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WO2024119103A9 - Lipid nanoparticles comprising nucleic acids and lipid-anchored polymers - Google Patents

Lipid nanoparticles comprising nucleic acids and lipid-anchored polymers Download PDF

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Publication number
WO2024119103A9
WO2024119103A9 PCT/US2023/082126 US2023082126W WO2024119103A9 WO 2024119103 A9 WO2024119103 A9 WO 2024119103A9 US 2023082126 W US2023082126 W US 2023082126W WO 2024119103 A9 WO2024119103 A9 WO 2024119103A9
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Prior art keywords
lipid
lnp
polymer
anchored
formula
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French (fr)
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WO2024119103A1 (en
Inventor
Matthew G. Stanton
Nolan GALLAGHER
Andrew MILSTEAD
Nathaniel SILVER
Constance MARTIN
Anshul Gupta
Christian J. SLUBOWSKI
Jimit G. RAGHAV
Randall Newton TOY
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Generation Bio Co
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Generation Bio Co
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Priority to EP23841115.1A priority Critical patent/EP4626402A1/en
Publication of WO2024119103A1 publication Critical patent/WO2024119103A1/en
Publication of WO2024119103A9 publication Critical patent/WO2024119103A9/en
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions

Definitions

  • nucleic acid therapeutics Unfortunately, effective targeted delivery of biologically active substances, such as therapeutic nucleic acids, represents a continuing medical challenge. This has severely limited broad applications of nucleic acids such as mRNA and DNA in non-viral gene replacement therapy, gene therapy, gene editing, and vaccination. Lack of effective methods and vehicles for non-viral delivery represents a major barrier to a broad use of nucleic acid therapeutics.
  • RNA and DNA molecules which typically range from 300 kDa to 5,000 kDa in size, or ⁇ 1-15 kb
  • mRNA and DNA molecules are significantly larger than other types of RNAs, such as small interfering RNAs or siRNA (which are typically ⁇ 14 kDa) or antisense oligonucleotides or ASOs (which typically range from 4 kDa to 10 kDa).
  • siRNA which are typically ⁇ 14 kDa
  • ASOs which typically range from 4 kDa to 10 kDa.
  • viral delivery of nucleic acid therapeutics to targeted cells is hindered greatly by the activation of the innate and/or adaptive immune responses.
  • RNA sensing by myeloid dendritic cells MDCs
  • RNA cargo e.g., with 1m ⁇ , 2’OMe, etc.
  • PRR pattern recognition receptor
  • An alternative approach to gene therapy is the recombinant adeno-associated virus (rAAV) vector platform that packages heterologous DNA in a viral capsid.
  • rAAV adeno-associated virus
  • LNPs lipid nanoparticles
  • LNP compositions comprising a therapeutic nucleic acid (TNA), e.g., a gene expression vector such as closed-ended DNA (ceDNA), single-stranded DNA (ssDNA) vector, or messenger RNA (mRNA), wherein the structural LNP components comprise an ionizable lipid; a structural lipid, e.g., a sterol; and one or more types of lipid-anchored polymers, and wherein the entire structural LNP components do not comprise a helper lipid.
  • TAA therapeutic nucleic acid
  • ceDNA closed-ended DNA
  • ssDNA single-stranded DNA
  • mRNA messenger RNA
  • the LNPs disclosed herein provide surprising and unexpected properties as compared to known LNPs comprising helper lipids. These properties include, e.g., improved tolerability and slower plasma clearance indicating an increased stealth property. Moreover, the disclosed LNPs and LNP compositions surprisingly reduce LNP related toxicity, as is evidenced by serum levels of immune response markers (see Examples herein). Further, the disclosed LNPs and LNP compositions comprising a certain molecular percentage of sterol (30%-45% molecular percentage of the total lipid) are characterized by a diameter of about 80 nm or less, making them particularly amenable and useful for therapeutic administration as most target organs require the size of LNP being smaller than 85 nm for efficient delivery.
  • the disclosure provides a lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; an ionizable lipid; a sterol; a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises: a polymer; a lipid moiety comprising at least one hydrophobic tail; and optionally a linker connecting the polymer to the lipid moiety; wherein the at least one hydrophobic tail comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; and wherein the LNP does not comprise a helper lipid.
  • TAA therapeutic nucleic acid
  • a sterol a first lipid-anchored polymer
  • the first lipid-anchored polymer comprises: a polymer; a lipid moiety comprising at least one hydrophobic tail; and optionally a linker connecting the polymer to the lipid moiety; wherein the at least one hydrophobic
  • the therapeutic nucleic acid is about 100 to about 10,000 base pairs or about 100 to about 10,000 nucleotides in length, or about 250 to about 10,000 base pairs or about 250 to about 10,000 nucleotides in length, or about 500 to about 10,000 base pairs or about 500 to about 10,000 nucleotides in length, or about 1,000 to about 10,000 base pairs or about 1,000 to about 10,000 nucleotides in length, or about 2,500 to about 10,000 base pairs or about 2,500 to about 10,000 nucleotides in length, or about 3,000 to about 10,000 base pairs or about 3,000 to about 10,000 nucleotides in length, or about 4,000 to about 10,000 base pairs or about 4,000 to about 10,000 nucleotides in length.
  • the therapeutic nucleic acid does not comprise short interfering RNA (siRNA). In some embodiments, the therapeutic nucleic acid (TNA) does not comprise microRNA (miRNA). In some embodiments, the therapeutic nucleic acid (TNA) does not comprise antisense oligonucleotide (ASO). 2 ME146907648v.1
  • the LNP has an average particle size of about 50-100 nm in diameter. In some embodiments, the LNP has an average particle size of about 50-90 nm in diameter. In some embodiments, the LNP has an average particle size of about 50-80 nm in diameter. In some embodiments, the LNP has an average particle size of about 50-70 nm in diameter.
  • the LNP has an average particle size of about 50-60 nm in diameter.
  • the lipid moiety comprises at least two hydrophobic tails.
  • the two hydrophobic tails each independently comprise 16, 17, 18, 19, 20, 21, or 22 carbon atoms.
  • the two hydrophobic tails each independently comprise 16, 17, 18, 19, 20, or 21 carbon atoms.
  • the two hydrophobic tails each independently comprise 16, 17, 18, 19 or 20 carbon atoms.
  • the two hydrophobic tails each independently comprise 16, 17, 18, or 19 carbon atoms.
  • the two hydrophobic tails each independently comprises 16, 17, or 18 carbon atoms.
  • the two hydrophobic tails each comprise 18, 19 or 20 carbon atoms. In some embodiments, the two hydrophobic tails each comprise 16 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 18 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 20 carbon atoms. In some embodiments, the at least one or two hydrophobic tails are each a fatty acid.
  • the at least one or two hydrophobic tails are each independently selected from the group consisting of octadecane, octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, ⁇ -linolenic acid, arachidonic acid, eicosapentaenoic acid, and a derivative thereof.
  • the at least two hydrophobic tails are each independently selected from the group consisting of lauric acid, myristic acid, myristoleic acid, and a derivative thereof.
  • the lipid moiety comprises two hydrophobic tails.
  • the two hydrophobic tails are each a fatty acid.
  • the two hydrophobic tails are each independently selected from the group consisting of octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, ⁇ -linolenic acid, arachidonic acid, eicosapentaenoic acid, and a derivative thereof.
  • the two hydrophobic tails are each independently selected from the group consisting of lauric acid, myristic acid, myristoleic acid, and a derivative thereof.
  • the first lipid-anchored polymer comprises a glycerolipid. In some embodiments, the first lipid-anchored polymer comprises a phospholipid.
  • the first lipid-anchored polymer comprises a linker-lipid moiety selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn- 3 ME146907648v.1 glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn- phosphatidylethanol
  • the first lipid- anchored polymer comprises a linker-lipid moiety selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, a derivative thereof, and a combination of any of the foregoing.
  • the polymer in the first lipid-anchored polymer is selected from the group consisting of polyethylene (PE), polypropylene (PP), polyethylene glycol (PEG), polyglycerol (PG), polyvinyl alcohol (PVOH), polysarcosine (pSar), and a combination thereof.
  • the polymer is selected from the group consisting of polyethylene glycol (PEG), polyglycerol (PG), polysarcosine (pSar), and a combination thereof.
  • the polymer has a molecular weight of between about 1000 Da and about 5000 Da. In some embodiments, the polymer has a molecular weight of between about 2000 Da and about 5000 Da. In some embodiments, the polymer has a molecular weight of about 2000 Da. In some embodiments, the polymer is polyethylene glycol (PEG).
  • the sterol is selected from the group consisting of cholesterol, beta- sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and a derivative of thereof, and a combination of any of the foregoing. In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is beta-sitosterol.
  • the ionizable lipid is a lipid represented by: a) Formula (A): Formula (A), or a pharmaceutically acceptable salt thereof, wherein: R 1 and R 1’ are each independently optionally substituted linear or branched C1-3 alkylene; 4 ME146907648v.1 R 2 and R 2’ are each independently optionally substituted linear or branched C1-6 alkylene; R 3 and R 3’ are each independently optionally substituted linear or branched C1-6 alkyl; or alternatively, when R 2 is optionally substituted branched C1-6 alkylene, R 2 and R 3 , taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; or alternatively, when R 2’ is optionally substituted branched C1-6 alkylene, R 2’ and R 3' , taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; R 4 and R 4’ are each independently –CR a , –C(
  • the LNP further comprises a targeting moiety.
  • the LNP comprises a second lipid-anchored polymer and the targeting moiety is conjugated to the second lipid-anchored polymer.
  • the second lipid- anchored polymer comprises a linker-lipid moiety selected from the group consisting of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- 7 ME146907648v.1 phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycer
  • DPPC 1,2- dipalmit
  • the second lipid-anchored polymer comprises a linker-lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, a derivative thereof, and a combination of any of the foregoing.
  • the first and the second lipid-anchored polymers are different lipid- anchored polymers; and the first and the second lipid-anchored polymers comprise one of the following combinations: DSG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DSPE (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DSG (the second
  • the first and the second lipid-anchored polymers are the same lipid- anchored polymers; and wherein the first and the second lipid-anchored polymers comprise one of the following combinations: DSG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DSPE (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); and DPG (the first lipid-anchored polymer) and DPG (the second lipid-anchored polymer).
  • the targeting moiety is conjugated to a DSPE-anchored polymer.
  • the DSPE-anchored polymer is DSPE-PEG or a derivative thereof.
  • the targeting moiety is conjugated to a DSG-anchored polymer.
  • the DSG-anchored polymer is DSG-PEG or a derivative thereof. 8 ME146907648v.1
  • the targeting moiety is capable of binding to a liver cell.
  • the liver cell is a hepatocyte.
  • the targeting moiety is N-acetyl galactosamine (GalNAc) or a GalNAc derivative.
  • the targeting moiety is a tri- antennary GalNAc conjugate or a tetra-antennary GalNAc conjugate.
  • the targeting moiety is an ApoE protein, an ApoE polypeptide, an ApoB protein, an ApoB polypeptide, a fragment thereof, or a derivative of any of the foregoing. In some embodiments, the targeting moiety is capable of binding to ASGPR1 protein. In some embodiments, the targeting moiety is an antibody, antibody fragment, or an antibody derivative. In some embodiments, the antibody, antibody fragment, or antibody derivative is selected from the group consisting of a full-length antibody, an Fab, an Fab’, a single-domain antibody, a single-chain antibody, a nanobody, and a VHH.
  • the LNP further comprises a dissociable lipid-conjugated polymer; wherein the dissociable lipid-conjugated polymer comprises: a polymer; a lipid moiety comprising at least one hydrophobic tail; and optionally a linker connecting the polymer to the lipid moiety; wherein the at least one hydrophobic tail comprises 10-15 carbon atoms in a single aliphatic chain backbone. In some embodiments, the at least one hydrophobic tail comprises 14 carbon atoms in a single aliphatic chain backbone.
  • the dissociable lipid-conjugated polymer is 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol (DMG-PEG) or mono-C18-PEG.
  • the mono-C18-PEG is mono-C18-PEG2000.
  • the dissociable lipid-conjugated polymer is present in the LNP in an amount of about 5%.
  • the ionizable lipid in an LNP of the present disclosure in accordance with any of the foregoing embodiments is Ionizable Lipid 87: , or a pharmaceutically acceptable salt thereof.
  • the ionizable lipid is present in the LNP in an amount of about 35 mol% to about 60 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of no less than about 30 mol% (i.e., ⁇ 30 mol%) of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 20 mol% to about 45 mol% of the total lipid present in the 9 ME146907648v.1 LNP. In some embodiments, the sterol is present in the LNP in an amount of about 35 mol% to about 45 mol% of the total lipid present in the LNP.
  • the sterol is present in the LNP in an amount of about 35 mol% to about 40 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 40 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 35 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 40 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 45 mol% of the total lipid present in the LNP.
  • the first lipid-anchored polymer is present in the LNP in an amount of about 0.005 mol% to about 5 mol% of the total lipid present in the LNP.
  • the second lipid-anchored polymer is present in the LNP in an amount of about 0.05 mol% to about 2 mol% of the total lipid present in the LNP.
  • the second lipid-anchored polymer is present in the LNP in an amount of about 0.1 mol% to about 1 mol% of the total lipid present in the LNP.
  • the second lipid-anchored polymer is present in the LNP in an amount of about 0.5 mol% of the total lipid present in the LNP.
  • the first lipid-anchored polymer and the second lipid anchored polymer are present in the LNP in an amount of about 2.5 mol% and 0.5 mol%, respectively, of the total lipid present in the LNP.
  • the LNP is suitable for intravenous administration.
  • the LNP is less immunogenic than a reference LNP that comprises a helper lipid; and/or does not comprise the first lipid-anchored polymer and comprises a reference lipid-anchored polymer comprising one or more hydrophobic tails that each independently comprises less than 16 carbon atoms in a single aliphatic chain backbone.
  • the LNP elicits lower pro-inflammatory cytokine response than the reference LNP.
  • the reference lipid-anchored polymer is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) or methoxypolyethyleneglycoloxy-N,N-ditetradecylacetamide (Ac-PEG).
  • DMG-PEG 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol
  • Ac-PEG methoxypolyethyleneglycoloxy-N,N-ditetradecylacetamide
  • the LNP elicits lower pro-inflammatory cytokine response than the reference LNP.
  • the LNP results in a lower uptake level of TNA by a blood cell than that of the reference LNP.
  • the blood cell is selected from the group consisting of a red blood cell, a macrophage, and a peripheral blood mononuclear cell.
  • the therapeutic nucleic acid is selected from the group consisting of a minigene, a plasmid, a minicircle, a small interfering RNA (siRNA), a microRNA (miRNA), a guide RNA (gRNA) an antisense oligonucleotide (ASO), a ribozyme, a closed-ended DNA (ceDNA), single-stranded DNA (ssDNA), a ministring, a doggyboneTM, a protelomere closed ended DNA, a dumbbell linear DNA, synthetic single stranded AAV vector, a dicer-substrate dsRNA, a small hairpin RNA (shRNA), an asymmetrical interfering RNA (aiRNA), mRNA, tRNA, rRNA, a DNA viral vector, a viral RNA vector, a non-viral vector and any combination thereof.
  • a small interfering RNA siRNA
  • miRNA microRNA
  • gRNA guide RNA
  • the TNA is greater than about 200 bp or greater than about 200 nt in length. In some embodiments, the TNA is greater than about 500 bp or greater than about 500 nt in length. In some embodiments, the TNA is greater than about 1000 bp or greater than about 1000 nt in length. In some embodiments, the TNA is greater than about 4000 bp or greater than about 4000 nt in length. In some embodiments, the TNA is a closed-ended DNA (ceDNA). In some embodiments, the TNA is a messenger RNA (mRNA). In one embodiment, the TNA is a single- stranded DNA (ssDNA).
  • mRNA messenger RNA
  • ssDNA single- stranded DNA
  • the TNA is a single-stranded (ss) nucleic acid. In some embodiments, the TNA is a double-stranded nucleic acid. In some aspects, the present disclosure also provides a pharmaceutical composition comprising the LNP of the present disclosure and a pharmaceutically acceptable carrier. In some aspects, the present disclosure also provides a method of treating a genetic disorder in a subject, the method comprising administering to the subject an effective amount of the LNP of the present disclosure or the pharmaceutical composition of the present disclosure. In some embodiments, the subject is a human.
  • the genetic disorder is selected from the group consisting of sickle cell anemia; melanoma; hemophilia A (clotting factor VIII (FVIII) deficiency); hemophilia B (clotting factor IX (FIX) deficiency); cystic fibrosis (CFTR); familial hypercholesterolemia (LDL receptor defect); hepatoblastoma; Wilson disease; phenylketonuria (PKU); congenital hepatic porphyria; an inherited disorder of hepatic metabolism; Lesch Nyhan syndrome; a thalassaemia; xeroderma pigmentosum; Fanconi’s anemia; retinitis pigmentosa; ataxia telangiectasia; Bloom’s syndrome; retinoblastoma; a mucopolysaccharide storage disease; a Niemann-Pick Disease; Fabry disease; Schindler disease; GM2-gangliosidosis Type II (Sandhoff Disease);
  • the genetic disorder is phenylketonuria (PKU). In some embodiments, the genetic disorder is hemophilia A (Factor VIII deficiency). In some embodiments, the genetic disorder is Wilson disease. In some embodiments, the genetic disorder is Gaucher disease. In some embodiments, the genetic disorder is Gaucher disease Type I, Gaucher disease Type II or Gaucher 11 ME146907648v.1 disease type III. In some embodiments, the genetic disorder is Leber congenital amaurosis (LCA). In some embodiments, the LCA is LCA10. In some embodiments, the genetic disorder is Stargardt disease. In some embodiments, the genetic disorder is wet macular degeneration (wet AMD).
  • PKU phenylketonuria
  • the genetic disorder is hemophilia A (Factor VIII deficiency). In some embodiments, the genetic disorder is Wilson disease. In some embodiments, the genetic disorder is Gaucher disease. In some embodiments, the genetic disorder is Gaucher disease Type I, Gaucher disease Type II or Gaucher 11 ME14690
  • the present disclosure also provides a method of providing anti-tumor immunity in a subject, the method comprising administering to the subject an effective amount of the LNP of the present disclosure or the pharmaceutical composition of the present disclosure.
  • the present disclosure also provides a method of treating a subject having a disease, disorder or condition associated with an elevated expression of a tumor antigen, the method comprising administering to the subject an effective amount of the LNP of the present disclosure or the pharmaceutical composition of the present disclosure.
  • the subject is a human.
  • the TNA is retained in the spleen for at least about 6 hours, or at least about 9 hours, or at least about 12 hours, or at least about 15 hours, or at least about 18 hours, or at least about 21 hours, or at least about 24 hours, or at least about 27 hours, or at least about 30 hours, or at least about 33 hours, or at least about 36 hours after dosing.
  • the concentration of the TNA at the start of a 12, 18, or 24-hour time window post-dosing and the concentration of the TNA at the end of the time window are within the same order of magnitude.
  • the TNA is a messenger RNA (mRNA).
  • ssDNA single-stranded DNA
  • the present disclosure further provides a method of treating a blood disease, disorder or condition in a subject, the method comprising administering to the subject an effective amount of the LNP of the present disclosure or the pharmaceutical composition of the present disclosure.
  • the blood disease, disorder or condition is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Hodgkin lymphoma (HL), multiple myeloma, a myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), adrenoleukodystrophy (ALD), Hurler syndrome, Krabbe disease (Globoid-cell leukodystrophy or GLD), metachromatic leukodystrophy (MLD), severe aplastic anemia (SAA), severe combined immunodeficiency (SCID), sickle cell disease (SCD), th
  • AML acute my
  • the TNA is a messenger RNA (mRNA). In one embodiment, the TNA is a single-stranded DNA (ssDNA). In some aspects, the present disclosure also provides a lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; wherein the TNA is a deoxyribonucleic acid (DNA) or a messenger ribonucleic acid (mRNA); an ionizable lipid; a sterol; 12 ME146907648v.1 a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises: a polymer; a lipid moiety comprising at least one or two hydrophobic tails; and optionally a linker connecting to the polymer to the lipid moiety; wherein each of the at least two hydrophobic tails independently comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; and wherein the LNP does
  • the present disclosure also provides a lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; an ionizable lipid; a sterol; a first lipid-anchored polymer and a second lipid-anchored polymer; wherein the first lipid- anchored polymer and the second lipid-anchored polymer each comprise: a polymer; a lipid moiety comprising at least one or two hydrophobic tails; wherein each of the at least two hydrophobic tails independently comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; and a linker connecting the polymer to the lipid moiety; wherein the second lipid-anchored polymer is conjugated to a targeting moiety; and wherein the LNP does not comprise a helper lipid.
  • TAA therapeutic nucleic acid
  • a sterol a first lipid-anchored polymer and a second lipid-anchored polymer
  • the present disclosure also provides a lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; an ionizable lipid; a sterol; a first lipid-anchored polymer and a second lipid-anchored polymer; wherein the first lipid- anchored polymer and the second lipid-anchored polymer each comprise: a polymer; a lipid moiety comprising at least one or two hydrophobic tails; wherein each of the at least two hydrophobic tails independently comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; and optionally a linker connecting the polymer to the lipid moiety; wherein the second lipid-anchored polymer is conjugated to a targeting moiety; and 13 ME146907648v.1 wherein the LNP does not comprise a helper lipid, and wherein the composition is prepared by a method comprising the following steps: a) adding the TAA) greater
  • the present disclosure also provides a lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; an ionizable lipid; a sterol; a first lipid-anchored polymer and a second lipid-anchored polymer; wherein the first lipid- anchored polymer and the second lipid-anchored polymer each comprise: a polymer; a lipid moiety comprising at least one or two hydrophobic tails; wherein each of the at least two hydrophobic tails independently comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; and optionally a linker connecting the polymer to the lipid moiety; wherein the second lipid-anchored polymer is conjugated to a targeting moiety; and wherein the LNP does not comprise a helper lipid, and wherein the composition is prepared by a method comprising the following steps: a) adding the TNA cargo to a solution comprising the
  • the targeting moiety is a tissue- and/or cell-type specific targeting moiety.
  • the targeting moiety is selected from the group consisting of a protein, a nucleic acid, and a sugar.
  • the targeting moiety is an antibody, antibody fragment, or antibody derivative.
  • the antibody, antibody fragment, or antibody derivative is selected from the group consisting of a full- length antibody, a Fab, a Fab’, a single-domain antibody, a single-chain antibody, and a VHH.
  • the antibody, antibody fragment, or antibody derivative is an scFv.
  • the antibody, antibody fragment, or antibody derivative is a VHH.
  • the VHH is a nanobody.
  • the targeting moiety is located on the exterior of the LNP.
  • the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative.
  • the targeting moiety is an aptamer.
  • the targeting moiety binds specifically to a T cell antigen.
  • the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, PD-1, and TCR.
  • the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD5, CD6, and CD7.
  • the LNP further comprises a linker between the second lipid-anchored polymer and the targeting moiety.
