 , l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, DMG-PEG, and one or more selective organ targeting (SORT) molecules.
  • DOPE l,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • SORT selective organ targeting
  • Embodiment 134 The method of embodiment 133, wherein the one or more SORT molecules comprises permanently positively charged moiety.
  • Embodiment 135 The method of embodiment 133 or 134, wherein the one or more SORT molecule is selected from the group consisting of 18:1 DOTMA (DOTMA); DORI, DC- 6-14; 12:0 EPC (Chloride Salt); 14:0 EPC (Chloride Salt); 16:0 EPC (Chloride Salt); 18:0 EPC (Chloride Salt); 18:1 EPC (Chloride Salt); 16:0-18:1 EPC (Chloride Salt); 14:1 EPC (Triflate Salt); 18:0 DDAB (Dimethyldioctadecylammonium (Bromide Salt)); 14:0 TAP; 16:0 TAP; 18:0 TAP; 18:1 TAP (DOTAP); 18:1 TAP (DOTAP, MS Salt); 18:1 DODAP, or 18:1 PA (1,2- dioleoyl-sn-glycero-3-phosphate (sodium salt)) (18PA).
  • DOTMA DOTMA
  • Embodiment 138 The method of any of embodiments 133-135, wherein the one or more SORT molecules comprises DODAP.
  • Embodiment 139 The method of embodiment 138, wherein the DODAP comprises about 20% molar ratio of the total lipids.
  • Embodiment 140 The method of embodiment 136, wherein the DOTAP comprises about 50% molar ratio of the total lipids.
  • Embodiment 143 The method of embodiment 133, 134, or 135, wherein the LNP comprises a ratio of DOPE:DOTMA of between 3:1 and 1:3.
  • Embodiment 144 The method of embodiment 143, wherein the ratio of DOPE:DOTMA is about 3:1.
  • Embodiment 146 The method of any of embodiments 133-145, wherein the one or more SORT molecules comprises from about 5% to about 60% molar percentage of the LNP.
  • Embodiment 153 The method of any of embodiments 114-152, wherein the guide RNA comprises a target sequence that is complementary with a target sequence of a cystic fibrosis transmembrane conductance regulator (CFTR) gene.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Embodiment 154 The method of any of embodiments 111-150, wherein the nucleotide sequence of the guide RNA is AAGTAAAACCTCTACAAATG (SEQ ID NO: 1) or TTGCTCATTGACCTCCACTC (SEQ ID NO: 2).
  • Embodiment 155 The method of any one of embodiments 114-154, wherein the composition comprises a pharmaceutically acceptable carrier.
  • Embodiment 156 The method of any one of embodiments 114-155, wherein the subject is a human.
  • Embodiment 157 The method of any one of embodiments 114-156, wherein the administration of the composition to the subject is by intravenous administration.
  • Embodiment 158 The method of any of embodiments 1-157, wherein the LNP is localized to the lungs of the subject.
  • Embodiment 159 The method of any of embodiments 1-157, wherein the LNP is capable of delivering the first and second nucleic acids to the lungs of the subject.
  • Embodiment 160 A lung cell type comprising a modified cystic fibrosis transmembrane conductance regulator (CFTR) gene, wherein the modification comprises the replacement of the thymine at 1789 base in exon 11 of the CFTR gene with cytosine.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Embodiment 161 A method of treating cystic fibrosis in a subject, the method comprising administering to the subject a composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises (i) a first nucleic acid encoding a base editor; and (ii) a second nucleic acid encoding a guide RNA, wherein the first nucleic acid and the second nucleic acid are delivered to a lung cell in the subject.
  • LNP lipid nanoparticle
  • Example 1 Lung SORT LNPs mediate stem cell delivery in vivo to achieve durable editing for over one year
  • mice were administrated with Cre mRNA Lung SORT LNPs (LNP-Cre) IV (2 mg/kg, 2 doses 48 hours apart) to evaluate the long-term efficacy of lung editing.
  • Lung tissues were collected at various intervals post the second injection, including baseline 48 hours, 7 days, 21 days, 42 days, 60 days, 120 days, 180 days, 270 days, and 360 days (FIG. 1A) for ex-vivo imaging and flow cytometry.
  • tdTom expression was uniformly spread throughout the mouse lung at every time point (FIG. IB).
  • Basal cells are regarded as the tissue- specific stem cells of the mouse and human airway epithelium due to their ability to self-renew and differentiate into multiple lineages, including ciliated cells, secretory cells, goblet cells and ionocytes (D. T. Montoro et al., A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature 560, 319-324 (2016); J. R. Rock et al., Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc. Natl. Acad. Sci. USA 106, 12771-12775 (2009); J. R. Rock, S. H. Randell, B. L.
  • Luo A global double- fluorescent Cre reporter mouse. Genesis 45, 593-605 (2007) was used to further visualize and quantify edited cells ( ⁇ 8% by tissue volume) two days after a single LNP-Cre administration that mediated excision to turn on eGFP expression replacing pre-existing tdTom fluorescent proteins (FIG. 8A, FIG. 8B). This further supports lung SORT LNP enabled genome editing in the majority of different lung cell types.
  • Example 5 Efficient editing of primary human CF patient-derived basal cells
  • the R553X mutation was corrected in primary CF patient-derived human bronchial epithelial (HBE) cells (D. C. Gruenert, W. E. Finkbeiner, J. H. Widdicombe, Culture and transformation of human airway epithelial cells. Am. J. Physiol. 268, L347-360 (1995)) carrying the R553X/F508del heterozygous mutation.
  • HBE human bronchial epithelial
  • the difficult-to-treat HBE model has been used to develop CFTR modulator small molecule therapies due to its capability of differentiation into a pseudostratified epithelium in air-liquid interface (ALI) culture that mimic characteristics of in vivo airway biology, allowing strong prediction of therapeutic efficacy in humans (J. P.