  • the first lipid-linker and the second lipid-linker are each independently selected from the group consisting of a non-ester-containing linker and an ester-containing linker.
  • the ester-containing linker is selected from the group consisting of an amide linker and a carbamate linker.
  • the targeting moiety is conjugated to the second lipid-anchored polymer via maleimide conjugation.
  • the targeting moiety is conjugated to the second lipid-anchored polymer via click chemistry.
  • FIG.1 shows the percent change in body weight of mice at Day 1 following administration of inventive LNP formulations LNP4, LNP5, LNP6, LNP7 (e.g., having no helper lipid) and Control LNP B formulation that contains the helper lipid distearoylphosphatidylcholine (DSPC)).
  • FIG.2C shows the whole blood and plasma levels of the ceDNA cargo of Control LNP B formulation and inventive LNP formulation LNP4 in CD-1 mice at 1 hour, 3 hours, and 6 hours post- dosing.
  • FIG.4A shows the in vitro expression of luciferase in primary mouse hepatocytes that were treated with inventive LNP formulations LNP8, LNP9, LNP10, LNP11 (e.g., having no helper lipid) that each carried an mRNA luciferase cargo.
  • FIG.4B shows the DiD signals that indicate the uptake of these inventive LNP formulations into the primary mouse hepatocytes.
  • FIG.5 shows and compares the 24-hour total IVIS fluorescence in the liver of CD-1 mice groups dosed with LNP101, LNP102, LNP103, LNP104, and Control LNP C, all of which carry luciferase mRNA as a nucleic acid cargo.
  • FIG.6A is a curve quantifying, via qPCR, concentrations of luciferase mRNA ( ⁇ g/mL) in whole blood at 2 minutes, 1 hour, 6 hours, and 24 hours after dosing for CD-1 mice groups dosed with LNP101, LNP102, LNP103, LNP104, and Control LNP C.
  • FIG.6B is a curve quantifying, via qPCR, copies of luciferase mRNA in the liver at 6 hours and 24 hours after dosing for CD-1 mice groups dosed with LNP101, LNP102, LNP103, LNP104, and Control LNP C.
  • FIG.6C is a curve quantifying, via qPCR, copies of luciferase mRNA) in the spleen at 6 hours and 24 hours after dosing for CD-1 mice groups dosed with LNP101, LNP102, LNP103, LNP104, and Control LNP C.
  • FIG.6D is a curve quantifying, via qPCR, copies of luciferase mRNA in the bone marrow at 6 hours and 24 hours after dosing for CD-1 mice groups dosed with LNP101, LNP102, LNP103, LNP104, and Control LNP C.
  • FIG.7A shows and compares the 7-day total IVIS fluorescence in the liver of CD-1 mice groups dosed with the LNPs of the present disclosure, as well as Control LNP 010, all of which carry ceDNA luciferase as a nucleic acid cargo.
  • FIG.7B shows and compares percent change in body weight at Day 1 of CD-1 mice groups dosed with the LNPs of the present disclosure, as well as Control LNP 010, all of which carry ceDNA luciferase as a nucleic acid cargo.
  • FIG.8A shows the IVIS expression data collected on Day 7 in the liver of CD-1 mice (5 per group) dosed at 1.0 mg/kg with the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different variants of Ionizable Lipid 87, and with or without a GalNAc-based targeting ligand.
  • FIG.8B shows the percent change in body weight of CD-1 mice (5 per group) at Day 1 following administration of the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different variants of Ionizable Lipid 87, and with or without a GalNAc-based targeting ligand.
  • FIG.9A shows the DiD signals that indicate the uptake of these inventive LNP formulations (e.g., having no helper lipid) into the primary mouse hepatocytes.
  • FIG.9B shows the in vitro expression of luciferase in primary mouse hepatocytes that were treated with inventive LNP formulations (e.g., having no helper lipid) that each carried an mRNA luciferase cargo.
  • FIG.10A schematically shows the formulation approach of adding/subtracting alternative polymer (e.g., 5% DMG-PEG or mono-C18-PEG) to/from total lipid content of the base LNP compositions (e.g., LNP Formulations 617, 618 and 619) of the present disclosure (e.g., having no helper lipid).
  • FIG.10B shows the IVIS expression data collected on Day 7 in the liver of CD-1 mice (5 per group) dosed at 2.0 mg/kg with the LNPs of the present disclosure (e.g., having no helper lipid) formulated according to the scheme as shown in FIG.10A.
  • FIG.10C shows the percent change in body weight of CD-1 mice (5 per group) at Day 1 following administration of the LNPs of the present disclosure (e.g., having no helper lipid) formulated according to the scheme as shown in FIG.10A.
  • FIGs.11A and 11B show the percent change in body weight of CD-1 mice (5 per group) at Day 1, and longitudinal body weight loss over the course of the study, following administration of the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different processes, and with or without a GalNAc-based targeting ligand.
  • FIGs.12A and 12B show the IVIS expression data collected on Days 4 and 7, respectively, in the liver of CD-1 mice (5 per group) dosed at 0.5, 2.0 and 4.0 mg/kg with the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different processes, and with or without a GalNAc-based targeting ligand.
  • FIGs.14A and 14B show the whole blood and plasma PK profiles of the ceDNA cargo of the stealth LNP formulations of the present disclosure prepared by Old Process (OP) or New Process (NP), with or without a GalNAc-based targeting ligand. Test articles were compared against a control LNP prepared with dissociable C14 tailed DMG-PEG2000.
  • FIGs.16D-16F show ISH staining of mouse livers collected at 6 hours post-administration of of the stealth LNP formulations of the present disclosure with higher amount of cholesterol, and with or without a GalNAc-based targeting ligand. Test articles were compared against a control LNP prepared with dissociable Ac-PEG.
  • FIGs.17A and 17B show the IVIS expression data collected on Days 4 and 7 in the liver of CD-1 mice (5 per group) dosed at 0.5 and 2.0 mg/kg with the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different amounts of cholesterol.
  • FIGs.18A and 18B show the percent change in body weight of CD-1 mice (5 per group) at Day 1, and longitudinal body weight loss over the course of the study, following administration of the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different amounts of cholesterol.
  • FIGs.21A and 21B show the results of a screening study of the LNP formulations of the present disclosure with antibody (VHH; “A05”) conjugation for targeting hepatic ASGPR1 protein, for their relative ability to gain entry into primary human hepatocytes after 24 hours, according to the workflow as depicted in FIG.20.
  • FIGs.22A and 22B show the results of a screening study of the LNP formulations of the present disclosure with antibody (VHH; “A05”) conjugation for targeting hepatic ASGPR1 protein, for their relative ability to express mLuc and rLuc cargo, according to the workflow as depicted in FIG.20.
  • FIG.23 depicts a workflow for using primary human hepatocytes to screen and compare various LNP formulations for their relative ability to enter cells, with an endocytosis inhibitor.
  • FIGs.24A and 24B show the results of a screening study of the LNP formulations of the present disclosure with antibody (VHH (“A05”) and scFv) conjugation for targeting hepatic ASGPR1 18 ME146907648v.1 protein, for their relative ability to gain entry into primary human hepatocytes after 24 hours, according to the workflow as depicted in FIG.23.
  • FIGs.25A and 25B show the results of a screening study of the LNP formulations of the present disclosure with antibody (VHH (“A05”) and scVc) conjugation for targeting hepatic ASGPR1 protein, for their relative ability to express mLuc cargo, with varying inhibition conditions, according to the workflow as depicted in FIG.23.
  • FIGs.26A and 26B show the results of a screening study of the LNP formulations of the present disclosure with antibody (VHH; “A05”) conjugation for targeting hepatic ASGPR1 protein, for their relative ability to gain entry into primary human hepatocytes after 24 hours, according to the workflow as depicted in FIG.23.
  • FIGs.27A and 27B show the results of a screening study of the LNP formulations of the present disclosure with antibody (VHH; “A05”) conjugation for targeting hepatic ASGPR1 protein, for their relative ability to express mLuc and rLuc cargo, according to the workflow as depicted in FIG.23.
  • the present disclosure provides lipid nanoparticles (LNPs) and LNP compositions (e.g., pharmaceutical compositions), wherein the LNP comprises a therapeutic nucleic acid (TNA), an ionizable lipid; a structural lipid, e.g., a sterol; and one or more types of lipid-anchored polymers, wherein the LNP does not comprise a helper lipid.
  • TAA therapeutic nucleic acid
  • a structural lipid e.g., a sterol
  • lipid-anchored polymers e.g., a helper lipid
  • the abbreviation “e.g.” is synonymous with the term “for example.”
  • the use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.
  • the term “about,” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • “comprise,” “comprising,” and “comprises” are meant to be synonymous with “include”, “including”, “includes” or “contain”, “containing”, “contains” and are inclusive or open- ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.
  • administering refers to introducing a composition or agent (e.g., nucleic acids, in particular ceDNA) into a subject and includes concurrent and sequential introduction of one or more compositions or agents.
  • administering can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods.
  • administering also encompasses in vitro and ex vivo treatments.
  • Administration includes self-administration and the administration by another. Administration can be carried out by any suitable route.
  • a suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.
  • immunogenicity of an LNP refers to the ability of a composition comprising LNPs of the present disclosure to stimulate an undesired immune response in a subject after the LNPs of the disclosure or a composition comprising the LNPs of the disclosure are administered to the subject.
  • the immune response e.g., before and after administration of a composition comprising LNPs of the present disclosure, may be measured by measuring levels of one or more pro- inflammatory cytokines.
  • Exemplary pro-inflammatory cytokines that may be used to determine immunogenicity of LNPs of the present disclosure or a composition comprising LNPs of the present disclosure include, but are not limited to, granulocyte colony stimulating factor (G-CSF), interleukin 1 alpha (IL-1 ⁇ ), interleukin 1 beta (IL-1 ⁇ ), interleukin 6 (IL-6), interleukin 8 (IL-8 or CXCL8), interleukin 11 (IL-11), interleukin 17 (IL-17), interleukin 18 (IL-18), interferon ⁇ (IFN- ⁇ ), interferon ⁇ (IFN- ⁇ ), interferon ⁇ (IFN- ⁇ ), C-X-C motif chemokine ligand 10 (CXCL10 or IP-10), monocyte chemoattractant protein 1 (MCP-1), CD40L, CCL2, CCL3, CCL4, CCL5, CCL11, tumor necrosis factor ⁇ (TNF- ⁇ ), and combinations thereof.
  • G-CSF granulocyte colony
  • off-target delivery refers to delivery of LNPs of the disclosure to non-target cells.
  • an LNP of the disclosure comprising GalNAc targets delivery of the LNP to hepatocytes
  • off-target delivery of the LNP refers to the delivery of the LNP to random, non-target cells that are not, for example, hepatocytes.
  • the non-target cell may be a blood cell, or macrophage,.
  • the non-target cell is a macrophage.
  • the non-target cell may be a liver sinusoidal endothelial cell (LSEC cell), a spleen cell or a Kupffer cell.
  • an LNP may be delivered to a non-target cell, e.g., one or more of blood cells listed above, and may result in expression of a therapeutic nucleic acid (TNA) in the non-target cell, or may be degraded once engulfed by, e.g., a macrophage.
  • a non-target cell e.g., one or more of blood cells listed above
  • TAA therapeutic nucleic acid
  • a reference LNP that comprises a helper lipid; and /or does not comprise the first lipid-anchored polymer and comprises a reference lipid-anchored polymer comprising one or more hydrophobic tails that each independently comprise less than 16 carbon atoms in a single aliphatic chain backbone. may be characterized by a higher rate of delivery to a non-target cell, e.g., one or more of blood cells listed above, as compared to an LNP of the present disclosure.
  • an LNP of the present disclosure results in an uptake level of TNA (e.g., ceDNA, ssDNA, or mRNA) in a blood cell that is lower than that of a reference LNP.
  • the reference LNP is an LNP that comprises a helper lipid; and /or does not comprise the first lipid-anchored polymer and comprises a reference lipid-anchored polymer comprising one or more hydrophobic tails that each independently comprise less than 16 carbon atoms in a single aliphatic chain backbone.
  • the blood cell is a cell selected from the group consisting of a red blood cell, a macrophage, and a peripheral blood mononuclear cell.
  • aqueous solution refers to a composition comprising in whole, or in part, water.
  • bases includes purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.
  • carrier and “excipient” are meant to include any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • dispersion media vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically- acceptable refers to molecular entities and compositions that do not produce a toxic, an allergic, or similar untoward reaction when administered to a host.
  • the term “ceDNA” refers to capsid-free closed-ended linear double stranded (ds) duplex DNA for non-viral gene transfer, synthetic or otherwise.
  • the ceDNA is a closed-ended linear duplex (CELiD) CELiD DNA.
  • the ceDNA is a DNA-based minicircle.
  • the ceDNA is a minimalistic immunological-defined gene expression (MIDGE)-vector.
  • the ceDNA is a ministering DNA.
  • the ceDNA is a 22 ME146907648v.1 dumbbell shaped linear duplex closed-ended DNA comprising two hairpin structures of ITRs in the 5’ and 3’ ends of an expression cassette.
  • the ceDNA is a doggyboneTM DNA.
  • ITR inverted terminal repeat
  • closed-ended DNA vector refers to a capsid-free DNA vector with at least one covalently closed end and where at least part of the vector has an intramolecular duplex structure.
  • the terms “ceDNA vector” and “ceDNA” are used interchangeably and refer to a closed-ended DNA vector comprising at least one terminal palindrome. In some embodiments, the ceDNA comprises two covalently-closed ends.
  • the term “ceDNA-bacmid” refers to an infectious baculovirus genome comprising a ceDNA genome as an intermolecular duplex that is capable of propagating in E. coli as a plasmid, and so can operate as a shuttle vector for baculovirus.
  • the term “ceDNA-baculovirus” refers to a baculovirus that comprises a ceDNA genome as an intermolecular duplex within the baculovirus genome.
  • ceDNA-baculovirus infected insect cell and “ceDNA-BIIC” are used interchangeably, and refer to an invertebrate host cell (including, but not limited to an insect cell (e.g., an Sf9 cell)) infected with a ceDNA-baculovirus.
  • ceDNA genome refers to an expression cassette that further incorporates at least one inverted terminal repeat (ITR) region.
  • ITR inverted terminal repeat
  • a ceDNA genome may further comprise one or more spacer regions.
  • the ceDNA genome is incorporated as an intermolecular duplex polynucleotide of DNA into a plasmid or viral genome.
  • DNA regulatory sequences As used herein, the terms “DNA regulatory sequences,” “control elements,” and “regulatory elements,” are used interchangeably herein, and refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., DNA-targeting RNA) or a coding sequence (e.g., site-directed modifying polypeptide, or Cas9/Csn1 polypeptide) and/or regulate translation of an encoded polypeptide.
  • a non-coding sequence e.g., DNA-targeting RNA
  • a coding sequence e.g., site-directed modifying polypeptide, or Cas9/Csn1 polypeptide
  • terminal repeat includes any viral or non-viral terminal repeat or synthetic sequence that comprises at least one minimal required origin of replication and a 23 ME146907648v.1 region comprising a palindromic hairpin structure.
  • a Rep-binding sequence (“RBS” or also referred to as Rep-binding element (RBE)) and a terminal resolution site (“TRS”) together constitute a “minimal required origin of replication” for an AAV and thus the TR comprises at least one RBS and at least one TRS.
  • TRs that are the inverse complement of one another within a given stretch of polynucleotide sequence are typically each referred to as an “inverted terminal repeat” or “ITR”.
  • ITRs In the context of a virus, ITRs plays a critical role in mediating replication, viral particle and DNA packaging, DNA integration and genome and provirus rescue. TRs that are not inverse complement (palindromic) across their full length can still perform the traditional functions of ITRs, and thus, the term ITR is used to refer to a TR in a viral or non-viral AAV vector that is capable of mediating replication of in the host cell. It will be understood by one of ordinary skill in the art that in a complex AAV vector configurations more than two ITRs or asymmetric ITR pairs may be present.
  • the “ITR” can be artificially synthesized using a set of oligonucleotides comprising one or more desirable functional sequences (e.g., palindromic sequence, RBS).
  • the ITR sequence can be an AAV ITR, an artificial non-AAV ITR, or an ITR physically derived from a viral AAV ITR (e.g., ITR fragments removed from a viral genome).
  • the ITR can be derived from the family Parvoviridae, which encompasses parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin that serves as the origin of SV40 replication can be used as an ITR, which can further be modified by truncation, substitution, deletion, insertion and/or addition.
  • Parvoviridae family viruses consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates.
  • Dependoparvoviruses include the viral family of the adeno-associated viruses (AAV) which are capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine and ovine species.
  • AAV adeno-associated viruses
  • ITR sequences can be derived not only from AAV, but also from Parvovirus, lentivirus, goose virus, B19, in the configurations of wildtype, “doggy bone” and “dumbbell shape”, symmetrical or even asymmetrical ITR orientation.
  • the ITRs are typically present in both 5’ and 3’ ends of an AAV vector, ITR can be present in only one of end of the linear vector. For example, the ITR can be present on the 5’ end only.
  • the ITR can be present on the 3’ end only in synthetic AAV vector.
  • an ITR located 5’ to (“upstream of”) an expression cassette in a synthetic AAV vector is referred to as a “5’ ITR” or a “left ITR”
  • an ITR located 3’ to (“downstream of”) an expression cassette in a vector or synthetic AAV is referred to as a “3’ ITR” or a “right ITR”.
  • a “wild-type ITR” or “WT-ITR” refers to the sequence of a naturally occurring ITR sequence in an AAV genome or other dependovirus that remains, e.g., Rep binding activity and Rep nicking ability.
  • WT-ITR sequences encompasses for use herein include WT-ITR sequences as result of naturally occurring changes (e.g., a replication error).
  • WT-ITR sequences as result of naturally occurring changes (e.g., a replication error).
  • the term “substantially symmetrical WT-ITRs” or a “substantially symmetrical WT-ITR pair” refers to a pair of WT-ITRs within a synthetic AAV vector that are both wild type ITRs that have an inverse complement sequence across their entire length.
  • an ITR can be considered to be a wild-type sequence, even if it has one or more nucleotides that deviate from the canonical naturally occurring canonical sequence, so long as the changes do not affect the physical and functional properties and overall three-dimensional structure of the sequence (secondary and tertiary structures).
  • the deviating nucleotides represent conservative sequence changes.
  • a sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical sequence (as measured, e.g., using BLAST at default settings), and also has a symmetrical three-dimensional spatial organization to the other WT-ITR such that their 3D structures are the same shape in geometrical space.
  • the substantially symmetrical WT-ITR has the same A, C-C’ and B-B’ loops in 3D space.
  • a substantially symmetrical WT-ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE’) and terminal resolution site (trs) that pairs with the appropriate Rep protein.
  • RBE or RBE operable Rep binding site
  • trs terminal resolution site
  • the phrases of “modified ITR” or “mod-ITR” or “mutant ITR” are used interchangeably and refer to an ITR with a mutation in at least one or more nucleotides as compared to the WT-ITR from the same serotype.
  • the mutation can result in a change in one or more of A, C, C’, B, B’ regions in the ITR, and can result in a change in the three-dimensional spatial organization (i.e. its 3D structure in geometric space) as compared to the 3D spatial organization of a WT-ITR of the same serotype.
  • the term “asymmetric ITRs” also referred to as “asymmetric ITR pairs” refers to a pair of ITRs within a single synthetic AAV genome that are not inverse complements across their full length. As one non-limiting example, an asymmetric ITR pair does not have a symmetrical three- dimensional spatial organization to their cognate ITR such that their 3D structures are different shapes in geometrical space.
  • an asymmetrical ITR pair have the different overall geometric structure, i.e., they have different organization of their A, C-C’ and B-B’ loops in 3D space (e.g., one ITR may have a short C-C’ arm and/or short B-B’ arm as compared to the cognate ITR).
  • the difference in sequence between the two ITRs may be due to one or more nucleotide addition, deletion, truncation, or point mutation.
  • one ITR of the asymmetric ITR pair may be a wild-type AAV ITR sequence and the other ITR a modified ITR as defined herein (e.g., a non-wild- type or synthetic ITR sequence).
  • neither ITRs of the asymmetric ITR pair is a wild-type AAV sequence and the two ITRs are modified ITRs that have different shapes in geometrical space (i.e., a different overall geometric structure).
  • one mod-ITRs of an asymmetric ITR pair can have a short C-C’ arm and the other ITR can have a different modification (e.g., a single arm, or a short B-B’ arm etc.) such that they have different three- dimensional spatial organization as compared to the cognate asymmetric mod-ITR.
  • symmetric ITRs refers to a pair of ITRs within a single stranded AAV genome that are wild-type or mutated (e.g., modified relative to wild-type) dependoviral ITR sequences and are inverse complements across their full length.
  • both ITRs are wild type ITRs sequences from AAV2.
  • neither ITRs are wild type ITR AAV2 sequences (i.e., they are a modified ITR, also referred to as a mutant ITR), and can have a difference in sequence from the wild type ITR due to nucleotide addition, deletion, substitution, truncation, or point mutation.
  • an ITR located 5’ to (upstream of) an expression cassette in a synthetic AAV vector is referred to as a “5’ ITR” or a “left ITR”
  • an ITR located 3’ to (downstream of) an expression cassette in a synthetic AAV vector is referred to as a “3’ ITR” or a “right ITR”.
  • the terms “substantially symmetrical modified-ITRs” or a “substantially symmetrical mod-ITR pair” refers to a pair of modified-ITRs within a synthetic AAV that are both that have an inverse complement sequence across their entire length.
  • the modified ITR can be considered substantially symmetrical, even if it has some nucleotide sequences that deviate from the inverse complement sequence so long as the changes do not affect the properties and overall shape.
  • a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical sequence (as measured using BLAST at default settings), and also has a symmetrical three-dimensional spatial organization to their cognate modified ITR such that their 3D structures are the same shape in geometrical space.
  • a substantially symmetrical modified-ITR pair have the same A, C-C’ and B-B’ loops organized in 3D space.
  • the ITRs from a mod-ITR pair may have different reverse complement nucleotide sequences but still have the same symmetrical three-dimensional spatial organization – that is both ITRs have mutations that result in the same overall 3D shape.
  • one ITR (e.g., 5’ ITR) in a mod-ITR pair can be from one serotype
  • the other ITR (e.g., 3’ ITR) can be from a different serotype, however, both can have the same corresponding mutation (e.g., if the 5’ITR has a deletion in the C region, the cognate modified 3’ITR from a different serotype has a deletion at the corresponding position in the C’ region), such that the modified ITR pair has the same symmetrical three- dimensional spatial organization.
  • each ITR in a modified ITR pair can be from different serotypes (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) such as the combination of AAV2 and AAV6, with the modification in one ITR reflected in the corresponding position in the cognate ITR from a different serotype.
  • a substantially symmetrical modified ITR pair refers to a pair of modified ITRs (mod-ITRs) so long as the difference in nucleotide sequences between the ITRs does not affect the properties or overall shape and they have substantially the same shape in 3D space.