  • Trikafta (elexacaftor/tezacaftor/ivacaftor, the standard of care for CF patients with a single F508del allele (P. G. Middleton et al., Elexacaftor-Tezacaftor-Ivacaftor for cystic fibrosis with a single Phe508del allele. New. Engl. J. Med. 381, 1809-1819 (2019))), further increased the expression of fully glycosylated CFTR by 7.8-fold (Band C, FIG. 13F).
  • Example 6 In vivo stem cell editing in CF mouse lungs
  • Example 7 Lung SORT LNPs mediate stem cell delivery to achieve durable editing for >1.5 years
  • Basal cells are regarded as the tissue- specific stem cells of the mouse and human airway epithelium due to their ability to self-renew and differentiate into various mature cell lineages, including ciliated cells, secretory cells, goblet cells and ionocytes (D. T. Montoro et al., A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature 560, 319- 324 (2016); J. R. Rock et al., Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc. Natl. Acad. Sci. USA 106, 12771-12775 (2009); J. R. Rock, S. H. Randell, B. L.
  • Airway basal stem cells a perspective on their roles in epithelial homeostasis and remodeling. Dis. Models Meeh. 3, 545-556 (2010); Y. Zhou et al., Airway basal cells show regionally distinct potential to undergo metaplastic differentiation. Elife 11, (2022)).
  • These cells in the mouse and human airway epithelium can be identified by the expression of nerve growth factor receptor (Ngfr) (J. R. Rock et al., Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc. Natl. Acad. Sci. USA 106, 12771-12775 (2009)) or cytoskeletal protein keratin 5 (Krt5) (W.
  • Ngfr nerve growth factor receptor
  • Krt5 cytoskeletal protein keratin 5
  • Editing level was consistently high at the 660-day end point (>93% in endothelial cells, >23% in immune cells, and >48% in epithelial cells) (FIG. 17I-FIG. 17K).
  • 660-day end point >93% in endothelial cells, >23% in immune cells, and >48% in epithelial cells
  • FIG. 17M Within EpCAM+KRT5+ stem cells, 27% displayed tdTom expression at 48 hours, peaking to 97% on day 120 and then remaining between 45 and 80% for up to 660 days.
  • FIG. 21A-FIG. 21B A more in-depth characterization revealed 13.5-41.7% editing in various immune cell types, including neutrophils (L. M. Yonker et al., Neutrophil dysfunction in cystic fibrosis. J. Cyst. Fibres. 20, 1062-1071 (2021)) and macrophages (J. L. Gillan et al., CAGE sequencing reveals CFTR-dependent dysregulation of type I IFN signaling in activated cystic fibrosis macrophages. Sci. Adv. 9, eadg5128(2023)) (FIG. 22A-FIG.
  • CD157 serves as a marker for tissue-resident endothelial progenitors in a range of organs, including the lungs (T. Wakabayashi et al., CD157 marks tissue-resident endothelial stem cells with homeostatic and regenerative properties. Cell Stem Cell 22, 384-397. e386 (2016)).
  • Flow cytometry was employed to track the expression of tdTom in lung tissue-resident endothelial progenitor cells (CD31 + CD157 + ) after an IV injection of LNP-Cre (FIG.
  • Example 9 Long-term editing of diverse lung epithelial cell types in vivo
  • the lung's epithelial lining is made up of a diverse assortment of cell types, each performing unique roles in preserving lung functionality and homeostasis (D. N. Kotton, E. E. Morrisey, Lung regeneration: mechanisms, applications and emerging stem cell populations. Nat. Med. 20, 822-832 (2014); Brigid et al., Repair and regeneration of the respiratory system: Complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell 15, 123-138 (2014)).
  • ATI alveolar type 1
  • AT2 type 2
  • goblet cells for mucus production
  • ciliated cells for mucus removal
  • club cells for bronchiolar epithelium protection
  • rare ionocytes that highly express CFTR
  • Tissue section imaging with a whole-slide scanner allowed for the identification of co-localization of tdTom expression and cell-specific markers.
  • the primary antibody staining conditions are listed below in Table E6. All primary antibodies are used at a 1:100 dilution and incubated for 20 minutes.
  • FIG. 27A Nearly all cell types displayed co-localization of tdTom and their respective cell marker, indicating widespread editing (FIG. 27A). Approximately 18% of ATI cells, 20% of AT2 cells, 10% of goblet cells, 6% of ciliated cells, and 2% of club cells showed evidence of editing, which persisted for up to 360 days (FIG. 27B). A total of 21.4% of ionocytes were edited by day 2, increasing to 58% at the 660-day mark (FIG. 28A- FIG. 28B), which is important because ionocytes control airway surface liquid absorption (Yuan et al., Transgenic ferret models define pulmonary ionocyte diversity and function. Nature.

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Abstract

La divulgation concerne des méthodes de traitement de la fibrose kystique chez un sujet. La divulgation concerne également des procédés de modification de la séquence d'acide nucléique dans le gène CFTR dans un type de cellule pulmonaire, des procédés d'augmentation de l'expression du gène CFTR dans un type de cellule pulmonaire et des procédés de modulation de l'activité d'un gène CFTR dans un type de cellule pulmonaire. La présente divulgation concerne en outre la méthode de traitement de la fibrose kystique comprenant l'administration d'une nanoparticule lipidique (LNP) et d'un système d'édition de gènes, l'administration de la LNP médiant l'édition du génome dans les cellules pulmonaires.
PCT/US2024/061579 2023-12-22 2024-12-20 Méthodes d'édition de gènes et compositions pour le traitement de la fibrose kystique Pending WO2025137646A1 (fr)

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