  • a mod-ITR that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the canonical mod-ITR as determined by standard means well known in the art such as BLAST (Basic Local Alignment Search Tool), or BLASTN at default settings, and also has a symmetrical three-dimensional spatial organization such that their 3D structure is the same 26 ME146907648v.1 shape in geometric space.
  • BLAST Basic Local Alignment Search Tool
  • a substantially symmetrical mod-ITR pair has the same A, C-C’ and B-B’ loops in 3D space, e.g., if a modified ITR in a substantially symmetrical mod-ITR pair has a deletion of a C-C’ arm, then the cognate mod-ITR has the corresponding deletion of the C-C’ loop and also has a similar 3D structure of the remaining A and B-B’ loops in the same shape in geometric space of its cognate mod-ITR.
  • an “effective amount” or “therapeutically effective amount” of an active agent or therapeutic agent, such as a therapeutic nucleic acid is an amount sufficient to produce the desired effect, e.g., inhibition of expression of a target sequence in comparison to the expression level detected in the absence of a therapeutic nucleic acid.
  • Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
  • the term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing.
  • expression products include RNA transcribed from a gene (e.g., transgene), and polypeptides obtained by translation of mRNA transcribed from a gene.
  • expression vector refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the host cell.
  • An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression vector may be a recombinant vector.
  • the term “flanking” refers to a relative position of one nucleic acid sequence with respect to another nucleic acid sequence. Generally, in the sequence ABC, B is flanked by A and C. The same is true for the arrangement AxBxC. Thus, a flanking sequence precedes or follows a flanked sequence but need not be contiguous with, or immediately adjacent to the flanked sequence.
  • spacer region refers to an intervening sequence that separates functional elements in a vector or genome. In some embodiments, spacer regions keep two functional elements at a desired distance for optimal functionality. In some embodiments, the spacer regions provide or add to the genetic stability of the vector or genome. In some embodiments, spacer regions facilitate ready genetic manipulation of the genome by providing a convenient location for cloning sites and a gap of design number of base pair.
  • expression cassette and “expression unit” are used interchangeably, and refer to a heterologous DNA sequence that is operably linked to a promoter or other DNA regulatory sequence sufficient to direct transcription of a transgene of a DNA vector, e.g., 27 ME146907648v.1 synthetic AAV vector.
  • Suitable promoters include, for example, tissue specific promoters. Promoters can also be of AAV origin.
  • the phrase “genetic disease” or “genetic disorder” refers to a disease, partially or completely, directly or indirectly, caused by one or more abnormalities in the genome, including and especially a condition that is present from birth. The abnormality may be a mutation, an insertion or a deletion in a gene.
  • the abnormality may affect the coding sequence of the gene or its regulatory sequence.
  • peptide polypeptide
  • protein protein
  • a polypeptide of the disclosure is an ApoE or an ApoB polypeptide.
  • the ApoE polypeptide is a functional fragment (or a functional portion) of the full length ApoE polypeptide.
  • the ApoE polypeptide is a functional fragment (or a functional portion) of the full length ApoB polypeptide.
  • the ApoE polypeptide is 30 amino acids in length or less.
  • the ApoB polypeptide is 30 amino acids in length or less.
  • lipid refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.
  • phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.
  • amphipathic lipids Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols, and ⁇ -acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipids described above can be mixed with other lipids including triglycerides and sterols. As used herein, the term “helper lipid” refers to a non-cationic lipid comprising at least one non-polar chain.
  • helper lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE
  • a reference LNP used herein contains a helper lipid and the reference LNP was compared with an LNP of the invention which contains no helper lipid.
  • the helper lipid as referred to herein is not conjugated to a polymer (e.g., PEG or PG).
  • the molecular structures of the representative helper lipids DSPC and DOPE having C18 hydrophobic tails are shown below. DOPE.
  • lipid-anchored polymer which may be used herein interchangeably with the term “lipid conjugate” or “lipid-conjugated polymer”, refers to a molecule comprising a lipid moiety covalently attached to a polymer, e.g., optionally via a linker.
  • lipid-anchored polymer can inhibit aggregation of LNPs and provide steric stabilization.
  • lipid-anchored polymers include, but are not limited to, PEGylated lipids such as, e.g., PEG coupled to dialkyloxypropyls (e.g., PEG- DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides (see, e.g., U.S.
  • PEGylated lipids such as, e.g., PEG coupled to dialkyloxypropyls (e.g., PEG- DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides (
  • Patent No.5,885,613 cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ- DAA conjugates; see, e.g., U.S. Provisional Application No.61/294,828, filed Jan.13, 2010, and U.S. 29 ME146907648v.1 Provisional Application No.61/295,140, filed Jan.14, 2010), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. Additional examples of POZ-lipid conjugates are described in International Patent Application Publication No. WO 2010/006282. PEG or POZ can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety.
  • linker moiety suitable for coupling the PEG or the POZ to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties.
  • non-ester containing linker moieties such as amides or carbamates, are used.
  • the term “dissociable lipid-conjugated polymer” refers to a lipid-conjugated polymer that comprises a lipid moiety comprising at least one or two hydrophobic tails, wherein the at least two hydrophobic tails each comprise 10-15 carbons atoms, e.g., 14 carbon atoms, and wherein the at least one hydrophobic tail comprises 16-22 carbon atoms, e.g., 18 carbon atoms, in a single aliphatic chain backbone.
  • lipid encapsulated refers to a lipid particle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., a ceDNA, ssDNA, mRNA), with full encapsulation, partial encapsulation, or both.
  • a nucleic acid e.g., a ceDNA, ssDNA, mRNA
  • the nucleic acid is fully encapsulated in the lipid particle (e.g., to form a nucleic acid containing lipid particle).
  • lipid particle or “lipid nanoparticle” (LNP) refer to a lipid formulation that can be used to deliver a therapeutic agent, such as therapeutic nucleic acid, to a target site of interest (e.g., cell, tissue, organ, and the like).
  • a therapeutic agent such as therapeutic nucleic acid
  • a target site of interest e.g., cell, tissue, organ, and the like.
  • the lipid nanoparticle of the disclosure is typically formed from an ionizable lipid (e.g., a pH-sensitive cationic lipid), sterol (e.g., cholesterol), a conjugated lipid (e.g., lipid-anchored polymer) that prevents aggregation of the particle.
  • an ionizable lipid e.g., a pH-sensitive cationic lipid
  • sterol e.g., cholesterol
  • a conjugated lipid e.g., lipid-anchored polymer
  • the lipid particle of the disclosure is a nucleic acid containing lipid particle, which is typically formed from a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle.
  • a therapeutic agent such as a therapeutic nucleic acid may be encapsulated in the lipid portion of the 30 ME146907648v.1 particle, thereby protecting it from enzymatic degradation.
  • the lipid particle comprises a nucleic acid (e.g., ceDNA, ssDNA, mRNA) and a lipid comprising one or more - tertiary amino groups, one or more phenyl ester bonds and a disulfide bond.
  • a nucleic acid e.g., ceDNA, ssDNA, mRNA
  • a lipid comprising one or more - tertiary amino groups, one or more phenyl ester bonds and a disulfide bond.
  • lipid particles of the disclosure typically have a mean diameter of from about 20 nm to about 75 nm, about 20 nm to about 70 nm, about 25 nm to about 75 nm, about 25 nm to about 70 nm, from about 30 nm to about 75 nm, from about 30 nm to about 70 nm, from about 35 nm to about 75 nm, from about 35 nm to about 70 nm, from about 40 nm to about 75 nm, from about 40 nm to about 70 nm, from about 45 nm to about 75 nm, from about 50 nm to about 75 nm, from about 50 nm to about 70 nm, from about 60 nm to about 75 nm, from about 60 nm to about 70 nm, from about 65 nm to about 75 nm, from about 65 nm to about 70 nm, or about 20 nm, about 25 nm, about 30 nm to about 75 n
  • the LNPs of the disclosure have a mean diameter selected to provide an intended therapeutic effect.
  • the LNPs of the disclosure have a mean diameter that is compatible with a target organ, such that the LNPs of the disclosure are able to diffuse through the fenestrations of a target organ (e.g., liver) or a target cell subpopulation (e.g., hepatocytes).
  • a target organ e.g., liver
  • a target cell subpopulation e.g., hepatocytes
  • the lipid particles of the disclosure typically have a mean diameter of less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, less than about 20 nm in size.
  • the term “cationic lipid” refers to any lipid that is positively charged at physiological pH.
  • the cationic lipid in the lipid particles may comprise, e.g., one or more cationic lipids such as 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), 1,2-di- ⁇ -linolenyloxy-N,N-dimethylaminopropane ( ⁇ - DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), “SS-cleavable lipid”, or a mixture thereof.
  • DLinDMA 1,2-dilinoleyloxy-N,N-dimethyla
  • a cationic lipid can also be an ionizable lipid, i.e., an ionizable cationic lipid. i.e.
  • the term “cationic lipids” also encompasses lipids that are positively charged at any pH, e.g., lipids comprising quaternary amine groups, i.e., quarternary lipids. Any cationic lipid described herein comprising a primary, secondary or tertiary amine group may be converted to a corresponding quaternary lipid, for example, by treatment with chloromethane (CH3Cl) in acetonitrile (CH3CN) and chloroform (CHCl3).
  • CH3Cl chloromethane
  • CH3CN acetonitrile
  • CHCl3 chloroform
  • the term “ionizable lipid” refers to a lipid, e.g., cationic lipid, having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below 31 ME146907648v.1 physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form.
  • ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7.
  • ionizable lipid may include “cleavable lipid” or “SS- cleavable lipid”.
  • neutral lipid refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.
  • non-cationic lipid refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid that can be used as a helper lipid in LNP formulation.
  • cleavable lipid or “SS-cleavable lipid” refers to an ionizable lipid comprising a disulfide bond cleavable unit.
  • Cleavable lipids may include cleavable disulfide bond (“ss”) containing lipid-like materials that comprise a pH-sensitive amine, e.g., a tertiary amine, and self-degradable phenyl ester.
  • a SS-cleavable lipid can be an ss-OP lipid (COATSOME ® SS-OP), an ss-M lipid (COATSOME ® SS-M), an ss-E lipid (COATSOME ® SS-E), an ss-EC lipid (COATSOME ® SS-EC), an ss-LC lipid (COATSOME ® SS-LC), an ss-OC lipid (COATSOME ® SS- OC), and an ss-PalmE lipid (see, for example, Formulae I-IV), or a lipid described by Togashi et al., (2016) Journal of Controlled Release “A hepatic pDNA delivery system based on an intracellular environment sensitive vitamin E -scaffold lipid-like material with the aid of an anti-inflammatory drug” 279:262-270.
  • cleavable lipids comprise a tertiary amine, which responds to an acidic compartment, e.g., an endosome or lysosome for membrane destabilization and a disulfide bond that can be cleaved in a reducing environment, such as the cytoplasm.
  • a cleavable lipid is a cationic lipid.
  • a cleavable lipid is an ionizable cationic lipid.
  • organic lipid solution refers to a composition comprising in whole, or in part, an organic solvent having a lipid.
  • liposome refers to lipid molecules assembled in a spherical configuration encapsulating an interior aqueous volume that is segregated from an aqueous exterior. Liposomes are vesicles that possess at least one lipid bilayer. Liposomes are typical used as carriers for drug/ therapeutic delivery in the context of pharmaceutical development. They work by fusing with a cellular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical ingredient.
  • Liposome compositions for such delivery are typically composed of 32 ME146907648v.1 phospholipids, especially compounds having a phosphatidylcholine group, however these compositions may also include other lipids.
  • local delivery refers to delivery of an active agent such as an interfering RNA (e.g., siRNA) directly to a target site within an organism.
  • an agent can be locally delivered by direct injection into a disease site such as a tumor or other target site such as a site of inflammation or a target organ such as the liver, heart, pancreas, kidney, and the like.
  • nucleic acid refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof.
  • DNA may be in the form of, e.g., antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups.
  • DNA may be in the form of minicircle, plasmid, bacmid, minigene, ministring DNA (linear covalently closed DNA vector), closed-ended linear duplex DNA (CELiD or ceDNA), single-stranded DNA (ssDNA), doggyboneTM DNA, dumbbell shaped DNA, minimalistic immunological-defined gene expression (MIDGE)-vector, viral vector or nonviral vectors.
  • RNA may be in the form of small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, gRNA, viral RNA (vRNA), and combinations thereof.
  • Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid.
  • analogs and/or modified residues include, without limitation, phosphorothioates, phosphorodiamidate morpholino oligomer (morpholino), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, locked nucleic acid (LNATM), and peptide nucleic acids (PNAs).
  • nucleic acid therapeutic refers to any modality of therapeutic using nucleic acids as an active component of therapeutic agent to treat a disease or disorder. As used herein, these phrases refer to RNA-based therapeutics and DNA-based therapeutics.
  • RNA-based therapeutics include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNAs (RNAi), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA).
  • RNAi interfering RNAs
  • shRNA small hairpin RNA
  • aiRNA asymmetrical interfering RNA
  • miRNA microRNA
  • Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigene, viral DNA (e.g., Lentiviral or AAV genome) or non-viral synthetic DNA 33 ME146907648v.1 vectors, closed-ended linear duplex DNA (ceDNA / CELiD), single-stranded DNA (ssDNA), plasmids, bacmids, DOGGYBONETM DNA vectors, minimalistic immunological-defined gene expression (MIDGE)-vector, nonviral ministring DNA vector (linear-covalently closed DNA vector), or dumbbell-shaped DNA minimal vector (“dumbbell DNA”).
  • MIDGE minimalistic immunological-defined gene expression
  • nonviral ministring DNA vector linear-covalently closed DNA vector
  • dumbbell DNA dumbbell-shaped DNA minimal vector
  • nucleotides contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
  • pharmaceutically acceptable carrier includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil, and various types of wetting agents.
  • receptor is meant a polypeptide, or portion thereof, present on a cell membrane that selectively binds one or more ligands.
  • receptor as used herein is intended to encompass the entire receptor or ligand-binding portions thereof. These portions of the receptor particularly include those regions sufficient for specific binding of the ligand to occur.
  • the term “subject” refers to a human or animal, to whom treatment, including prophylactic treatment, with the therapeutic nucleic acid according to the present disclosure, is provided.
  • the animal is a vertebrate such as, but not limited to a primate, rodent, domestic animal or game animal.
  • Primates include but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus.
  • Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • domestic and game animals include, but are not limited to, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is a mammal, e.g., a primate or a human.
  • a subject can be male or female.
  • a subject can be an infant or a child.
  • the subject can be a neonate or an unborn subject, e.g., the subject is in utero.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of diseases and disorders.
  • the methods and compositions described herein can be used for domesticated animals and/or pets.
  • a human subject can be of any age, gender, race or ethnic group, e.g., Caucasian (white), Asian, African, black, African American, African European, Hispanic, Mideastern, etc.
  • the subject can be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment.
  • the subject is an embryo, a fetus, neonate, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, human neonate, human infant, human child, human 34 ME146907648v.1 adolescent, or human adult. In some embodiments, the subject is an animal embryo, or non-human embryo or non-human primate embryo. In some embodiments, the subject is a human embryo.
  • the phrase “subject in need” refers to a subject that (i) will be administered a ceDNA, mRNA or ssDNA-containing lipid particle (or pharmaceutical composition comprising a ceDNA lipid particle) according to the described disclosure, (ii) is receiving a ceDNA, mRNA, or ssDNA-containing lipid particle (or pharmaceutical composition comprising aceDNA lipid particle) according to the described disclosure; or (iii) has received a ceDNA, mRNA, ssDNA-containing lipid particle (or pharmaceutical composition comprising a ceDNA, mRNA, or ssDNA-containing lipid particle) according to the described disclosure, unless the context and usage of the phrase indicates otherwise.
  • the term “suppress,” “decrease,” “interfere,” “inhibit” and/or “reduce” generally refers to the act of reducing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition.
  • the term “systemic delivery” refers to delivery of lipid particles that leads to a broad biodistribution of an active agent such as an interfering RNA (e.g., siRNA) within an organism. Some techniques of administration can lead to the systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body.
  • Systemic delivery of LNPs can be by any means known in the art including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of LNPs is by intravenous delivery.
  • the terms “effective amount”, which may be used interchangeably with the terms “therapeutic amount”, “therapeutically effective amount”, an “amount effective”, or “pharmaceutically effective amount” of an active agent refers to an amount that is sufficient to provide the intended benefit of treatment or effect, e.g., expression or inhibition of expression of a target sequence in comparison to the expression level detected in the absence of a therapeutic nucleic acid.
  • Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art. Dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular active agent employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods.
  • the terms “effective amount”, 35 ME146907648v.1 “therapeutic amount”, “therapeutically effective amounts” and “pharmaceutically effective amounts” include prophylactic or preventative amounts of the compositions of the described invention.
  • pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and/or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes presenting during development of the disease, disorder or condition.
  • a maximum dose that is, the highest safe dose according to some medical judgment.
  • dose and “dosage” are used interchangeably herein.
  • therapeutic amount refers to non-prophylactic or non-preventative applications.
  • therapeutic effect refers to a consequence of treatment, the results of which are judged to be desirable and beneficial.
  • a therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation.
  • a therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.
  • therapeutically effective amount may be initially determined from preliminary in vitro studies and/or animal models.
  • a therapeutically effective dose may also be determined from human data.
  • the applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan.
  • General principles for determining therapeutic effectiveness which may be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below.
  • Pharmacokinetic principles provide a basis for modifying a dosage regimen to obtain a desired degree of therapeutic efficacy with a minimum of unacceptable adverse effects. In situations where the drug's plasma concentration can be measured and related to therapeutic window, additional guidance for dosage modification can be obtained.
  • the terms “treat,” “treating,” and/or “treatment” include abrogating, inhibiting, slowing or reversing the progression of a condition, ameliorating clinical symptoms of a condition, or preventing the appearance of clinical symptoms of a condition, obtaining beneficial or desired clinical results.
  • Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); 36 ME146907648v.1 and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).
  • the terms “treat,” “treating,” and/or “treatment” include abrogating, inhibiting, slowing or reversing the progression of a condition, or ameliorating clinical symptoms of a condition.
  • Beneficial or desired clinical results include, but are not limited to, preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization (i.e., not worsening) of the disease, disorder or condition, preventing spread of the disease, disorder or condition, delaying or slowing of the disease, disorder or condition progression, amelioration or palliation of the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.
  • proliferative treatment preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization (i.e., not worsening) of
  • the term “combination therapy” refers to treatment regimens for a clinical indication that comprise two or more therapeutic agents.
  • the term refers to a therapeutic regimen in which a first therapy comprising a first composition (e.g., active ingredient) is administered in conjunction with a second therapy comprising a second composition (active ingredient) to a patient, intended to treat the same or overlapping disease or clinical condition.
  • the first and second compositions may both act on the same cellular target, or discrete cellular targets.
  • the phrase “in conjunction with,” in the context of combination therapies, means that therapeutic effects of a first therapy overlaps temporarily and/or spatially with therapeutic effects of a second therapy in the subject receiving the combination therapy.
  • the combination therapies may be formulated as a single formulation for concurrent administration, or as separate formulations, for sequential administration of the therapies.
  • alkyl refers to a saturated monovalent hydrocarbon radical of 1 to 20 carbon atoms (i.e., C 1-20 alkyl). “Monovalent” means that alkyl has one point of attachment to the remainder of the molecule. In one embodiment, the alkyl has 1 to 12 carbon atoms (i.e., C 1-12 alkyl) or 1 to 10 carbon atoms (i.e., C 1-10 alkyl).
  • Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2- pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2- hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2- methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like.
  • a linear or branched alkyl such as a “linear or branched C1-6 alkyl,” “linear or branched C1-4 alkyl,” or “linear or branched C1-3 alkyl” means that the saturated monovalent hydrocarbon radical is a linear or 37 ME146907648v.1 branched chain.
  • the term “linear” as referring to aliphatic hydrocarbon chains means that the chain is unbranched.
  • alkylene refers to a saturated divalent hydrocarbon radical of 1 to 20 carbon atoms (i.e., C1-20 alkylene), examples of which include, but are not limited to, those having the same core structures of the alkyl groups as exemplified above.
  • “Divalent” means that the alkylene has two points of attachment to the remainder of the molecule.
  • the alkylene has 1 to 12 carbon atoms (i.e., C1-12 alkylene) or 1 to 10 carbon atoms (i.e., C1-10 alkylene).
  • the alkylene has 1 to 8 carbon atoms (i.e., C1-8 alkylene), 1 to 7 carbon atoms (i.e., C1-7 alkylene), 1 to 6 carbon atoms (i.e., C1-6 alkylene), 1 to 4 carbon atoms (i.e., C1-4 alkylene), 1 to 3 carbon atoms (i.e., C1-3 alkylene), ethylene, or methylene.
  • a linear or branched alkylene such as a “linear or branched C1-6 alkylene,” “linear or branched C1-4 alkylene,” or “linear or branched C1-3 alkylene” means that the saturated divalent hydrocarbon radical is a linear or branched chain.
  • alkenyl refers to straight or branched aliphatic hydrocarbon radical with one or more (e.g., one or two) carbon-carbon double bonds, wherein the alkenyl radical includes radicals having “cis” and “trans” orientations, or by an alternative nomenclature, “E” and “Z” orientations.
  • Alkenylene refers to aliphatic divalent hydrocarbon radical of 2 to 20 carbon atoms (i.e., C 2-20 alkenylene) with one or two carbon-carbon double bonds, wherein the alkenylene radical includes radicals having “cis” and “trans” orientations, or by an alternative nomenclature, “E” and “Z” orientations. “Divalent” means that alkenylene has two points of attachment to the remainder of the molecule. In one embodiment, the alkenylene has 2 to 12 carbon atoms (i.e., C 2-16 alkenylene), 2 to 10 carbon atoms (i.e., C 2-10 alkenylene).
  • a linear or branched alkenylene such as a “linear or branched C 2-6 alkenylene,” “linear or branched C 2-4 alkenylene,” or “linear or branched C 2-3 alkenylene” means that the unsaturated divalent hydrocarbon radical is a linear or branched chain.
  • Cycloalkylene refers to a divalent saturated carbocyclic ring radical having 3 to 12 carbon atoms as a monocyclic ring, or 7 to 12 carbon atoms as a bicyclic ring. “Divalent” means that the cycloalkylene has two points of attachment to the remainder of the molecule. In one embodiment, the cycloalkylene is a 3- to 7-membered monocyclic or 3- to 6-membered monocyclic.
  • Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, cyclononylene, cyclodecylene, cycloundecylene, cyclododecylene, and the like.
  • the cycloalkylene is cyclopropylene.
  • heterocycle refers to a cyclic group which contains at least one N atom has a heteroatom and optionally 1-3 additional heteroatoms selected from N and S, and are non-aromatic (i.e., partially or fully saturated). It can be monocyclic or bicyclic (bridged or fused).
  • Examples of 38 ME146907648v.1 heterocyclic rings include, but are not limited to, aziridinyl, diaziridinyl, thiaziridinyl, azetidinyl, diazetidinyl, triazetidinyl, thiadiazetidinyl, thiazetidinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, isothiazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, azepanyl, azocanyl, and the like.
  • the heterocycle contains 1 to 4 heteroatoms, which may be the same or different, selected from N and S. In one embodiment, the heterocycle contains 1 to 3 N atoms. In another embodiment, the heterocycle contains 1 or 2 N atoms. In another embodiment, the heterocycle contains 1 N atom.
  • a “4- to 8-membered heterocyclyl” means a radical having from 4 to 8 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring.
  • a “5- or 6-membered heterocyclyl” means a radical having from 5 or 6 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring.
  • the term “heterocycle” is intended to include all the possible isomeric forms. Heterocycles are described in Paquette, Leo A., Principles of Modern Heterocyclic Chemistry (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J.
  • the heterocyclyl groups may be carbon (carbon-linked) or nitrogen (nitrogen-linked) attached to the rest of the molecule where such is possible. If a group is described as being “optionally substituted,” the group may be either (1) not substituted, or (2) substituted. If a carbon of a group is described as being optionally substituted with one or more of a list of substituents, one or more of the hydrogen atoms on the carbon (to the extent there are any) may separately and/or together be replaced with an independently selected optional substituent.
  • Suitable substituents for an alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl are those which do not significantly adversely affect the biological activity of the molecule.
  • the substituent for the optionally substituted alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl described above is selected from the group consisting of halogen, -CN, -NR101R102, -CF3, -OR100, aryl, heteroaryl, heterocyclyl, -SR101, -SOR101, -SO2R101, and -SO3M.
  • the suitable substituent is selected from the group consisting of halogen, -OH, -NO2, -CN, C1-4 alkyl, -OR100, NR101R102, -NR101COR102, - SR100, -SO2R101, -SO2NR101R102, -COR101, -OCOR101, and -OCONR101R102, wherein R100, R101, and R102 are each independently -H or C1-4 alkyl.
  • Halogen as used herein refers to F, Cl, Br or I.
  • Cyano is –CN.
  • pharmaceutically acceptable salt refers to pharmaceutically acceptable organic or inorganic salts of an ionizable lipid of the disclosure.
  • Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate “mesylate,” ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1’-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth
  • a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion.
  • the counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
  • a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counter ion.
  • One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
  • the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes. 40 ME146907648v.1 Other terms are defined herein within the description of the various aspects of the disclosure.
  • LNPs Lipid Nanoparticles
  • LNPs lipid nanoparticles
  • TAA therapeutic nucleic acid
  • a structural lipid e.g., a sterol
  • one or more lipid-anchored polymers e.g., a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the LNP does not comprise a helper lipid.
  • LNPs consisting essentially of a therapeutic nucleic acid (TNA); an ionizable lipid; a structural lipid (e.g., a sterol); and one or more lipid-anchored polymers, e.g., a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the LNP does not comprise a helper lipid.
  • TAA therapeutic nucleic acid
  • structural lipid e.g., a sterol
  • lipid-anchored polymers e.g., a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the LNP does not comprise a helper lipid.
  • LNPs consisting of a therapeutic nucleic acid (TNA); an ionizable lipid; a structural lipid (e.g., a sterol); and one or more lipid-anchored polymers, e.g., a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the LNP does not comprise a helper lipid.
  • the helper lipids as referred to herein are not conjugated to a polymer.
  • An LNP of the present disclosure does not comprise a helper lipid, such as distearoylphosphatidylcholine (DSPC).
  • DSPC distearoylphosphatidylcholine
  • an LNP of the present disclosure does not comprise 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, an LNP of the present disclosure does not comprise a phosphatidylcholine that is not conjugated to a polymer. In some embodiments, an LNP of the present disclosure does not comprise 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE).
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
  • DOPE 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine
  • the ionizable lipid is present in the LNP provided by the present disclosure in an amount of about 20 mol% to about 70 mol%, about 20 mol% to about 65 mol%, about 20 mol% to about 60 mol%, about 20 mol% to about 55 mol%, about 20 mol% to about 50 mol%, about 25 mol% to about 70 mol%, about 25 mol% to about 65 mol%, about 25 mol% to about 60 mol%, about 25 mol% to about 55 mol%, about 25 mol% to about 50 mol%, about 30 mol% to about 70 mol%, about 30 mol% to about 65 mol%, about 30 mol% to about 60 mol%, about 30 mol% to about 55 mol%, about 30 mol% to about 50 mol%, about 35 mol% to about 70 mol%, about 35 mol% to about 65 mol%, about 35 mol% to about 60 mol%, about 35 mol% to about 60 mol%, about 35
  • the LNPs provided by the present disclosure comprise an ionizable lipid.
  • Exemplary ionizable lipids in the LNPs of the present disclosure are described in International Patent Application Publication Nos. WO2015/095340, WO2015/199952, WO2018/011633, WO2017/049245, WO2015/061467, WO2012/040184, WO2012/000104, WO2015/074085, WO2016/081029, WO2017/004143, WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541, WO2013/116126, WO2011/153120, WO2012/044638, WO2012/054365, 42 ME146907648v.1 WO2011/090965, WO2013/016058, WO2012/162210, WO2008/042973, WO2010/129709, WO2010/144740 , WO2012/099755, WO2013
  • the ionizable lipid in the LNPs of the present disclosure is represented by Formula (A): or a pharmaceutically acceptable salt thereof, wherein: R 1 and R 1’ are each independently C 1-3 alkylene; R 2 and R 2’ are each independently linear or branched C 1-6 alkylene, or C 3-6 cycloalkylene; 43 ME146907648v.1 R 3 and R 3’ are each independently optionally substituted C1-6 alkyl or optionally substituted C3-6 cycloalkyl; or alternatively, when R 2 is branched C1-6 alkylene and when R 3 is C1-6 alkyl, R 2 and R 3 , taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; or alternatively, when R 2’ is branched C1-6 alkylene and when R 3’ is C1-6 alkyl, R 2’ and R 3' , taken together with their intervening N atom, form a 4- to 8-membered heterocycly
  • R 2 and R 2’ are each independently C1-3 alkylene.
  • the linear or branched C 1-3 alkylene represented by R 1 or R 1’ , the linear or branched C 1-6 alkylene represented by R 2 or R 2’ , and the optionally substituted linear or branched C1-6 alkyl are each optionally substituted with one or more halo and cyano groups.
  • R 1 and R 2 taken together are C 1-3 alkylene and R 1’ and R 2’ taken together are C 1-3 alkylene, e.g., ethylene.
  • R 3 and R 3’ are each independently optionally substituted C 1-3 alkyl, e.g., methyl.
  • R 4 and R 4’ are each –CH.
  • R 2 is optionally substituted branched C 1-6 alkylene; and R 2 and R 3 , taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl.
  • R 2’ is optionally substituted branched C 1-6 alkylene; and R 2’ and R 3’ , taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl, such as pyrrolidinyl or piperidinyl.
  • R 4 is –C(R a ) 2 CR a , or –[C(R a ) 2 ] 2 CR a and R a is C 1-3 alkyl; and R 3 and R 4 , taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl.
  • R 4’ is –C(R a ) 2 CR a , or –[C(R a ) 2 ] 2 CR a and R a is C 1-3 alkyl; and R 3’ and R 4’ , taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl, such as pyrrolidinyl or piperidinyl.
  • R 5 and R 5’ are each independently C1-10 alkylene or C2-10 alkenylene. In one embodiment, R 5 and R 5’ are each independently C1-8 alkylene or C1-6 alkylene.
  • R 6 and R 6’ are independently C1-10 alkylene, C3-10 cycloalkylene, or C2-10 alkenylene.
  • the C3-10 cycloalkylene or the C3-6 cycloalkylene is cyclopropylene.
  • m and n are each 3. 44 ME146907648v.1
  • the ionizable lipid in the LNPs of the present disclosure may be selected from any one of the lipids listed in Table 1 below, or a pharmaceutically acceptable salt thereof: Table 1.
  • ionizable lipids of Formula (A) 45 ME146907648v.1 46 ME146907648v.1 47 ME146907648v.1 48 ME146907648v.1 49 ME146907648v.1 50 ME146907648v.1 51 ME146907648v.1 52 ME146907648v.1 53 ME146907648v.1 54 ME146907648v.1 Formula (B)
  • the ionizable lipid in the LNPs of the present disclosure is represented by Formula (B): or a pharmaceutically acceptable salt thereof, wherein: a is an integer ranging from 1 to 20 (e.g., a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); b is an integer ranging from 2 to 10 (e.g., b is 2, 3, 4, 5, 6, 7, 8, 9, or 10); 55 ME146907648v.1 R 1 is absent or is selected from (C2-C20)alkenyl, -C
  • the ionizable lipid of Formula (B) is represented by Formula (B-1): or a pharmaceutically acceptable salt thereof, wherein c and d are each independently integers ranging from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and wherein the remaining variables are as described for Formula (B).
  • c and d in Formula (B-1) are each independently integers ranging from 2 to 8, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 8, 4 to 7, 4 to 6, 5 to 8, 5 to 7, or 6 to 8, wherein the remaining variables are as described for Formula (B-1).
  • c in Formula (B-1) is 2, 3, 4, 5, 6, 7, or 8, wherein the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B).
  • c and d in Formula (B-1) are each independently 1, 3, 5, or 7, wherein the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B).
  • d in the cationic lipid of Formula (B-1) is 2, 3, 4, 5, 6, 7, or 8, wherein the remaining variables are as described for Formula (B), or the second, third or fourth embodiments of Formula (B).
  • At least one of c and d in Formula (B-1) is 7, wherein the remaining variables are as described for Formula (B), or the second, third or fourth embodiments of Formula (B).
  • the ionizable lipid of Formula (B) or Formula (B-1) is represented by Formula (B-2): (B-2); or a pharmaceutically acceptable salt thereof, wherein the remaining variables are as described for Formula (B) or Formula (B-1).
  • b in Formula (B), (B-1), or (B-2) is an integer ranging from 3 to 9, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth or sixth embodiments of Formula (B).
  • b in Formula (B), (B-1), or (B-2) is an integer ranging from 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 9, 5 to 8, 5 to 7, 6 to 9, 6 to 8, or 7 to 9, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth or sixth embodiments of Formula (B).
  • b in Formula (B), (B-1), or (B-2) is 3, 4, 5, 6, 7, 8, or 9, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth or sixth embodiments of Formula (B).
  • a in Formula (B), (B-1), or (B-2) is an integer ranging from 2 to 18, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth or seventh embodiment of Formula (B).
  • a in Formula (B), (B- 1), or (B-2) is an integer ranging from 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 25 to 8, 5 to 7, 6 to 18, 6 to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 8 to 18, 8
  • a in Formula (B), (B-1), or (B-2) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, , wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth or seventh embodiment of Formula (B).
  • R 1 in Formula (B), Formula (B-1), or Formula (B-2) is absent or is selected from (C 5 -C 15 )alkenyl, -C(O)O(C 4 -C 18 )alkyl, and cyclopropyl substituted with (C 4 -C 16 )alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B).
  • R 1 in Formula (B), Formula (B-1), or Formula (B-2) is absent or is selected from (C5-C15)alkenyl, -C(O)O(C4-C16)alkyl, and cyclopropyl substituted with (C4-C16)alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B).
  • R 1 in Formula (B), Formula (B-1), or Formula (B-2) is absent or is selected from (C5- C12)alkenyl, -C(O)O(C4-C12)alkyl, and cyclopropyl substituted with (C4-C12)alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B).
  • R 1 in the cationic lipid of Formula (B), 57 ME146907648v.1 Formula (B-1), or Formula (B-2) is absent or is selected from (C5-C10)alkenyl, -C(O)O(C4-C10)alkyl, and cyclopropyl substituted with (C4-C10)alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B).
  • R 1 is C10 alkenyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B).
  • the alkyl in C(O)O(C2-C20)alkyl, -C(O)O(C4- C18)alkyl, -C(O)O(C4-C12)alkyl, or -C(O)O(C4-C10)alkyl of R 1 in Formula (B), Formula (B-1), or Formula (B-2) is an unbranched alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, eighth or ninth embodiments of Formula (B).
  • R 1 is -C(O)O(C9 alkyl).
  • the alkyl in -C(O)O(C4-C18)alkyl, - C(O)O(C4-C12)alkyl, or -C(O)O(C4-C10)alkyl of R 1 in Formula (B), Formula (B-1), or Formula (B-2) is a branched alkyl, wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth or ninth embodiments of Formula (B).
  • R 1 is -C(O)O(C17 alkyl), wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth or ninth embodiments of Formula (B).
  • R 1 in Formula (B), Formula (B-1), or Formula (B-2) is selected from any group listed in Table 2 below, wherein the wavy bond in each of the groups indicates the point of attachment of the group to the rest of the ionizable lipid molecule, and wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B).
  • the present disclosure further contemplates the combination of any one of the R 1 groups in Table 2 with any one of the R 2 groups in Table 3 in Formula (B), wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B). Table 2.
  • R 1 groups in Formula (B), Formula (B-1), or Formula (B-2) 58 ME146907648v.1
  • R 2 in Formula (B) or a pharmaceutically acceptable salt thereof is selected from any group listed in Table 3 below, wherein the wavy bond in each of the groups indicates the point of attachment of the group to the rest of the ionizable lipid molecule, and wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh or eighth, ninth, tenth, eleventh or twelfth embodiments of Formula (B).
  • ionizable lipid in the LNPs of the present disclosure are represented by Formula (C): or a pharmaceutically acceptable salt thereof, wherein: R 1 and R 1’ are each independently (C1-C6)alkylene optionally substituted with one or more groups selected from R a ; R 2 and R 2’ are each independently (C1-C2)alkylene; R 3 and R 3’ are each independently (C1-C6)alkyl optionally substituted with one or more groups selected from R
  • R 1 and R 1 are each independently (C1-C6)alkylene, wherein the remaining variables are as described above for Formula (C).
  • R 1 and R 1’ are each independently (C1-C3)alkylene, wherein the remaining variables are as described above for Formula (C).
  • the ionizable lipid of the Formula (C) is represented by Formula (C-1): or a pharmaceutically acceptable salt thereof, wherein R 2 and R 2’ , R 3 and R 3’ , R 4 and R 4 ’ and R 5 and R 5 ’ are as described above for Formula (C) or the second embodiment of Formula (C).
  • the ionizable lipid of Formula (C) is represented by Formula (C-2) or Formula or a pharmaceutically acceptable salt thereof, wherein R 4 and R 4 ’ and R 5 and R 5 ’ are as described above for Formula (C).
  • the ionizable lipid of Formula (C) is represented by Formula (C-4) or (C-5): or a pharmaceutically acceptable salt thereof, wherein R 5 and R 5 ’ are as described above for Formula (C).
  • the ionizable lipid of Formula (C) is represented by Formula (C-6), (C-7), (C-8), or (C-9): 65 ME146907648v.1 or a pharmaceutically acceptable salt thereof, wherein R 5 and R 5 ’ are as described above for Formula (XV).
  • R 5 and R 5 ’ are as described above for Formula (XV).
  • at least one of R 5 and R 5’ in Formula (C), (C-1), (C- 2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5 and R 5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C 6 -C 26 )alkyl or (C 6 -C 26 )alkenyl, each of which are optionally interrupted with –C(O)O- or (C 3 -C 6 )cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5 in Formula (C), (C-1), (C- 2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C 6 -C 26 )alkyl or (C 6 -C 26 )alkenyl, each of which are optionally interrupted with –C(O)O- or (C 3 -C 5 )cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C 7 -C 26 )alkyl or (C 7 - C 26 )alkenyl, each of which are optionally interrupted with –C(O)O- or (C 3 -C 5 )cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C 8 -C 26 )alkyl or (C 8 -C 26 )alkenyl, each of which are optionally interrupted with –C(O)O- or (C 3 - C5)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C- 6), (C-7), (C-8), or (C-9) is a (C 6 -C 24 )alkyl or (C 6 -C 24 )alkenyl, each of which are optionally 66 ME146907648v.1 interrupted with –C(O)O- or cyclopropyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5 in Formula (C), (C-1), (C- 2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C8-C24)alkyl or (C8-C24)alkenyl, wherein said (C8-C24)alkyl is optionally interrupted with –C(O)O- or cyclopropyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C8-C10)alkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C14-C16)alkyl interrupted with cyclopropyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C10-C24)alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C 16 -C 18 )alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • R 5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C- 8), or (C-9) is a (C 15 -C 28 )alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C).
  • R 5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C 17 -C 28 )alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C).
  • R 5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C 19 -C 28 )alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C).
  • R 5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C17-C26)alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C).
  • R 5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C19-C26)alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C).
  • R 5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C20-C26)alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C).
  • R 5’ is a (C22-C24)alkyl interrupted with –C(O)O-, and the remaining 67 ME146907648v.1 variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C).
  • R 5’ is –(CH2)5C(O)OCH[(CH2)7CH3]2, –(CH2)7C(O)OCH[(CH2)7CH3]2, – (CH2)5C(O)OCH(CH2)2[(CH2)7CH3]2, or –(CH2)7C(O)OCH(CH2)2[(CH2)7CH3]2, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C).
  • the ionizable lipid of Formula (C), (C-1), (C-3), (C-3), (C-4), (C-5), (C-7), (C-8), or (C-9) may be selected from any of the lipids listed in Table 5 below, or pharmaceutically acceptable salts thereof. Table 5.
  • the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (D): or a pharmaceutically acceptable salt thereof, wherein: R’ is absent, hydrogen, or C 1 -C 6 alkyl; provided that when R’ is hydrogen or C 1 -C 6 alkyl, the nitrogen atom to which R’, R 1 , and R 2 are all attached is positively charged; R 1 and R 2 are each independently hydrogen, C1-C6 alkyl, or C2-C6 alkenyl; R 3 is C1-C12 alkylene or C2-C12 alkenylene; R 4 is C 1 -C 18 unbranched alkyl, C 2 -C 18 unbranched
  • X 1 and X 2 are the same; and all other remaining variables are as described for Formula (C).
  • the ionizable lipid e.g., cationic lipid, in the LNPs of the present disclosure, is represented by Formula (D-1): (D-1) or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, 3, and 4; and all other remaining variables are as described for Formula (D) or the second or third embodiments of Formula (D).
  • the ionizable lipid e.g., cationic lipid, in the LNPs of the present disclosure, is represented by Formula (D-2): (D-2) or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, and 3; and all other remaining variables are as described for Formula (D) or the second or third embodiments of Formula (D).
  • the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (D-3): or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (D) or the second or third embodiments of Formula (D).
  • R 1 and R 2 are each independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl, or C1-C5 alkyl or C2-C5 alkenyl, or C1-C4 alkyl or C2-C4 alkenyl, or C6 alkyl, or C5 alkyl, or C4 alkyl, or C3 alkyl, or C2 alkyl, or C1 alkyl, or C6 alkenyl, or C5 alkenyl, or C4 alkenyl, or C3 alkenyl, or C2 alkenyl; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3) or the second or third embodiments of Formula (D).
  • the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (D-4): or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3) or the second, third or seventh embodiments of Formula (D).
  • R 3 is C1-C9 alkylene or C2-C9 alkenylene, C1-C7 alkylene or C2- C7 alkenylene, C1-C5 alkylene or C2-C5 alkenylene, or C2-C8 alkylene or C2-C8 alkenylene, or C3-C7 alkylene or C3-C7 alkenylene, or C5-C7 alkylene or C5-C7 alkenylene; or R 3 is C12 alkylene, C11 alkylene, C10 alkylene, C9 alkylene, or C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C3 alkylene
  • R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; or R 5 is absent, C1-C4 alkylene, or C2-C4 alkenylene; or R 5 is absent; or R 5 is C8 alkylene, C7 alkylene, C6 alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, C1 alkylene, C8 alkenylene, C7 alkenylene, C6 alkenylene, C5 alkenylene, C4 alkenylene, C3 alkenylene, or C2 alkenylene; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula
  • R 4 is C 1 -C 14 unbranched alkyl, C 2 - C14 unbranched alkenyl, or R 4a and R 4b are each independently C1-C12 unbranched alkyl or C2-C12 unbranched alkenyl; or R 4 is C2-C12 unbranched alkyl or C2-C12 unbranched alkenyl; or R 4 is C5-C7 unbranched alkyl or C5-C7 unbranched alkenyl; or R 4 is C16 unbranched alkyl, C15 unbranched alkyl, C14 unbranched alkyl, C13 unbranched alkyl, C12 unbranched alkyl, C11 unbranched alky
  • R 6a and R 6b are each independently C6-C14 alkyl or C6- C14 alkenyl; or R 6a and R 6b are each independently C8-C12 alkyl or C8-C12 alkenyl; or R 6a and R 6b are each independently C16 alkyl, C15 alkyl, C14 alkyl, C13 alkyl, C12 alkyl, C11 alkyl, C10 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C16 alkenyl, C15 alkenyl, C14 alkenyl, C13 alkenyl, C12 alkenyl, C11 alkenyl, C10
  • R 6a and R 6b contain an equal number of carbon atoms with each other; or R 6a and R 6b are the same; or R 6a and R 6b are both C 16 alkyl, C 15 alkyl, C 14 alkyl, C 13 alkyl, C 12 alkyl, C 11 alkyl, C 10 alkyl, C 9 alkyl, C 8 alkyl, C 7 alkyl, C 16 alkenyl, C 15 alkenyl, C 14 alkenyl, C 13 alkenyl, C 12 alkenyl, C 11 alkenyl, C 10 alkenyl, C 9 alkenyl,
  • R 6a and R 6b as defined in any one of the preceding embodiments each contain a different number of carbon atoms with each other; or the number of carbon atoms R 6a and R 6b differs by one or two carbon atoms; or the number of carbon atoms R 6a and R 6b differs by one carbon atom; or R 6a is C7 alkyl and R 6a is C8 alkyl, R 6a is C8 alkyl and R 6a is C7 alkyl, R 6a is C8 alkyl and R 6a is C9 alkyl, R 6a is C9 alkyl and R 6a is C8 alkyl, R
  • R 4 is C1-C16 unbranched alkyl, C2-C16 unbranched alkenyl, , wherein R 4a and R 4b are as described above for the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth or fourteenth embodiments of Formula (D).
  • the ionizable lipid e.g., cationic lipid, of the present disclosure or the ionizable lipid of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or Formula (D-4) is any one lipid selected from the lipids listed in Table 6 below, or a pharmaceutically acceptable salt thereof: Table 6.
  • the ionizable lipid in the LNPs of the present disclosure comprises Lipid No.87: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.
  • the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E): 78 ME146907648v.1 (E) or a pharmaceutically acceptable salt thereof, wherein: R’ is absent, hydrogen, or C1-C3 alkyl; provided that when R’ is hydrogen or C1-C3 alkyl, the nitrogen atom to which R’, R 1 , and R 2 are all attached is positively charged; R 1 and R 2 are each independently hydrogen or C1-C3 alkyl; R 3 is C3-C10 alkylene or C3-C10 alkenylene; R 4 is C1-C16 unbranched alkyl, C2-C16 unbranched alkenyl, or ; wherein: R 4a and R 4b are each independently C1-C16 unbranched alkyl or C2-C16 unbranched alkenyl; R 5 is absent, C1-C6 alkylene, or C2-C
  • the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E-1): (E-1) or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, 3, and 4; and all other remaining variables are as described for Formula (E) or the second embodiment of Formula (E). Alternatively, n is an integer selected from 1, 2, and 3; and all other remaining variables are as described for Formula (E) or the second embodiment of Formula (E).
  • the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E-2): 79 ME146907648v.1 (E-2) or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-1) or the second embodiment of Formula (E).
  • R 1 and R 2 are each independently hydrogen or C 1 -C 2 alkyl, or C 2 -C 3 alkenyl; or R’, R 1 , and R 2 are each independently hydrogen, C 1 -C 2 alkyl; and all other remaining variables are as described for Formula (E), Formula (E-1) or the second embodiment of Formula (E).
  • the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E-3): (E-3) or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2) or the second or fifth embodiments of Formula (E).
  • R 5 is absent or C1-C8 alkylene; or R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; or R 5 is absent, C1-C4 alkylene, or C2-C4 alkenylene; or R 5 is absent; or R 5 is C8 alkylene, C7 alkylene, C6 alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, C1 alkylene, C8 alkenylene, C7 alkenylene, C6 alkenylene, C5 alkenylene, C4 alkenylene, C3 alkenylene, or C2 alkenylene; and all other remaining variables are as described for Formula (E), Formula (E), Formula (E)
  • he ionizable lipid e.g., cationic lipid
  • LNPs of the present disclosure is represented by Formula (E-4): 80 ME146907648v.1 (E-4) or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3) or the second, fifth or seventh embodiments of Formula (E).
  • R 4 is C 1 -C 14 unbranched alkyl, C 2 -C 14 unbranched alkenyl, or , wherein R 4a and R 4b are each independently C 1 -C 12 unbranched alkyl or C 2 -C 12 unbranched alkenyl; or R 4 is C 2 -C 12 unbranched alkyl or C 2 -C 12 unbranched alkenyl; or R 4 is C 5 -C 12 unbranched alkyl or C 5 -C 12 unbranched alkenyl; or R 4 is C 16 unbranched alkyl, C 15 unbranched alkyl, C 14 unbranched alkyl, C 13 unbranched alkyl, C 12 unbranched
  • R 3 is C3-C8 alkylene or C3-C8 alkenylene, C3-C7 alkylene or C3-C7 alkenylene, or C3-C5 alkylene or C3-C5 alkenylene,; or R 3 is C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C1 alkylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E
  • R 6a and R 6b are each independently C7-C12 alkyl or C7-C12 alkenyl; or R 6a and R 6b are each independently C 8 -C 10 alkyl or C 8 -C 10 alkenyl; or R 6a and R 6b are each independently C 12 alkyl, C 11 alkyl, C 10 alkyl, C 9 alkyl, C 8 alkyl, C 7 alkyl, C 12 alkenyl, C 11 alkenyl, C 10 alkenyl, C 9 alkenyl, C 8 alkenyl, or C 7 alkenyl; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (
  • R 6a and R 6b contain an equal number of carbon atoms with each other; or R 6a and R 6b are the same; or R 6a and R 6b are both C 12 alkyl, C 11 alkyl, C 10 alkyl, C 9 alkyl, C 8 alkyl, C 7 alkyl, C 12 alkenyl, C 11 alkenyl, C 10 alkenyl, C 9 alkenyl, C 8 alkenyl, or C 7 alkenyl; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth or eleventh embodiments of Formula (E), R 6a and R 6b contain an equal number of carbon atoms with each other; or R 6a and R 6b are the same; or R 6a and R 6b are both C 12 alkyl, C 11 alkyl, C 10 alkyl, C 9 alkyl, C 8 alkyl, C 7 alkyl
  • R 6a and R 6b as defined in any one of the preceding embodiments each contain a different number of carbon atoms with each other; or the number of carbon atoms R 6a and R 6b differs by one or two carbon atoms; or the number of carbon atoms R 6a and R 6b differs by one carbon atom; or R 6a is C7 alkyl and R 6a is C8 alkyl, R 6a is C8 alkyl and R 6a is C7 alkyl, R 6a is C8 alkyl and R 6a is C9 alkyl, R 6a is C9 alkyl and R 6a is C8 alkyl, R 6a is C9 alkyl and R 6a is C10 alkyl, R 6a is C10 alkyl and R 6a is C9 alkyl
  • ionizable lipid e.g., cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth, eleventh, twelfth or thirteenth embodiments of Formula (E), R’ is absent; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth, eleventh, twelfth or thirteenth embodiments of Formula (E).
  • the ionizable lipid e.g., cationic lipid, in the LNPs of the present disclosure or the cationic lipid of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) is any one lipid selected from the lipids in Table 7 or a pharmaceutically acceptable salt thereof: Table 7.
  • Exemplary lipids of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E- 4) 83 ME146907648v.1 84 ME146907648v.1 Specific examples are provided in the exemplification section below and are included as part of the cationic or ionizable lipids described herein. Pharmaceutically acceptable salts as well as neutral forms are also included.
  • the LNPs provided by the present disclosure comprise an ionizable lipid that is also a cleavable lipid.
  • cleavable lipid which may be used interchangeably with the term “SS-cleavable lipid” refers to an ionizable lipid comprising a disulfide bond (“SS”).
  • the SS in the cleavable lipid is a cleavable unit.
  • a cleavable lipid comprises an amine, e.g., a tertiary amine, e.g.and a disulfide bond.
  • Cleavable lipids also include pH-activated lipid-like materials, such as ss-OP lipids, ssPalm lipids, ss-M lipids, ss-E lipids, ss-EC lipids, ss-LC lipids and ss-OC lipids, etc.
  • SS-cleavable lipids are described in International Patent Application Publication No. WO2019188867, incorporated by reference in its entirety herein.
  • a cleavable lipid may comprise three components: an amine head group, a linker group, and a hydrophobic tail(s).
  • the cleavable lipid comprises one or more phenyl ester bonds, one of more tertiary amino groups, and a disulfide bond.
  • the tertiary amine groups provide pH responsiveness and induce endosomal escape, the phenyl ester bonds enhance the degradability of the structure (self- degradability) and the disulfide bond becomes cleaved in a reductive environment.
  • the cleavable lipid is an ss-OP lipid.
  • an ss-OP lipid comprises the structure of Lipid A shown below: Lipid A .
  • the SS-cleavable lipid is an SS-cleavable and pH-activated lipid-like material (ssPalm).
  • ssPalm lipids are well known in the art. For example, see Togashi et al., Journal of Controlled Release, 279 (2016) 262-270, the entire contents of which are incorporated herein by reference.
  • the ssPalm is an ssPalmM lipid comprising the structure of Lipid B shown below: Lipid B .
  • the ssPalmE lipid is a ssPalmE-P4-C2 lipid comprising the structure of Lipid C below: Lipid C .
  • the ssPalmE lipid is a ssPalmE-Paz4-C2 lipid, comprising the structure of Lipid D below: Lipid D 86 ME146907648v.1
  • the cleavable lipid is an ss-M lipid.
  • an ss-M lipid comprises the structure shown in Lipid E below: Lipid E
  • the cleavable lipid is an ss-E lipid.
  • an ss-E lipid comprises the structure shown in Lipid F below: Lipid F .
  • the cleavable lipid is an ss-EC lipid.
  • an ss-EC lipid comprises the structure shown for Lipid G below: Lipid G
  • the cleavable lipid is an ss-LC lipid.
  • an ss-LC lipid comprises the structure shown for Lipid H below: Lipid H 87 ME146907648v.1
  • the cleavable lipid is an ss-OC lipid.
  • an ss-OC lipid comprises the structure shown for Lipid J below: Lipid J .
  • the ionizable lipid in the LNPs of the present disclosure is selected from the group consisting of N-[1-(2,3-dioleyloxy)propyll-N,N,N-trimethylammonium chloride (DOTMA); N-[1-(2,3-dioleoyloxy)propyll-N,N,N-trimethylammonium chloride (DOTAP); 1,2- dioleoyl-sn-glycero -3-ethylphosphocholine (DOEPC); 1,2-dilauroyl-sn-glycero-3- ethylphosphocholine (DLEPC); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2- dimyristoleoyl- sn-glycero-3-ethylphosphocholine (14:1), N1- [2-((1S)-1-[(3-aminopropyl)
  • DOTAP 1,2-
  • the condensing agent e.g. a cationic lipid
  • the ionizable lipid in the LNP of the present disclosure is represented by the following structure: 88 ME146907648v.1 5 , 89 ME146907648v.1 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.
  • the LNPs provided by the present disclosure comprise a structural lipid. Without wishing to be bound by a specific theory, it is believed that a structural lipid, when present in an LNP, contributes to membrane integrity and stability of the LNP.
  • the structural lipid is a sterol, e.g., cholesterol, or a derivative thereof.
  • the structural lipid is cholesterol.
  • the structural lipid is a derivative of cholesterol.
  • cholesterol derivatives include polar analogues such as 5 ⁇ -cholestanol, 5 ⁇ -coprostanol, cholesteryl-(2’-hydroxy)-ethyl ether, cholesteryl-(4’- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5 ⁇ -cholestane, cholestenone, 5 ⁇ -cholestanone, 5 ⁇ -cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the cholesterol derivative is a polar analogue such as cholesteryl-(4’-hydroxy)- butyl ether.
  • cholesterol derivative is cholestryl hemisuccinate (CHEMS). 90 ME146907648v.1 Exemplary cholesterol derivatives are described in International Patent Application Publication No. WO2009/127060 and U.S. Patent Application Publication No. US2010/0130588, contents of both of which are incorporated herein by reference in their entirety.
  • the sterol in the LNPs of the present disclosure is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and derivatives thereof, and any combination thereof.
  • the sterol is cholesterol.
  • the sterol is beta-sitosterol.
  • the structural lipid constitutes about 20 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid constitutes about 25 mol% to about 45 mol% of the total lipid content of the LNP. In some embodiments, the structural lipid constitutes about 30 to about 45% of the total lipid present in the LNP. In some embodiments, the structural lipid constitutes about 30 mol% to about 40 mol% of the total lipid present in the LNP. In some embodiments, such a component is about 40 mol% of the total lipid present in the LNP.
  • the structural lipid e.g., a sterol
  • the structural lipid constitutes about 20 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid, e.g., a sterol, constitutes about 30 mol% to about 40 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 30 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 35 mol% to about 45 mol% of the total lipid present in the LNP.
  • the structural lipid is cholesterol and constitutes about 40 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 40 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 40 mol% to about 45 mol% of the total lipid present in the LNP, wherein the encapsulation efficiency (“Enc. Eff.”) of TNA is greater than 95% and/or the average size of the LNP ranges about 70 nm to 90 nm in diameter.
  • Enc. Eff. encapsulation efficiency
  • the LNPs provided by the present disclosure comprise at least one type of lipid-anchored polymer, i.e., a first lipid-anchored polymer.
  • lipid- anchored polymer refers to a molecule comprising a lipid moiety covalently attached to a polymer, 91 ME146907648v.1 e.g., via a linker. Without wishing to be bound by a specific theory, it is believed that a lipid-anchored polymer can inhibit aggregation of LNPs and provide steric stabilization.
  • the LNPs provided by the present disclosure comprise two lipid-anchored polymers, i.e., a first lipid- anchored polymer and a second lipid-anchored polymer.
  • a lipid-anchored polymer e.g., a first lipid-anchored polymer
  • a lipid-anchored polymer in accordance with the present disclosure comprises: (i) a polymer; (ii) a lipid moiety comprising at least two hydrophobic tails (which may be linear or branched); and (iii) a linker connecting the polymer to the lipid moiety; wherein the at least two hydrophobic tails (which may be linear or branched) comprise 16 to 22 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, 21, or 22 carbon atoms in a single aliphatic chain backbone.
  • the at least two hydrophobic tails are each a fatty acid.
  • the lipid-anchored polymer e.g., a first lipid-anchored polymer, comprises a lipid moiety comprising two hydrophobic tails, wherein the two hydrophobic tails each independently comprise 16 to 22 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, 21, or 22 carbon atoms in a single aliphatic chain backbone.
  • the two hydrophobic tails each independently comprise 16 to 21 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, or 21 carbon atoms in a single aliphatic chain backbone.
  • the two hydrophobic tails each independently comprise 16 to 20 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, or 20 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 to 19 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, or 19 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 to 18 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, or 18 carbon atoms in a single aliphatic chain backbone.
  • the two hydrophobic tails each independently comprise 16 or 18 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 or 20 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 18 or 20 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 16 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 17 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 18 carbon atoms in a single aliphatic chain backbone.
  • the two hydrophobic tails each comprise 19 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 20 carbon atoms in a single aliphatic chain backbone.
  • linker-lipid moiety refers to a lipid moiety comprising at least two hydrophobic tails, e.g., two hydrophobic tails, covalently attached to a linker. In some embodiments, the linker-lipid moiety may be a part of a lipid-anchored polymer.
  • derivative when used herein in reference to hydrophobic tails in a lipid-anchored polymer, refers to a hydrophobic tail that has been modified as compared to the original or native hydrophobic tail.
  • the derivative contains one or more of the following modifications as compared to the original or native hydrophobic tail: a) carboxylate group has been replaced with an amine group, an amide group, an ether group, or a carbonate group; b) one or more points of saturation, e.g., double bonds, have been introduced into (e.g., via dehydrogenation) the hydrophobic tail; c) one or more points of saturation, e.g., double bonds, have been removed from (e.g., via hydrogenation) the hydrophobic tail; and d) configuration of one or more double bonds, if present, has been changed, e.g., from a cis configuration to a trans configuration, or from a trans configuration to a cis configuration.
  • the derivative contains the same number of carbon atoms as its original or native hydrophobic tail.
  • a single aliphatic chain backbone when referring to a hydrophobic tail in a lipid-anchored polymer refers to the main linear aliphatic chain or carbon chain, i.e., the longest continuous linear aliphatic chain or carbon chain.
  • the alkyl chain below that has several branchings contains 18 carbon atoms in a single aliphatic chain backbone, i.e., the longest continuous linear alkyl chain contains 18 carbon atoms. Note that the one or two carbon atoms (all indicated with *) in the several branching points are not included in the carbon atom count in the single aliphatic chain backbone.
  • Linkers in lipid-anchored polymers in a lipid-anchored polymer of the present disclosure, a lipid moiety is covalently attached to a polymer via a linker.
  • the linker in the lipid-anchored polymer of the present disclosure is a glycerol linker, a phosphate linker, an ether linker, an amide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, or any combination thereof.
  • the linker in the lipid-anchored polymer in the LNPs of the present disclosure a glycerol linker.
  • the lipid-anchored polymer in the LNPs of the present disclosure is a glycerolipid, wherein the glycerolipid comprises glycerol as a linker and one or more lipid moieties as described above, e.g., distearoyl-rac-glycerol (DSG). 93 ME146907648v.1
  • the linker in the lipid-anchored polymer in the LNPs of the present disclosure is a phosphate linker.
  • the lipid-anchored polymer in the LNPs of the present disclosure is a phospholipid, wherein the phospholipid comprises a phosphate group as a linker and one or more lipid moieties as described above.
  • the lipid-anchored polymer in an LNP of the present disclosure is both a glycerolipid and a phospholipid, such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).
  • the first lipid-anchored polymer comprises a linker-lipid moiety (i.e., with one or more hydrophobic tails containing 16 to 22 carbon atoms in a single aliphatic chain) selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1- palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn-phosphatidylethanol
  • the term “derivative” when used in reference to a linker-lipid moiety means a linker-lipid moiety containing one or more of the following modifications: a) a phosphatidylethanolamine (PE) head group, if present, is modified to convert an amino group into a methylamino group or a dimethylamino group; b) the modified linker-lipid moiety comprises one or more additional functional groups or moieties, such as -OH, -OCH 3 , -NH 2 , a maleimide, an azide or a cyclooctyne such as dibonzeocyclooctyne (DBCO).
  • PE phosphatidylethanolamine
  • the first lipid-anchored polymer comprises a linker-lipid moiety (i.e., with one or more hydrophobic tails containing 16 to 22 carbon atoms in a single aliphatic chain) selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, a derivative thereof, and a combination of any of the foregoing.
  • a linker-lipid moiety i.e., with one or more hydrophobic tails containing 16 to 22 carbon atoms in a single aliphatic chain
  • Polymers in lipid-anchored polymers In some embodiments, the polymer in the lipid-anchored polymer is selected from the group consisting of polyethylene (PE), polypropylene (PP), polyethylene glycol (PEG), polyglycerol (PG), polyvinyl alcohol (PVOH), polysarcosine (pSar), and a combination thereof.
  • the polymer is selected from the group consisting of polyethylene glycol (PEG), polyglycerol (PG), polysarcosine (pSar), and a combination thereof.
  • the polymer is polyethyelene glycol (PEG).
  • the polymer is polyglycerol (PG).
  • the polymer in the lipid-anchored polymer has a molecular weight of about 5000 Da or less, e.g., about 4500 Da or less, about 4000 Da or less, about 3500 Da or less, about 3200 Da or less, about 3000 Da or less, about 2500 Da or less, about 2000 Da or less, about 1500 Da or less, about 1000 Da or less, about 500 Da or less, about 100 Da or less or about 50 Da or less.
  • the polymer in the lipid-anchored polymer has an average molecular weight of about 20 Da to about 100 Da, about 50 Da to about 500 Da, about 500 Da to about 2000 Da, about 1000 Da to about 5000 Da, e.g., about 2000 Da to about 5000 Da, about 1000 Da to about 3000 Da, about 1500 Da to about 2500 Da, about 2000 Da to about 4000 Da or about 2000 Da to about 5000 Da.
  • the polymer in the lipid-anchored polymer has an average molecular weight of about 1000 Da, about 1500 Da, about 2000 Da, about 2500 Da, about 3000 Da, about 3200 Da, about 3300 Da, about 3350 Da, about 3400 Da, about 3500 Da, about 4000 Da, about 4500 Da or about 5000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 2000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 2000 Da. In some embodiments, the polymer in the lipid- anchored polymer has an average molecular weight of about 3200 Da to about 3500 Da.
  • an LNP of the present disclosure further comprises one or more targeting moieties.
  • the targeting moiety targets the LNP for delivery to a specific site or a tissue in a subject, e.g., liver.
  • the targeting moiety is capable of binding to specific liver cells, such as hepatocytes.
  • the targeting moiety is capable of binding to the asialoglycoprotein receptor (ASGPR), e.g., hepatocyte-specific ASGPR.
  • the targeting moiety comprises an N-acetylgalactosamine molecule (GalNAc) or a GalNAc derivative thereof.
  • GalNAc derivative refers to a modified GalNAc molecule or a conjugate of one or more GalNAc molecules (modified or unmodified) covalently linked to, for example, a lipid-anchored polymer as defined herein.
  • the targeting moiety is a tri-antennary or tri-valent GalNAc conjugate (i.e., GalNAc3) which is a ligand conjugate having three GalNAc molecules or three GalNAc derivatives.
  • the targeting moiety is a tri-antennary GalNAc represented by the following structural formula: 95 ME146907648v.1
  • the targeting moiety is a tetra-antennary GalNAc conjugate. In one embodiment, the targeting moiety is a tetra-antennary or tetra-valent GalNAc conjugate (i.e., GalNAc4) which is a ligand having four GalNAc molecules or four GalNAc derivatives. In one embodiment, the targeting moiety is capable of binding to low-density lipoprotein receptors (LDLRs), e.g., hepatocyte-specific LDLRs.
  • LDLRs low-density lipoprotein receptors
  • the targeting moiety comprises an apoliprotein E (ApoE) protein, an ApoE polypeptide (or peptide), an apoliprotein B (ApoB) protein, an ApoB polypeptide (or peptide), a fragment of any of the foregoing, or a derivative of any of the foregoing.
  • the ApoE polypeptide, ApoB polypeptide, or a fragment thereof is a ApoE polypeptide, ApoB polypeptide, or a fragment thereof as disclosed in International Patent Application Publication No. WO2022/261101, which is incorporated herein by reference in its entirety.
  • the ApoE protein is a modified ApoE protein and the ApoB protein is a modified ApoB protein.
  • the ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98, or at least about 99% sequence identity to the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQE LRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYR GEVQAMLGQSTEELRVR
  • the ApoE protein comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 1.
  • the ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98, or at least about 99% sequence identity to the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQE LRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYR 96 ME146907648v.1 GEVQA
  • the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98, or at least about 99% sequence identity to the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQE LRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMED
  • the ApoE protein comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 3.
  • the ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98, or at least about 99% sequence indentity to the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQE LRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYR GEVQAMLGQSTEELRVR
  • the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO: 4. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO: 4.
  • sequence identity refers to the ratio of the number of identical amino acids between the 2 aligned sequences over the aligned length, expressed as a percentage. In some embodiments, the 2 aligned sequences are identical in length, i.e., have the same number of amino acids.
  • the targeting moiety in an LNP of the present disclosure is an ApoE protein conjugate in an ApoB protein conjugate, which is a conjugate of one or more ApoE and/or ApoB protein molecules (native or modified) or a fragment thereof covalently linked to, for example, a lipid-anchored polymer as defined herein.
  • the targeting moiety in an LNP of the present disclosure is an ApoE polypeptide conjugate in an ApoB polypeptide conjugate, which is a conjugate of one or more ApoE and/or ApoB polypeptide molecules or a fragment thereof covalently linked to, for example, a lipid-anchored polymer as defined herein.
  • an LNP of the present disclosure comprises a second lipid-anchored polymer and the targeting moiety as defined herein (and including GalNAc, ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide) is conjugated to the second lipid-anchored polymer.
  • the second lipid-anchored polymer is structurally similar to the first lipid-anchored polymer as described herein in that the second lipid-anchored polymer also contains a lipid moiety covalently attached to a polymer via a linker.
  • the second lipid- anchored polymer comprises a linker-lipid moiety selected from the group consisting of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn- glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn- phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE),
  • the second lipid-anchored polymer comprises a linker-lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and a derivative of thereof, and a combination of any of the foregoing.
  • the ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, or a fragment thereof is covalently linked to a lipid-anchored polymer (e.g., second lipid-anchored polymer) or to an LNP of the present disclosure via strain promoted alkyne-azide cycloaddition (SPAAC) chemistry, such as via an azide-modified lipid-anchored polymer (e.g., DSG-PEG2000- azide, DSPE-PEG2000-azide, DSG-PEG3400-azide, DSPE-PEG3400-azide, DSG-PEG5000-azide, DSPE-PEG5000-azide) and a dibenzocycl
  • SPAAC strain
  • the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer.
  • the LNPs of the present disclosure may comprise a first lipid-anchored polymer that does not comprise a targeting moiety, and a second type of lipid-anchored polymer that comprises a targeting moiety, such as GalNAc.
  • the LNPs of the present disclosure may comprise DSG-PEG2000 modified to comprise an additional OCH3 group (DSG-PEG2000-OMe) as a first lipid-anchored polymer and DSPE-PEG2000-GalNAc3 as a second lipid-anchored polymer.
  • the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety.
  • the second lipid-anchored polymer comprses a lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and a derivative of 98 ME146907648v.1 thereof.
  • the first lipid-anchored polymer is any lipid-anchored polymer as described hereinabove.
  • the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety, and the first lipid-anchored polymer and the second lipid-anchored polymer are different lipid-anchored polymers and are selected from one of the following combinations: DSG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DSPE (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DSG (
  • the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety, and the first lipid-anchored polymer and the second lipid-anchored polymer are the same lipid-anchored polymers and are selected from one of the following combinations: DSG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DSPE (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); and DPG (the first lipid-anchored polymer) and DPG (the second lipid-anchored polymer).
  • DSG the first lipid-anchored polymer
  • DSG the second lipid-anchored polymer
  • DSPE the first lipid-anchored polymer
  • DSPE the second lipid-anchored polymer
  • DODA the first lipid-anchored polymer
  • DODA the
  • the targeting moiety is conjugated to a DSPE-anchored polymer.
  • the DSPE-anchored polymer is DSPE-PEG or a derivative thereof.
  • the targeting moiety is conjugated to a DSG-anchored polymer.
  • the DSG-anchored polymer is DSG-PEG or a derivative thereof.
  • the lipid-anchored polymers (first and second lipid-anchored polymers in combination) constitute about 0.1 mol% to about 20 mol% of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 0.5 mol% to about 10 mol% present in the LNP.
  • the lipid-anchored polymers constitute about 1 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 2 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute more than about 2 mol% (e.g., 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, 2.5 mol%, 2.6 99 ME146907648v.1 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%) to about 10 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 3 mol% to about 8 mol% present in the LNP.
  • the lipid-anchored polymers constitute about 3 mol% to about 7 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 3 mol% to about 5 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 2 mol% to about 4 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 2% to about 3% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 2 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 2.5 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 3 mol% present in the LNP.
  • the lipid-anchored polymers constitute about 3.5 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 4 mol% present in the LNP. In some embodiments, the first lipid-anchored polymer is present in about 0.1 mol% to about 10 mol% of the total lipid present in the LNP, or about 0.2 mol% to about 8 mol%, or about 0.2 mol% to about 7 mol%, or about 0.2% mol% to about 5 mol%, or about 0.3 mol to about 4 mol%, or about 0.4 mol% to about 4 mol%, or about 0.5 mol% to about 5 mol%, or about 0.5 mol% to about 4 mol%, or about 0.5 mol% to about 3.5 mol%, or about 0.5 mol% to about 3 mol%, or about 0.7 mol% to about 5 mol%, or about 0.7 mol% to about 4 mol%, or about 0.7 mol% to about 3.5 mol%, or about
  • the second lipid-anchored polymer if present, is present in about 0.005 mol% to about 5 mol% of the total lipid present in the LNP, or about 0.005 mol% to about 3 mol%, or about 0.005 mol% to about 2 mol%, or about 0.005 mol% to about 1 mol%, or about 0.005 mol% to about 0.5 mol%, or about 0.01 mol% to about 3 mol%, or about 0.01 mol% to about 2 mol%, or about 0.01 mol% to about 1 mol%, or about 0.01 mol% to about 0.5 mol%, or about 0.025 mol% to about 3 mol%, or about 0.025 mol% to about 2 mol%, or about 0.025 mol% to about 1 mol%, or about 0.025 mol% to about 0.5 mol%, or about 0.05 mol% to about 3 mol%, or about 0.05 mol% to about 2 mol%, or about 0.05 mol% to
  • LNPs Lipid nanoparticles comprising ceDNA are disclosed in International Patent Application No. PCT/US2018/050042, filed on September 7, 2018, which is incorporated herein in its entirety and envisioned for use in the methods and compositions as disclosed herein.
  • the size of LNPs can be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK).
  • LNPs of the present disclosure have a mean diameter as determined by light scattering of less than about 90 nm, e.g., less than about 80 nm or less than about 75 nm.
  • the pKa of each cationic lipid is determined in lipid nanoparticles using an assay based on fluorescence of 2-(p-toluidino)-6- napthalene sulfonic acid (TNS).
  • LNPs in PBS at a concentration of 0.4 mM total lipid can be prepared using the in-line process as described herein and elsewhere.
  • TNS can be prepared as a 100 mM stock solution in distilled water.
  • Vesicles can be diluted to 24 mM lipid in 2 mL of buffered solutions containing, 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where the pH ranges from 2.5 to 11.
  • TNS solution An aliquot of the TNS solution can be added to give a final concentration of 1 mM and following vortex mixing fluorescence intensity is measured at room temperature in a SLM Aminco Series 2 Luminescence Spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best fit analysis can be applied to the fluorescence data and the pKa is measured as the pH giving rise to half-maximal fluorescence intensity.
  • relative activity can be determined by measuring luciferase expression in the liver 4 hours following administration via tail vein injection. The activity is compared at a dose of 0.3 and 1.0 mg ceDNA/kg and expressed as ng luciferase/g liver measured 4 hours after administration.
  • LNP of the present disclosure includes a lipid formulation that can be used to deliver a capsid-free, non-viral DNA vector to a target site of interest (e.g., cell, tissue, organ, and the like).
  • the LNP comprises capsid-free, non-viral DNA vector and a cationic lipid or a salt thereof.
  • lipid-anchored polymers include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide -lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof.
  • the conjugated lipid molecule is a PEGylated lipid, for example, a (methoxy polyethylene glycol)- conjugated lipid.
  • PEG-diacylglycerol DAG
  • PEG-DMG PEG-diacylglycerol
  • DAA PEG-dialkyloxypropyl
  • PEG-phospholipid PEG-phospholipid
  • PEG-PE PEG-PE
  • PEG succinate diacylglycerol PGS-DAG
  • PEG dialkoxypropylcarbam such as 4-0- (2’,3’-di(tetradecanoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)
  • PEG dialkoxypropylcarbam N-(carbonyl-methoxypoly ethylene glycol 2000)-
  • PEG-lipid conjugates are described, for example, in International Patent Application Publication Nos. WO 1996/010392, WO1998/051278, W02002/087541, W02005/026372, WO2008/147438, W02009/086558, W02012/000104, WO2017/117528, WO2017/099823, WO2015/199952, W02017/004143, WO2015/095346, WO2012/000104, WO2012/000104, and WO2010/006282, U.S. Patent Application Publication Nos.
  • PEG-DAA PEGylated lipids include, for example, PEG- dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl.
  • the PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG- disterylglycamide, PEG-cholesterol (l-[8’-(Cholest-5-en-3[beta]- oxy)carboxamido-3’,6’-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-Ditetradecoxylbenzyl-[omega]- methyl-poly(ethylene glycol) ether), and l,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(poly
  • the PEG-lipid can be selected from the group consisting of PEG-DMG, l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], , 102 ME146907648v.1 .
  • lipid-anchored polymers include N-(Carbonyl- methoxypo1yethy1eneg1yco1n)-1,2-dimyristoyl-sn-glycero-3 -phosphoethanolamine (DMPE-PEG n , where n is 350, 500, 750, 1000 or 2000), N-(Carbonyl-methoxypolyethyleneglycol n )-1,2-distearoyl- sn-glycero-3-phosphoethanolamine (DSPE-PEG n , where n is 350, 500, 750, 1000 or 2000), DSPE- polyglycelin-cyclohexyl-carboxylic acid, DSPE-polyglycelin-2-methylglutar-carboxylic acid, 1,2- Distearoyl-sn-Glycero-3-Phosphoethanolamine (DSPE) conjugated Polyethylene Glycol (DSPE-PEG- OH), polyethylene glycol-dimyristolg
  • the PEG- lipid is N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE-PEG 2,000).
  • DSPE-PEG n where n is 350, 500, 750, 1000 or 2000, the PEG-lipid is N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl- sn-glycero-3-phosphoethanolamine (DSPE-PEG 2,000).
  • the PEG-lipid is DSPE-PEG-OH. In some embodiments, the PEG-lipid is PEG-DMG having two C 14 hydrophobic tails and PEG2000. D. Therapeutic Nucleic Acids
  • the LNPs provided by the present disclosure also comprise a therapeutic nucleic acid (TNA). According to embodiments, also provided are pharmaceutical compositions comprising the LNPs of the disclosure.
  • Illustrative therapeutic nucleic acids in the LNPs of the present disclosure can include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, closed ended double stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA (“ministring”), single-stranded DNA (ssDNA), doggyboneTM, protelomere closed ended DNA, or dumbbell linear DNA), single stranded DNA (e.g., synthetically made AAV vectors), dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), mRNA, tRNA, rRNA, gRNA and DNA viral vectors, viral RNA vector, and any combination thereof.
  • minigenes plasmids, minicircles, small interfering RNA (
  • the therapeutic nucleic acid can be a therapeutic DNA.
  • Said therapeutic DNA can be ceDNA, CELiD, linear covalently closed DNA 103 ME146907648v.1 (“ministring” or otherwise), ssDNA, doggyboneTM, protelomere closed ended DNA, dumbbell linear DNA, minigenes, plasmids, single stranded DNA (e.g., synthetically made AAV vectors) or minicircles.
  • the TNA is selected from the group consisting of RNA, DNA, and derivatives and analogues thereof.
  • the TNA encodes a therapeutic gene and/or a therapeutic protein.
  • the e TNA is selected from the group consisting of mRNA, siRNA, synthetic ribozymes, antisense RNA, and gRNA.
  • the TNA is mRNA.
  • the TNA is selected from the group consisting of single-stranded-DNA (ssDNA) and double-stranded DNA (dsDNA).
  • the TNA is ssDNA.
  • the TNA is linear ssDNA. According to some embodiments of any of the above aspects and embodiments, the TNA is dsDNA. According to some embodiments of any of the above aspects and embodiments, the TNA is a non-viral capsid-free DNA vector with covalently-closed ends (ceDNA vector).
  • RNAi RNA interference
  • siRNA or miRNA that can downregulate the intracellular levels of specific proteins through a process called RNA interference (RNAi) are also contemplated by the present disclosure to be nucleic acid therapeutics. After siRNA or miRNA is introduced into the cytoplasm of a host cell, these double-stranded RNA constructs can bind to a protein called RISC.
  • the sense strand of the siRNA or miRNA is removed by the RISC complex.
  • the RISC complex when combined with the complementary mRNA, cleaves the mRNA and release the cut strands. RNAi is by inducing specific destruction of mRNA that results in downregulation of a corresponding protein.
  • Antisense oligonucleotides (ASO) and ribozymes that inhibit mRNA translation into protein can be nucleic acid therapeutics.
  • ASO antisense oligonucleotides
  • ribozymes that inhibit mRNA translation into protein can be nucleic acid therapeutics.
  • these single stranded deoxy nucleic acids have a complementary sequence to the sequence of the target protein mRNA, and Watson - capable of binding to the mRNA by Crick base pairing.
  • the therapeutic nucleic acid can be a therapeutic RNA.
  • Said therapeutic RNA can be messenger RNA (mRNA) encoding a protein or peptide, an inhibitor of mRNA translation, agent of RNA interference (RNAi), catalytically active RNA molecule (ribozyme), transfer RNA (tRNA) or an RNA that binds an mRNA transcript (ASO), protein or other molecular ligand (aptamer).
  • the agent of RNAi can be a double-stranded RNA, single-stranded RNA, microRNA, short interfering RNA, short hairpin RNA, or a triplex-forming oligonucleotide.
  • ssDNA Single-Stranded DNA
  • the disclosure provides an isolated, linear single stranded deoxyribonucleic acid (ssDNA) molecule comprising at least one nucleic acid sequence of interest flanked by at least one stem-loop structure at the 3’ end.
  • the at least one stem-loop structure at the 3’ end is sufficient to prime replication and/or transcription.
  • the stem structure at the 3’ end comprises a partial DNA duplex of between 4- 500 nucleotides. According to a further embodiment, the stem structure at the 3’ end comprises a partial DNA duplex of between 4-5 nucleotides. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 3’ end comprises between 3-500 unbound nucleotides. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 3’ end comprises a minimum of 3 unbound nucleotides. According to one embodiment of any of the aspects and embodiments herein, the ssDNA comprises at least two stem- loop structures at the 3’ end.
  • the ssDNA comprises at least three stem-loop structures at the 3’ end. According to one embodiment of any of the aspects and embodiments herein, the ssDNA comprises at least four or more stem-loop structures at the 3’ end. According to one embodiment of any of the aspects and embodiments herein, the at least one stem-loop structure at the 3’ end comprises a hairpin DNA structure.
  • the at least one stem-loop structure at the 3’ end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadraplex DNA structure, a bulged DNA structure, and a multibranched loop structure.
  • the at least one stem-loop structure at the 3’ end does not comprise the A, A’, D, and D’ regions that would be present in a wild-type AAV ITR.
  • the at least one stem-loop structure at the 3’ end does not comprise the A, A’, B, B’, C, C’, D, and D’ regions that would be present in a wild- type AAV ITR.
  • the at least one stem-loop structure at the 3’ end does not comprise a rep binding element (RBE) that would be present in a wild-type ITR.
  • the at least one stem-loop structure at the 3’ end does not comprise a terminal resolution site (trs) that would be present in a wild-type ITR.
  • the stem structure at the 3’ end comprises four or more nucleotides that are modified to be exonuclease resistant.
  • the nucleotides are phosphorothioate-modified nucleotides.
  • at least one stem-loop structure at the 3’ end further comprises a functional moiety.
  • the ssDNA molecule further comprises a 5’ end, comprising at least one stem-loop structure.
  • the ssDNA comprises at least two stem-loop structures at the 5’ end.
  • the ssDNA comprises at least three stem-loop structures at the 5’ end. According to one embodiment of any of the aspects and embodiments herein, the ssDNA comprises at least four or more stem-loop structures at the 5’ end. According to one embodiment of any of the aspects and embodiments herein, the at least one stem- loop structure at the 5’ end comprises a hairpin DNA structure.
  • the at least one stem-loop structure at the 5’ end comprises a DNA structure selected from the group consisting of: a cruciform DNA structure, a hammerhead DNA structure, a quadraplex DNA structure, a bulged DNA structure, and a multibranched loop structure.
  • the at least one stem-loop structure at the 5’ end does not comprise the A, A’, D, and D’ regions that would be present in a wild-type AAV ITR.
  • the at least one stem-loop structure at the 5’ end does not comprise the A, A’, B, B’, C, C’, D, and D’ regions that would be present in a wild-type AAV ITR.
  • the at least one stem-loop structure at the 5’ end does not comprise a rep binding element (RBE) that would be present in a wild-type ITR.
  • the at least one stem- loop structure at the 5’ end does not comprise a terminal resolution site (trs) that would be present in a wild-type ITR.
  • the stem structure at the 5’ end comprises four or more nucleotides that are modified to be exonuclease resistant.
  • the nucleotides are phosphorothioate-modified nucleotides.
  • the loop structure at the 5’ end further comprises one or more nucleic acids to stabilize the ends.
  • the loop structure at the 5’ end further comprises one or more nucleic acids that are chemically modified.
  • the loop structure at the 5’ end further comprises one or more aptamers.
  • the loop structure at the 5’ end further comprises one or more synthetic ribozymes. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more antisense oligonucleotides (ASOs). According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more short-interfering RNAs (siRNAs). According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more antiviral nucleoside analogues (ANAs).
  • ASOs antisense oligonucleotides
  • siRNAs short-interfering RNAs
  • ANAs antiviral nucleoside analogues
  • the loop structure at the 5’ end further comprises one or more triplex forming oligonucleotides. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more gRNAs or gDNAs. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more 106 ME146907648v.1 molecular probes. According to one embodiment of any of the aspects and embodiments herein, the ssDNA molecule is devoid of any viral capsid protein coding sequences. According to one embodiment of any of the aspects and embodiments herein, the ssDNA molecule is synthetically produced in vitro.
  • the ssDNA molecule is synthetically produced in vitro in a cell-free environment. According to one embodiment of any of the aspects and embodiments herein, the ssDNA molecule does not activate or minimally activates an immune pathway. According to a further embodiment, the immune pathway is an innate immune pathway. According to another further embodiment, the innate immune pathway is selected from the group consisting of the cGAS/STING pathway, the TLR9 pathway, an inflammasome-mediated pathway, and a combination thereof. Closed-ended DNA (ceDNA) Vectors In some embodiments, LNPs provided by the present disclosure comprise closed-ended DNA (ceDNA).
  • the TNA comprises closed-ended linear duplexed (ceDNA) vectors that can express a transgene (e.g., a therapeutic nucleic acid (TNA)).
  • a transgene e.g., a therapeutic nucleic acid (TNA)
  • the ceDNA vectors as described herein have no packaging constraints imposed by the limiting space within the viral capsid.
  • ceDNA vectors represent a viable eukaryotically-produced alternative to prokaryote-produced plasmid DNA vectors, as opposed to encapsulated AAV genomes. This permits the insertion of control elements, e.g., regulatory switches as disclosed herein, large transgenes, multiple transgenes etc.
  • ceDNA vectors preferably have a linear and continuous structure rather than a non- continuous structure.
  • the linear and continuous structure is believed to be more stable from attack by cellular endonucleases, as well as less likely to be recombined and cause mutagenesis.
  • a ceDNA vector in the linear and continuous structure is a preferred embodiment.
  • the continuous, linear, single strand intramolecular duplex ceDNA vector can have covalently bound terminal ends, without sequences encoding AAV capsid proteins.
  • These ceDNA vectors are structurally distinct from plasmids (including ceDNA plasmids described herein), which are circular duplex nucleic acid molecules of bacterial origin.
  • ceDNA vectors can be produced without DNA base methylation of prokaryotic type, unlike plasmids. Therefore, the ceDNA vectors and ceDNA-plasmids are different both in term of structure (in particular, linear versus circular) and also in view of the methods used for producing and purifying these different objects, and also in view of their DNA methylation which is of prokaryotic type for ceDNA-plasmids and of eukaryotic type for the ceDNA vector.
  • non-viral, capsid-free ceDNA molecules with covalently closed ends can be produced in permissive host cells from an expression construct (e.g., a ceDNA-plasmid, a ceDNA-bacmid, a ceDNA-baculovirus, or an integrated cell-line) containing a heterologous gene (e.g., a transgene, in particular a therapeutic transgene) positioned between two different inverted terminal repeat (ITR) sequences, where the ITRs are different with respect to each other.
  • an expression construct e.g., a ceDNA-plasmid, a ceDNA-bacmid, a ceDNA-baculovirus, or an integrated cell-line
  • a heterologous gene e.g., a transgene, in particular a therapeutic transgene
  • one of the ITRs is modified by deletion, insertion, and/or substitution as compared to a wild-type ITR sequence (e.g., AAV ITR); and at least one of the ITRs comprises a functional terminal resolution site (trs) and a Rep binding site.
  • the ceDNA vector is preferably duplex, e.g., self-complementary, over at least a portion of the molecule, such as the expression cassette (e.g., ceDNA is not a double stranded circular molecule).
  • a ceDNA vector has covalently closed ends, and thus is resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III), e.g., for over an hour at 37°C.
  • a ceDNA vector comprises, in the 5’ to 3’ direction: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (for example an expression cassette as described herein) and a second AAV ITR.
  • the first ITR (5’ ITR) and the second ITR (3’ ITR) are asymmetric with respect to each other - that is, they have a different 3D-spatial configuration from one another.
  • the first ITR can be a wild-type ITR and the second ITR can be a mutated or modified ITR, or vice versa, where the first ITR can be a mutated or modified ITR and the second ITR a wild- type ITR.
  • the first ITR and the second ITR are both modified but are different sequences, or have different modifications, or are not identical modified ITRs, and have different 3D spatial configurations.
  • a ceDNA vector with asymmetric ITRs have ITRs where any changes in one ITR relative to the WT-ITR are not reflected in the other ITR; or alternatively, where the asymmetric ITRs have a the modified asymmetric ITR pair can have a different sequence and different three-dimensional shape with respect to each other.
  • a ceDNA vector comprises, in the 5’ to 3’ direction: a first adeno- associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (for example an expression cassette as described herein) and a second AAV ITR, where the first ITR (5’ ITR) and the second ITR (3’ ITR) are symmetric, or substantially symmetrical with respect to each other - that is, a ceDNA vector can comprise ITR sequences that have a symmetrical three-dimensional spatial organization such that their structure is the same shape in geometrical space, or have the same A, C- C’ and B-B’ loops in 3D space.
  • AAV adeno- associated virus
  • ITR inverted terminal repeat
  • a symmetrical ITR pair, or substantially symmetrical ITR pair can be modified ITRs (e.g., mod-ITRs) that are not wild-type ITRs.
  • a mod- ITR pair can have the same sequence which has one or more modifications from wild-type ITR and are reverse complements (inverted) of each other.
  • a modified ITR pair are substantially symmetrical as defined herein, that is, the modified ITR pair can have a different sequence but have corresponding or the same symmetrical three-dimensional shape.
  • the symmetrical ITRs, or substantially symmetrical ITRs can be wild type ITRs (WT- ITRs) as described herein.
  • both ITRs have a wild-type sequence from the same AAV serotype.
  • the two wild-type ITRs are from different AAV serotypes.
  • one WT-ITR can be derived from one AAV serotype, and the other WT-ITR can be derived from a different AAV serotype.
  • a WT-ITR pair are substantially symmetrical as defined herein, that is, they can have one or more conservative nucleotide modification while still retaining the symmetrical three-dimensional spatial organization.
  • the wild-type or mutated or otherwise modified ITR sequences provided herein represent DNA sequences included in the expression construct (e.g., ceDNA-plasmid, ceDNA Bacmid, ceDNA- baculovirus) for production of the ceDNA vector.
  • ITR sequences actually contained in the ceDNA vector produced from the ceDNA-plasmid or other expression construct may or may not be identical to the ITR sequences provided herein as a result of naturally occurring changes taking place during the production process (e.g., replication error).
  • a ceDNA vector in the LNPs of the present disclosure comprising the expression cassette with a transgene which is a therapeutic nucleic acid sequence, can be operatively linked to one or more regulatory sequence(s) that allows or controls expression of the transgene.
  • the polynucleotide comprises a first ITR sequence and a second ITR sequence, wherein the nucleotide sequence of interest is flanked by the first and second ITR sequences, and the first and second ITR sequences are asymmetrical relative to each other, or symmetrical relative to each other.
  • an expression cassette is located between two ITRs in the following order with one or more of: a promoter operably linked to a transgene, a posttranscriptional regulatory element, and a polyadenylation and termination signal.
  • the promoter is regulatable - inducible or repressible.
  • the promoter can be any sequence that facilitates the transcription of the transgene.
  • the promoter is a CAG promoter, or variation thereof.
  • the posttranscriptional regulatory element is a sequence that modulates expression of the transgene, as a non-limiting example, any sequence that creates a tertiary structure that enhances expression of the transgene which is a therapeutic nucleic acid sequence.
  • the posttranscriptional regulatory element comprises WPRE.
  • the polyadenylation and termination signal comprise BGHpolyA.
  • any cis regulatory element known in the art, or combination thereof, can be additionally used e.g., SV40 late polyA signal upstream enhancer sequence (USE), or other posttranscriptional processing elements including, but not limited to, the thymidine kinase gene of herpes simplex virus, or hepatitis B virus (HBV).
  • the expression cassette length in the 5’ to 3’ direction is greater than the maximum length known to be encapsidated in an AAV virion. In one embodiment, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb.
  • Various expression cassettes are exemplified herein.
  • the expression cassette can comprise more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides or 50,000 nucleotides, or any range between about 4000-10,000 nucleotides or 10,000-50,000 nucleotides, or more than 50,000 nucleotides.
  • the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 50,000 nucleotides in length.
  • the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 75,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 10,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 1000 to 10,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 5,000 nucleotides in length.
  • the ceDNA vectors do not have the size limitations of encapsidated AAV vectors, and thus enable delivery of a large-size expression cassette to the host.
  • the ceDNA vector is devoid of prokaryote-specific methylation.
  • the rigid therapeutic nucleic acid can be a plasmid.
  • the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., for medical, diagnostic, or veterinary uses) or immunogenic polypeptides.
  • the expression cassette can comprise any transgene which is a therapeutic nucleic acid sequence.
  • the ceDNA vector comprises any gene of interest in the subject, which includes one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen binding fragments, or any combination thereof.
  • the ceDNA expression cassette can include, for example, an expressible exogenous sequence (e.g., open reading frame) that encodes a protein that is either absent, inactive, or insufficient activity in the recipient subject or a gene that encodes a protein having a desired biological or a therapeutic effect.
  • the exogenous sequence such as a donor sequence can encode a gene product that can function to correct the expression of a defective gene or transcript.
  • the expression cassette can also encode corrective DNA strands, encode polypeptides, sense or antisense oligonucleotides, or coding RNAs or non-coding RNAs (e.g., siRNAs, guide RNAs, shRNAs, micro-RNAs, and their antisense counterparts (e.g., antagoMiR)).
  • expression cassettes can include an exogenous sequence that encodes a reporter protein to be used for experimental or diagnostic purposes, such as b-lactamase, b-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.
  • the expression cassette can include any gene that encodes a protein, polypeptide or RNA that is either reduced or absent due to a mutation or which conveys a therapeutic benefit 110 ME146907648v.1 when overexpressed is considered to be within the scope of the disclosure.
  • the ceDNA vector may comprise a template or donor nucleotide sequence used as a correcting DNA strand to be inserted after a double-strand break (or nick) provided by a nuclease.
  • the ceDNA vector may include a template nucleotide sequence used as a correcting DNA strand to be inserted after a double-strand break (or nick) provided by a guided RNA nuclease, meganuclease, or zinc finger nuclease.
  • ceDNA vector as described herein comprising an asymmetrical ITR pair or symmetrical ITR pair as defined herein is described in section IV of PCT/US 18/49996 filed September 7, 2018, which is incorporated herein in its entirety by reference.
  • the ceDNA vector can be obtained, for example, by the process comprising the steps of: a) incubating a population of host cells (e.g.
  • insect cells harboring the polynucleotide expression construct template (e.g., a ceDNA-plasmid, a ceDNA-Bacmid, and/or a ceDNA- baculovirus), which is devoid of viral capsid coding sequences, in the presence of a Rep protein under conditions effective and for a time sufficient to induce production of the ceDNA vector within the host cells, and wherein the host cells do not comprise viral capsid coding sequences; and b) harvesting and isolating the ceDNA vector from the host cells.
  • the presence of Rep protein induces replication of the vector polynucleotide with a modified ITR to produce the ceDNA vector in a host cell.
  • the following is provided as a non-limiting example.
  • synthetic ceDNA is produced via excision from a double- stranded DNA molecule.
  • Synthetic production of the ceDNA vectors is described in Examples 2-6 of International Application PCT/US19/14122, filed January 18, 2019, which is incorporated herein in its entirety by reference.
  • a ceDNA vector can be generated using a double stranded DNA construct, e.g., see FIGS.7A-8E of PCT/US19/14122.
  • the double stranded DNA construct is a ceDNA plasmid, e.g., see, e.g., FIG.6 in International patent application PCT/US2018/064242, filed December 6, 2018).
  • a construct to make a ceDNA vector comprises additional components to regulate expression of the transgene, for example, regulatory switches, to regulate the expression of the transgene, or a kill switch, which can kill a cell comprising the vector.
  • a molecular regulatory switch is one which generates a measurable change in state in response to a signal. Such regulatory switches can be usefully combined with the ceDNA vectors described herein to control the output of expression of the transgene.
  • the ceDNA vector comprises a regulatory switch that serves to fine tune expression of the transgene.
  • the switch can serve as a biocontainment function of the ceDNA vector.
  • the switch is an “ON/OFF” switch that is designed to start or stop (i.e., shut down) expression of the gene of interest in the ceDNA vector in a controllable and regulatable fashion.
  • the 111 ME146907648v.1 switch can include a “kill switch” that can instruct the cell comprising the synthetic ceDNA vector to undergo cell programmed death once the switch is activated.
  • Exemplary regulatory switches encompassed for use in a ceDNA vector can be used to regulate the expression of a transgene, and are more fully discussed in International application PCT/US18/49996, which is incorporated herein in its entirety by reference and described herein.
  • Another exemplary method of producing a ceDNA vector using a synthetic method that involves assembly of various oligonucleotides is provided in Example 3 of PCT/US19/14122, where a ceDNA vector is produced by synthesizing a 5’ oligonucleotide and a 3’ ITR oligonucleotide and ligating the ITR oligonucleotides to a double-stranded polynucleotide comprising an expression cassette.
  • FIG.11B of PCT/US19/14122 shows an exemplary method of ligating a 5’ ITR oligonucleotide and a 3’ ITR oligonucleotide to a double stranded polynucleotide comprising an expression cassette.
  • Example 4 of PCT/US19/14122 incorporated by reference in its entirety herein, and uses a single- stranded linear DNA comprising two sense ITRs which flank a sense expression cassette sequence and are attached covalently to two antisense ITRs which flank an antisense expression cassette, the ends of which single stranded linear DNA are then ligated to form a closed-ended single-stranded molecule.
  • One non-limiting example comprises synthesizing and/or producing a single-stranded DNA molecule, annealing portions of the molecule to form a single linear DNA molecule which has one or more base-paired regions of secondary structure, and then ligating the free 5’ and 3’ ends to each other to form a closed single-stranded molecule.
  • the disclosure provides for host cell lines that have stably integrated the DNA vector polynucleotide expression template (ceDNA template) described herein, into their own genome for use in production of the non-viral DNA vector. Methods for producing such cell lines are described in Lee, L. et al. (2013) Plos One 8(8): e69879, which is herein incorporated by reference in its entirety.
  • the Rep protein is added to host cells at an MOI of 3.
  • the host cell line is an invertebrate cell line, preferably insect Sf9 cells.
  • the host cell line is a mammalian cell line, preferably 293 cells the cell lines can have polynucleotide vector template stably integrated, and a second vector, such as herpes virus can be used to introduce Rep protein into cells, allowing for the excision and amplification of ceDNA in the presence of Rep.
  • the capsid free non-viral DNA vector comprises or is obtained from a plasmid comprising a polynucleotide template comprising in this order: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (for example an expression cassette of an exogenous DNA) and a modified AAV ITR, wherein said template nucleic acid molecule is devoid of AAV capsid protein coding.
  • the nucleic acid template of the disclosure is devoid of viral capsid protein coding sequences (i.e., it is devoid of AAV capsid genes but also of capsid genes of other viruses).
  • the template 112 ME146907648v.1 nucleic acid molecule is also devoid of AAV Rep protein coding sequences. Accordingly, in a preferred embodiment, the nucleic acid molecule of the disclosure is devoid of both functional AAV cap and AAV rep genes.
  • ceDNA can include an ITR structure that is mutated with respect to the wild type AAV2 ITR disclosed herein, but still retains an operable RBE, TRS and RBE ⁇ portion.
  • ceDNA Plasmid A ceDNA-plasmid is a plasmid that contains ceDNA sequences and can be used for later production of a ceDNA vector.
  • a ceDNA-plasmid can be constructed using known techniques to provide at least the following as operatively linked components in the direction of transcription: (1) a modified 5’ ITR sequence; (2) an expression cassette containing a cis-regulatory element, for example, a promoter, inducible promoter, regulatory switch, enhancers and the like; and (3) a modified 3’ ITR sequence, where the 3’ ITR sequence is symmetric relative to the 5’ ITR sequence.
  • the expression cassette flanked by the ITRs comprises a cloning site for introducing an exogenous sequence.
  • the expression cassette replaces the rep and cap coding regions of the AAV genomes and ceDNA can be used as an expression vector.
  • a ceDNA vector is obtained from a plasmid, referred to herein as a “ceDNA-plasmid” encoding in this order: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), an expression cassette comprising a transgene, and a mutated or modified AAV ITR, wherein said ceDNA-plasmid is devoid of AAV capsid protein coding sequences.
  • AAV adeno-associated virus
  • ITR inverted terminal repeat
  • the ceDNA-plasmid encodes in this order: a first (or 5’) modified or mutated AAV ITR, an expression cassette comprising a transgene, and a second (or 3’) modified AAV ITR, wherein said ceDNA-plasmid is devoid of AAV capsid protein coding sequences, and wherein the 5’ and 3’ ITRs are symmetric relative to each other.
  • the ceDNA-plasmid encodes in this order: a first (or 5’) modified or mutated AAV ITR, an expression cassette comprising a transgene, and a second (or 3’) mutated or modified AAV ITR, wherein said ceDNA-plasmid is devoid of AAV capsid protein coding sequences, and wherein the 5’ and 3’ modified ITRs have the same modifications (i.e., they are inverse complement or symmetric relative to each other).
  • the ceDNA-plasmid system is devoid of viral capsid protein coding sequences (i.e., it is devoid of AAV capsid genes but also of capsid genes of other viruses).
  • the ceDNA-plasmid is also devoid of AAV Rep protein coding sequences.
  • ceDNA-plasmid is devoid of functional AAV cap and AAV rep genes GG-3’ for AAV2) plus a variable palindromic sequence allowing for hairpin formation.
  • LNPs Lipid Nanoparticles
  • LNPs can form spontaneously upon mixing of ceDNA and the lipid(s).
  • the resultant nanoparticle mixture can be extruded 113 ME146907648v.1 through a membrane (e.g., 100 nrn cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc).
  • a thermobarrel extruder such as Lipex Extruder (Northern Lipids, Inc).
  • the extrusion step can be omitted.
  • Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration.
  • the lipid nanoparticles are formed from mixing a high concentration organic (EtOH) mixture of lipid components and TNA with an aqueous buffer as described in WO 2022/016089, the content of which is incorporated herein by reference in its entirety.
  • EtOH organic
  • LNPs can be formed by any method known in the art.
  • the LNPs can be prepared by the methods described, for example, in US2013/0037977, US2010/0015218, US2013/0156845, US2013/0164400, US2012/0225129, and US2010/0130588, content of each of which is incorporated herein by reference in its entirety.
  • LNPs can be prepared using a continuous mixing method, a direct dilution process, or an in-line dilution process.
  • the processes and apparatuses for apparatuses for preparing lipid nanoparticles using direct dilution and in-line dilution processes are described in US2007/0042031, the content of which is incorporated herein by reference in its entirety.
  • the processes and apparatuses for preparing lipid nanoparticles using step-wise dilution processes are described in US2004/0142025, the content of which is incorporated herein by reference in its entirety.
  • the disclosure provides for an LNP comprising a DNA vector, including a ceDNA vector as described herein and an ionizable lipid.
  • lipid nanoparticle formulation that is made and loaded with therapeutic nucleic acid like ceDNA obtained by the process as disclosed in International Patent Application No. PCT/US2018/050042, filed on September 7, 2018, which is incorporated by reference in its entirety herein.
  • the LNPs can be prepared by an impinging jet process.
  • the particles are formed by mixing lipids dissolved in alcohol (e.g., ethanol) with ceDNA dissolved in a buffer, e.g., a citrate buffer, a sodium acetate buffer, a sodium acetate and magnesium chloride buffer, a malic acid buffer, a malic acid and sodium chloride buffer, or a sodium citrate and sodium chloride buffer.
  • a buffer e.g., a citrate buffer, a sodium acetate buffer, a sodium acetate and magnesium chloride buffer, a malic acid buffer, a malic acid and sodium chloride buffer, or a sodium citrate and sodium chloride buffer.
  • the mixing ratio of lipids to ceDNA can be about 45-55% lipid and about 65-45% ceDNA.
  • the lipid solution can contain an ionizable lipid, a lipid-anchored polymer and a sterol (e.g., cholesterol) at a total lipid concentration of 5-30 mg/mL, more likely 5-15 mg/mL, most likely 9-12 mg/mL in an alcohol, e.g., in ethanol.
  • a sterol e.g., cholesterol
  • mol ratio of the lipids can range from about 25-98% for the cationic lipid, preferably about 35-65%; about 0-15% for the non-ionic lipid, preferably about 0-12%; about 0-15% for the PEG or PEG conjugated lipid molecule, preferably about 1-6%; and about 0-75% for the sterol, preferably about 30-50%.
  • the ceDNA solution can comprise the ceDNA at a concentration range from 0.3 to 1.0 mg/mL, preferably 0.3-0.9 mg/mL in buffered solution, with pH in the range of 3.5-5.
  • the two liquids are heated to a temperature in the range of about 15-40°C, preferably about 30-40°C, and then mixed, for example, in an impinging jet mixer, instantly forming the LNP.
  • the mixing flow rate can range from 114 ME146907648v.1 10-600 mL/min.
  • the tube ID can have a range from 0.25 to 1.0 mm and a total flow rate from 10-600 mL/min.
  • the combination of flow rate and tubing ID can have the effect of controlling the particle size of the LNPs between 30 and 200 nm.
  • the solution can then be mixed with a buffered solution at a higher pH with a mixing ratio in the range of 1:1 to 1:3 vol:vol, preferably about 1:2 vol:vol. If needed this buffered solution can be at a temperature in the range of 15-40°C or 30-40°C.
  • the mixed LNPs can then undergo an anion exchange filtration step. Prior to the anion exchange, the mixed LNPs can be incubated for a period of time, for example 30mins to 2 hours. The temperature during incubating can be in the range of 15-40°C or 30-40°C. After incubating the solution is filtered through a filter, such as a 0.8 ⁇ m filter, containing an anion exchange separation step.
  • This process can use tubing IDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000 mL/min.
  • the LNPs can be concentrated and diafiltered via an ultrafiltration process where the alcohol is removed and the buffer is exchanged for the final buffer solution, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
  • PBS phosphate buffered saline
  • the ultrafiltration process can use a tangential flow filtration format (TFF) using a membrane nominal molecular weight cutoff range from 30-500 kD.
  • the membrane format is hollow fiber or flat sheet cassette.
  • the TFF processes with the proper molecular weight cutoff can retain the LNP in the retentate and the filtrate or permeate contains the alcohol; citrate buffer and final buffer wastes.
  • the TFF process is a multiple step process with an initial concentration to a ceDNA concentration of 1-3 mg/mL. Following concentration, the LNPs solution is diafiltered against the final buffer for 10-20 volumes to remove the alcohol and perform buffer exchange. The material can then be concentrated an additional 1-3-fold. The concentrated LNP solution can be sterile filtered. V.
  • Pharmaceutical Compositions and Formulations The present disclosure also provides a pharmaceutical composition comprising the LNPs of the present disclosure and at least one pharmaceutically acceptable excipient.
  • the TNA (e.g., ceDNA) is encapsulated in the LNP.
  • the LNPs of the disclosure are provided with full encapsulation, partial encapsulation of the therapeutic nucleic acid.
  • the nucleic acid therapeutics is fully encapsulated in the LNPs to form a nucleic acid containing lipid particle.
  • the nucleic acid may be encapsulated within the lipid portion of the particle, thereby protecting it from enzymatic degradation.
  • encapsulation of TNA (e.g., ceDNA) in the LNPs of the present disclosure can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid, for example, an Oligreen® assay or PicoGreen® assay.
  • encapsulation is determined by adding the dye to the lipid particle formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent.
  • Detergent- 115 ME146907648v.1 mediated disruption of the lipid bilayer releases the encapsulated TNA (e.g., ceDNA), allowing it to interact with the membrane-impermeable dye.
  • the proportions of the components can vary and the delivery efficiency of a particular formulation can be measured using, for example, an endosomal release parameter (ERP) assay.
  • the TNA e.g., ceDNA
  • the lipid portion of the particle or encapsulated in the lipid position of the lipid particle e.g., lipid nanoparticle.
  • the TNA can be fully encapsulated in the lipid position of the lipid particle (e.g., lipid nanoparticle), thereby protecting it from degradation by a nuclease, e.g., in an aqueous solution.
  • the TNA in the lipid particle e.g., lipid nanoparticle
  • the TNA in the lipid particle is not substantially degraded after exposure of the lipid particle (e.g., lipid nanoparticle) to a nuclease at 37°C. for at least about 20, 30, 45, or 60 minutes.
  • the TNA in the lipid particle is not substantially degraded after incubation of the particle in serum at 37°C.
  • the LNPs are substantially non-toxic to a subject, e.g., to a mammal such as a human.
  • the pharmaceutical composition comprising LNPs of the disclosure is an aqueous solution.
  • the pharmaceutical compostion comprising LNPs of the disclosure is a lyophilized powder.
  • the at least one pharmaceutically acceptable excipient in the pharmaceutical compositions of the present disclosure is a sucrose, tris, trehalose and/or glycine.
  • the pharmaceutical compositions comprising LNPs of the disclosure are suitable for administration to a subject for in vivo delivery to cells, tissues, or organs of the subject.
  • the pharmaceutical composition is suitable for a desired route of therapeutic administration (e.g., parenteral administration).
  • the pharmaceutical compositions for therapeutic purposes can be formulated as a solution, microemulsion, dispersion, liposomes, or other ordered structure suitable for high TNA (e.g., ceDNA) vector concentration.
  • Sterile injectable solutions can be prepared by incorporating the TNA (e.g., ceDNA) vector compound in the required amount in an appropriate buffer with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • compositions comprising LNPs of the disclosure are suitable for topical, systemic, intra-amniotic, intrathecal, intracranial, intraarterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intra-tissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (e.g., extra-orbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, sub-choroidal, intrastromal, intracameral and 116 ME146907648v.1 intravitreal), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration.
  • intra-tissue e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral
  • intrathecal e.g., intramuscular, intracardiac, intra
  • LNPs are solid core particles that possess at least one lipid bilayer.
  • the LNPs have a non-bilayer structure, i.e., a non-lamellar (i.e., non-bilayer) morphology.
  • the non-bilayer morphology can include, for example, three dimensional tubes, rods, cubic symmetries, etc.
  • the non-lamellar morphology (i.e., non-bilayer structure) of the LNPs can be determined using analytical techniques known to and used by those of skill in the art. Such techniques include, but are not limited to, Cryo-Transmission Electron Microscopy (“Cryo-TEM”), Differential Scanning calorimetry (“DSC”), X-Ray Diffraction, and the like.
  • Cryo-TEM Cryo-Transmission Electron Microscopy
  • DSC Differential Scanning calorimetry
  • X-Ray Diffraction and the like.
  • the morphology of the lipid particles (lamellar vs. non-lamellar) can readily be assessed and characterized using, e.g., Cryo-TEM analysis as described in US2010/0130588, the content of which is incorporated herein by reference in its entirety.
  • the LNPs having a non-lamellar morphology are electron dense.
  • the LNPs provided by the present disclosure is either unilamellar or multilamellar in structure.
  • the pharmaceutical composition of the disclosure comprises multi-vesicular particles and/or foam-based particles. By controlling the composition and concentration of the lipid components, one can control the rate at which the lipid conjugate exchanges out of the lipid particle and, in turn, the rate at which the LNP becomes fusogenic. In addition, other variables including, for example, pH, temperature, or ionic strength, can be used to vary and/or control the rate at which the LNP becomes fusogenic.
  • RNA-ligand conjugates and nanoparticle-ligand conjugates may be combined with ophthalmologically acceptable preservatives, co-solvents, surfactants, viscosity enhancers, penetration enhancers, buffers, sodium chloride, or water to form an aqueous, sterile ophthalmic suspension or solution.
  • ophthalmologically acceptable preservatives co-solvents
  • surfactants surfactants
  • viscosity enhancers penetration enhancers
  • buffers sodium chloride
  • sodium chloride sodium chloride
  • the pharmaceutical compositions can be presented in unit dosage form.
  • a unit dosage form will typically be adapted to one or more specific routes of administration of the pharmaceutical composition.
  • the unit dosage form is adapted for administration by inhalation.
  • the unit dosage form is adapted for administration by a vaporizer.
  • the unit dosage form is adapted for administration by a nebulizer.
  • the unit dosage form is adapted for 117 ME146907648v.1 administration by an aerosolizer.
  • the unit dosage form is adapted for oral administration, for buccal administration, or for sublingual administration.
  • the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration.
  • the unit dosage form is adapted for intrathecal or intracerebroventricular administration.
  • the pharmaceutical composition is formulated for topical administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • the present disclosure provides methods of treating a disorder in a subject that comprise administering to the subject an effective amount of an LNP of the disclosure of the pharmaceutical composition comprising the LNP of the disclosure.
  • the disorder is a genetic disorder.
  • the term “genetic disease” or “genetic disorder” refers to a disease, partially or completely, directly or indirectly, caused by one or more abnormalities in the genome, including and especially a condition that is present from birth.
  • the abnormality may be a mutation, an insertion or a deletion in a gene.
  • the abnormality may affect the coding sequence of the gene or its regulatory sequence.
  • Provided herein are methods for treating genetic disorders by administering the LNP of the disclosure or the pharmaceutical composition comprising LNPs of the disclosure.
  • inherited diseases in which defective genes are known, and typically fall into two classes: deficiency states, usually of enzymes, which are generally inherited in a recessive manner, and unbalanced states, which may involve regulatory or structural proteins, and which are typically but not always inherited in a dominant manner.
  • the LNPs and LNP compositions of the disclosure can be used to deliver transgenes to bring a normal gene into affected tissues for replacement therapy, as well, in some embodiments of any of the aspects and embodiments herein, to create animal models for the disease using antisense mutations.
  • the LNPs and LNP compositions of the disclosure can be used to create a disease state in a model system, which could then be used in efforts to counteract the disease state.
  • the LNPs or LNP compositions of the disclosure and methods disclosed herein permit the treatment of genetic diseases.
  • a disease state is treated by partially or wholly remedying the deficiency or imbalance that causes the disease or makes it more severe.
  • the ceDNA vectors as disclosed herein can be advantageously used in the treatment of individuals with metabolic disorders (e.g., ornithine transcarbamylase deficiency).
  • the LNPs of the disclosure or the pharmaceutical compositions comrpsing the LNPs of the disclosure can be used to treat, ameliorate, and/or prevent a disease or disorder caused by mutation in a gene or gene product.
  • Exemplary diseases or disorders that can be treated with the LNPs or the LNP compositions of the disclosure include, but are not limited to, metabolic diseases or disorders (e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage disease); urea cycle diseases or disorders (e.g., ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy (MLD), mucopolysaccharidosis Type II (MPSII; Hunter syndrome)); liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis (PFIC); blood diseases or disorders (e.g., hemophilia A and B, thalassemia, and anemia); cancers and tumors, and genetic diseases or disorders (e.g., cystic fibrosis).
  • metabolic diseases or disorders e.g., Fabry disease, Gaucher disease, phenylket
  • the LNPs or LNP compositions of the disclosure may be employed to deliver a heterologous nucleotide sequence in situations in which it is desirable to regulate the level of transgene expression (e.g., transgenes encoding hormones or growth factors).
  • the LNPs or LNP compositions of the disclosure can be used to correct an abnormal level and/or function of a gene product (e.g., an absence of, or a defect in, a protein) that results in the disease or disorder.
  • the LNPs or LNP compositions of the disclosure can produce a functional protein and/or modify levels of the protein to alleviate or reduce symptoms resulting from, or confer benefit to, a particular disease or disorder caused by the absence or a defect in the protein.
  • treatment of OTC deficiency can be achieved by producing functional OTC enzyme;
  • treatment of hemophilia A and B can be achieved by modifying levels of Factor VIII, Factor IX, and Factor X;
  • treatment of PKU can be achieved by modifying levels of phenylalanine hydroxylase enzyme;
  • treatment of Fabry or Gaucher disease can be achieved by producing functional alpha galactosidase or beta glucocerebrosidase, respectively;
  • treatment of MFD or MPSII can be achieved by producing functional arylsulfatase A or iduronate-2-sulfatase, respectively;
  • treatment of cystic fibrosis can be achieved by producing functional cystic fibrosis transmembrane conductance regulator;
  • the LNPs or LNP compositions of the disclosure can be used to provide a DNA-based therapeutic to a cell in vitro or in vivo.
  • DNA-based therapeutics include, but are not limited to, minicircle DNA, minigene, viral DNA (e.g., Lentiviral or AAV genome) or non-viral synthetic DNA vectors, closed-ended linear duplex DNA (ceDNA/CELiD), plasmids, bacmids, doggyboneTM DNA vectors, minimalistic immunological-defined gene expression (MIDGE)-vector, nonviral ministring DNA vector (linear-covalently closed DNA vector), or dumbbell-shaped DNA minimal vector (“dumbbell DNA”).
  • minicircle DNA minigene
  • minigene viral DNA
  • non-viral synthetic DNA vectors closed-ended linear duplex DNA
  • MIDGE minimalistic immunological-defined gene expression
  • nonviral ministring DNA vector linear-covalently closed DNA vector
  • dumbbell DNA dumbbell-shaped DNA minimal vector
  • exemplary transgenes encoded by ceDNA in the LNPs or LNP compositions of the disclosure include, but are not limited to: X, lysosomal enzymes (e.g., hexosaminidase A, associated with Tay-Sachs disease, or iduronate sulfatase, associated, with Hunter Syndrome/MPS II), erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, as well as cytokines (e.g., a interferon, ⁇ -interferon, interferon- ⁇ , interleukin-2, interleukin-4, interleukin 12, granulocyte- macrophage colony stimulating factor, lymphotoxin, and the like), peptide growth factors and hormones (e.g., somatotropin, insulin, insulin-
  • lysosomal enzymes e.
  • the transgene encodes a monoclonal antibody specific for one or more desired targets. In some exemplary embodiments, more than one transgene is encoded by the ceDNA vector. In some exemplary embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments of any of the aspects and embodiments herein, the transgene encodes an antibody, including a full-length antibody or antibody fragment, as defined herein. In some embodiments of any of the aspects and embodiments herein, the antibody is an antigen-binding domain or an immunoglobulin variable domain sequence, as that is defined herein.
  • transgene sequences encode suicide gene products (thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, and tumor necrosis factor), proteins conferring resistance to a drug used in cancer therapy, and tumor suppressor gene products.
  • this disclosure provides a method of providing anti-tumor immunity in a subject, the method comprising administering to the subject an effective amount of any embodiment of an LNP contemplated herein or any embodiment of a pharmaceutical composition comprising an LNP contemplated herein.
  • this disclosure provides a method of treating a subject having a disease, disorder or condition associated with an elevated expression of a tumor antigen, the method comprising administering to the subject an 120 ME146907648v.1 effective amount of any embodiment of an LNP contemplated herein or any embodiment of a pharmaceutical composition comprising an LNP contemplated herein.
  • the TNA is retained in the spleen for at least about 6 hours, or at least about 9 hours, or at least about 12 hours, or at least about 15 hours, or at least about 18 hours, or at least about 21 hours, or at least about 24 hours, or at least about 27 hours, or at least about 30 hours, or at least about 33 hours, or at least about 36 hours after dosing of an LNP of this disclosure, for example, via intravenous or intratumoral administration.
  • the amount (i.e., number of copies) of the TNA at the start of a 12, 18, or 24-hour time window post-dosing and the amount of the TNA at the end of the time window are within the same order of magnitude (e.g., 10 -7 copies, 10 -6 copies, 10 -5 copies, 10 -4 copies, 10 -3 copies, 10 -2 copies, 10 -1 copies, 10 0 copies, 10 1 copies, 10 2 copies, 10 3 copies, etc. or any other suitable therapeutic levels).
  • the TNA is a messenger RNA (mRNA).
  • solid tumors treatable with an LNP disclosed herein or a pharmaceutical composition comprising the same include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g., colon), genitourinary tract (e.g., renal, urothelial cells), prostate and pharynx.
  • Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • the tumor or cancer is a melanoma, e.g., an advanced stage melanoma.
  • Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the disclosure.
  • examples of other solid tumors or cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood,
  • the present disclosure provides a method of treating a blood disease, disorder or condition in a subject, the method comprising administering to the subject an effective amount of any embodiment of an LNP contemplated herein or any embodiment of a pharmaceutical composition comprising an LNP contemplated herein.
  • Non-limiting examples of the blood disease, 121 ME146907648v.1 disorder or condition include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Hodgkin lymphoma (HL), multiple myeloma, a myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), adrenoleukodystrophy (ALD), Hurler syndrome, Krabbe disease (Globoid-cell leukodystrophy or GLD), metachromatic leukodystrophy (MLD), severe aplastic anemia (SAA), severe combined immunodeficiency (SCID), sickle cell disease (SCD), thalassemia, Wiskott-Aldrich syndrome, Diamond-Blackfan anemia, essential thrombocytosis, Fanconi anemia, hemophagocytic lymphohistiscytosis (HL
  • the TNA is a messenger RNA (mRNA).
  • the TNA is retained in the bone marrow for at least about 6 hours, or at least about 9 hours, or at least about 12 hours, or at least about 15 hours, or at least about 18 hours, or at least about 21 hours, or at least about 24 hours, or at least about 27 hours, or at least about 30 hours, or at least about 33 hours, or at least about 36 hours after dosing of an LNP of this disclosure, for example, via intravenous or intratumoral administration.
  • the amount i.e.
  • the number of copies) of the TNA at the start of a 12, 18, or 24-hour time window post-dosing and the number of the TNA at the end of the time window are within the same order of magnitude (e.g., 10 -7 copies, 10 -6 copies, 10 -5 copies, 10 -4 copies, 10 -3 copies, 10 -2 copies, 10 -1 copies, 10 0 copies, 10 1 copies, 10 2 copies, 10 3 copies, etc. or any other suitable therapeutic levels) or are reduced for less than one order of magnitude.
  • an LNP or an LNP composition of the disclosure can be administered to an organism for transduction of cells in vivo.
  • an LNP or an LNP composition of the disclosure can be administered to an organism for transduction of cells ex vivo.
  • administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Exemplary modes of administration of an LNP or an LNP composition of the disclosure include oral, rectal, transmucosal, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intraendothelial, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular [including administration to skeletal, diaphragm and/or cardiac muscle], intrapleural, intracerebral, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, and the like, as well as direct tissue or organ injection (e.g., to liver, eye, skeletal muscle, cardiac muscle, diaphragm muscle or brain).
  • parenteral e.g., intravenous, subcutaneous, intradermal, intracranial,
  • LNP or LNP compositions of the disclosure can be to any site in a subject, including, without limitation, a site selected from the group consisting of the brain, a skeletal muscle, a smooth muscle, the heart, the diaphragm, the airway epithelium, the liver, the kidney, the spleen, the pancreas, the skin, and the eye.
  • a site selected from the group consisting of the brain, a skeletal muscle, a smooth muscle, the heart, the diaphragm, the airway epithelium, the liver, the kidney, the spleen, the pancreas, the skin, and the eye.
  • ceDNA permits one to administer more than one transgene in a single vector, or multiple ceDNA vectors (e.g., a ceDNA cocktail).
  • the LNPs or LNP compositions of the disclosure can be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intra-ocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra-aural, intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon’s region) delivery as well as intramuscular delivery with retrograde delivery to motor neurons.
  • intrathecal intra-ocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra-aural, intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon’s region
  • the LNPs of the disclosure or the pharmaceutical compositions comprising the LNPs of the disclosure when administered to a subject, is characterized by a lower immunogenicity than a reference LNP or a pharmaceutical composition comprising a reference LNP.
  • the immunogenicity of the LNP of the disclosure or the pharmaceutical composition comprising the LNP of the disclosure may be measured by measuring levels of one or more proinflammatory cytokines. Accordingly, in some embodiments, the LNPs of the disclosure or the pharmaceutical compositions comprising the LNPs of the disclosure, when administered to a subject, elicits a lower pro-inflammatory cytokine respose than a reference LNP or a pharmaceutical composition comprising a reference LNP.
  • the term “elicits a lower pro-inflammatory cytokine response than a reference LNP or a pharmaceutical composition comprising a reference LNP”, as used herein, means that the LNP of the disclosure or the pharmaceutical composition comprising the LNP of the disclosure, when administered to a subject, causes a smaller increase in the levels of one or more pro-inflammatory cytokines as compared to a reference LNP or a pharmaceutical composition comprising a reference LNP.
  • pro-inflammatory cytokines include, but are not limited to, granulocyte colony stimulating factor (G-CSF), interleukin 1 alpha (IL-1 ⁇ ), interleukin 1 beta (IL-1 ⁇ ), interleukin 6 (IL-6), interleukin 8 (IL-8 or CXCL8), interleukin 11 (IL-11), interleukin 17 (IL-17), interleukin 18 (IL-18), interferon ⁇ (IFN- ⁇ ), interferon ⁇ (IFN- ⁇ ), interferon ⁇ (IFN- ⁇ ), C-X-C motif chemokine ligand 10 (CXCL10 or IP-10), monocyte chemoattractant protein 1 (MCP-1), CD40L, CCL2, CCL3, CCL4, CCL5, CCL11, tumor necrosis factor ⁇ (TNF- ⁇ ), and combinations thereof.
  • G-CSF granulocyte colony stimulating factor
  • IL-1 ⁇ interleukin 1 alpha
  • IL-1 ⁇ interleukin 1 beta
  • IL-6
  • the reference LNP is an LNP that comprises a helper lipid; and /or does not comprise the first lipid-anchored polymer and comprises a reference lipid-anchored polymer comprising one or more hydrophobic tails that each independently comprise less than 16 carbon atoms in a single aliphatic chain backbone.
  • the reference LNP comprises an ionizable lipid, DSPC, cholesterol, a lipid-anchored polymer comprising PEG attached to a lipid 123 ME146907648v.1 moiety which has two hydrophobic tails, each comprising of 14 carbon atoms (e.g., DMG-PEG2000, see below for the structure)
  • DMG-PEG2000 see below for the structure
  • LNP formulations containing no helper lipid and containing C18 lipid-anchored polymer have physicochemical properties that are suitable for therapeutic use
  • Exemplary LNP formulations of the present disclosure e.g., containing no helper lipid and containing one or two C18 lipid-anchored polymers, were prepared using the methods as described herein, using a ceDNA vector having a luciferase transgene as the nucleic acid cargo.
  • Control LNP A that contains DSPC as a helper lipid were also prepared and the physicochemical properties of all control LNP and test LNP formulations (i.e., LNP1, LNP2, LNP3, LNP4, LNP5) with varying amounts of cholesterol were compared (see Table 8).
  • Table 8 Table 8.
  • LNP4 and LNP5 had average diameters that were equivalent or smaller than the average diameter of the Control LNP A, and both LNP4 and LNP5 exhibited the highest encapsulation efficiencies among the test formulations that were comparable to the encapsulation efficiency of Control LNP A.
  • LNP4 When formulated as a different batch, LNP4 had an average diameter of ⁇ 80 nm (see Table 10 in Example 2).
  • Example 2 LNP formulations containing no helper lipid and containing C18 lipid- anchored polymer reduced immunogenicity in mice The goal of this experiment was to demonstrate tolerability of the exemplary LNPs of the present disclosure, which are devoid of helper lipid.
  • LNP formulations (with ceDNA vector having a luciferase transgene as the nucleic acid cargo) were prepared using ionizable lipid (e.g., Ionizable Lipid 87 and another Ionizable Lipid Z, cholesterol, and first lipid-anchored polymer (DSG-PEG- OMe or DODA-PG or bis-DSG-PEG which are all C18, i.e., having two hydrophobic tails that each contain 18 carbon atoms) to examine the effect of helper lipids on tolerability.
  • ionizable lipid e.g., Ionizable Lipid 87 and another Ionizable Lipid Z, cholesterol, and first lipid-anchored polymer (DSG-PEG- OMe or DODA-PG or bis-DSG-PEG which are all C18, i.e., having two hydrophobic tails that each contain 18 carbon atoms
  • the structure of Ionizable Lipid 87 is shown in Table 6.
  • Ionizable Lipid Z belongs to a different class of ionizable lipids compared to Ionizable Lipid 87, where both the headgroup and lipid tail moieties are structurally different from those of Ionizable Lipid 87.
  • LNPs having DSPC as a helper lipid were employed.
  • Table 9 provides formulation analytics of the resultant LNPs. Table 9. Physicochemical properties of Control LNP B and test LNP formulations having different ionizable lipids 125 ME146907648v.1 * C14 PEG2000 Lipid is a lipid-anchored polymer having two hydrophobic tails that each contain 14 carbon atoms, conjugated to PEG2000.
  • the immunogenicity profiles of LNP4, LNP6, LNP7, and Control LNP B were compared by analyzing, at 6 hours post-dosing, the blood serum levels of multiple types of cytokines implicated in the regulation of innate immune response, i.e., IFN- ⁇ , IL-6, IFN- ⁇ , TNF- ⁇ , IL-18, and IP-10.
  • the LNP formulations that were devoid of helper lipid and that contained C18 lipid-anchored polymers exhibited an overall reduction in cytokine levels as compared to the DSPC- and C14 PEG2000 Lipid-containing Control LNP B.
  • LNP4 that contains DSG-PEG2000-OMe as a lipid-anchored polymer.
  • LNP4 exhibited reduction in all six tested cytokines, as compared to Control LNP B.
  • FIGs.2A-2F indicate that LNP formulations of this disclosure that contain no helper lipid and contain a lipid-anchored polymer having hydrophobic tails that are longer than 14 carbon atoms may mitigate pro-inflammatory immune responses as compared to a conventional LNP formulation that contains a lipid-anchored polymer having hydrophobic tails that are 14 carbon atoms in length.
  • the mitigated pro-inflammatory 126 ME146907648v.1 immune response is often characteristic of a stealth LNP formulation, in which the nucleic acid cargo is protected from degradation prior to delivery to a target organ.
  • Example 3 LNP formulations containing no helper lipid and containing C18 lipid-anchored polymer have higher retention in blood
  • the pharmacokinetics properties of DSPC- and C14 PEG2000 Lipid-containing Control LNP B and LNP4 i.e., no DSPC or no helper lipid and with DSG-PEG2000-OMe as lipid-anchored polymer
  • the LNPs were formulated as disclosed above.
  • Control LNP B and LNP4 formulations were injected as an IV bolus injection via the tail vein of CD-1 mice.
  • Whole blood samples were collected with K2EDTA as anticoagulant 150 ⁇ L/aliquot for qPCR quantitative analysis at 2-min, 1-hour, 3-hour and 6-hour timepoints.
  • the plasma portion of the blood was separated and tested for the ceDNA amount using qPCR.
  • whole blood and plasma concentrations of ceDNA collected from the mice group treated with LNP4 showed a much higher level of plasma retention than the control group treated with Control LNP B in the first hour post-dosing (FIG.3).
  • LNP formulations with mRNA cargo exhibit in vitro expression and hepatocyte uptake All of the LNP formulations described in the foregoing Examples 1-3 were prepared using ceDNA vector as the nucleic acid cargo.
  • LNP formulations that contain no helper lipid (e.g., no DSPC), and that contain lipid-anchored polymers having hydrophobic tails that are longer than 14 carbon atoms and mRNA as the nucleic acid cargo.
  • mRNA structurally differs from a ceDNA vector at least in that mRNA is single-stranded and is likely less negatively charged than the covalently closed-ended and double-stranded ceDNA vector.
  • mRNA is also known to be less stable than DNA and is less rigid than DNA.
  • the LNP formulations listed in Table 10 were prepared using luciferase mRNA as the nucleic cargo.
  • DiD DiD (DiIC18(5); 1,1′-dioctadecyl- 3,3,3′,3′- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt) for the purpose of measuring and analyzing the uptake of the particles by primary mouse hepatocytes.
  • mRNA/LNP formulations of this disclosure i.e., containing no helper lipid, and containing lipid-anchored polymers having hydrophobic tails that are longer than 14 carbon atoms
  • 127 ME146907648v.1 exhibited average diameters and encapsulation efficiencies that are considered to be suitable for therapeutic use, including delivery to the hepatocytes.
  • Table 10 Physicochemical properties of test LNP formulations having mRNA cargo and with or without GalNAc3 targeting moiety The in vitro expression of luciferase encoding mRNA as delivered in the exemplary LNP formulations of the disclosure was also investigated.
  • FIG.4A indicates that the luciferase activity was significantly higher in LNP9, LNP10, and LNP11 that were each formulated with DSPE-PEG2000-GalNAc3, as compared to LNP8 that was formulated without GalNAc3.
  • FIG.4B indicates that the presence of GalNAc3 resulted in significantly higher uptake by the cells.
  • Example 5 LNP formulations with mRNA cargo exhibit in vivo expression, improved half-life in whole blood, and superior cargo concentration and retention in certain organs
  • the following LNP formulations as listed in Table 11 were prepared using luciferase mRNA as the nucleic acid cargo.
  • Table 11 Physicochemical properties of LNP formulations having mRNA-luciferase cargo CD-1 mice were injected IV bolus via the tail vein at a dose of 0.3 mg/kg of any one of the LNP formulations listed in Table 11.
  • Whole blood samples were collected with K2EDTA as anticoagulant 150 uL/aliquot for qPCR at 2 min, 1-hour and 6-hour timepoints and also at the 24-hour terminal timepoint.
  • LNP101 which is an exemplary LNP formulation of the invention, i.e., LNP formulation that does not include a helper lipid (e.g., DSPC) and includes a lipid- anchored polymer having two hydrophobic tails that each comprises 16 to 22 carbon atoms in a single aliphatic chain backbone (e.g., DSG-PEG2000-OMe), showed robust in vivo luciferase expression in mice.
  • helper lipid e.g., DSPC
  • a lipid- anchored polymer having two hydrophobic tails that each comprises 16 to 22 carbon atoms in a single aliphatic chain backbone e.g., DSG-PEG2000-OMe
  • PK pharmacokinetic
  • PK parameters of luciferase mRNA LNP formulations Overall, the various PK parameters and values shown in Table 12 indicate that LNP101 (having no helper lipid, and having the C18 DSG-PEG2000-OMe as a lipid-anchored polymer) and Control LNP C having the combination of DSPC as a helper lipid and C14 DMG-PEG2000 as a lipid- anchored polymer possessed opposite PK profiles: longer half-life (t1/2), higher blood exposure at the terminal timepoint which is 24 hours (AUC last ) and slower clearance from the systemic circulation in the inventive LNP formulations, as opposed to shorter half-life (t 1/2 ), lower blood exposure at the terminal timepoint (AUC last ) and faster clearance from the systemic circulation in Control LNP C.
  • LNP101 exhibited excellent stealth properties in that the half-life (t 1/2 ) of LNP101 is greater than 5 hours.
  • Control LNP C that incorporates C14 tailed DMG-PEG2000 as a lipid-anchored polymer exhibited a half-life (t 1/2 ) of about 2.5 hours in whole blood.
  • LNP101 exhibited Cl rates of about 10-40 mL/min/kg, whereas Control LNP C had a significantly higher Cl rate of 307 ml/min/kg.
  • a higher clearance rate (Cl) is indicative of a quicker rate of the drug substance (i.e., luciferase mRNA) being cleared from the systemic circulation.
  • the Cl rates indicated that the luciferase mRNA delivered by Control LNP C was rapidly cleared from the bloodstream.
  • FIG.6A indicates that in LNP101, the luciferase mRNA concentrations as detected by qPCR steadily dropped from 10 -1 ⁇ g/mL to 10 -3 ⁇ g/mL over the 24-hour period; whereas in Control LNP C the luciferase mRNA concentrations dropped from 10 -1 ⁇ g/mL to almost 10 -4 ⁇ g/mL within the first hour, which continued to significantly drop to 10 -6 ⁇ g/mL at 6 hours and further, to almost 10 -7 ⁇ g/mL at 24 hours.
  • LNP101 exhibited slightly higher amounts of mRNA copies of luciferase and at least equivalent or higher retention rates of luciferase mRNA from 6 hours to 24 hours post-dosing, as compared to Control LNP C.
  • the observation of higher luciferase mRNA amounts in the inventive LNP formulation is surprising and unexpected, considering that the IVIS fluorescence data as shown in FIG.5 suggested that the luciferase mRNA expression levels of these inventive LNP formulations were lower than that of Control LNP C.
  • T-cells including CD8+ T-cells are primed to generate precursors with an enhanced ability to differentiate into long-lived, stem-like memory T cells.
  • Stem-like T-cells are a subpopulation of mature T-cells that display stem cell-like properties, maintaining long-lasting immune effect even among exhausting clones.
  • the amounts of luciferase mRNA throughout the 6-24 hour post-dosing period, as delivered by LNP101 were consistently found to be about 2 orders of magnitude higher than the amounts of luciferase mRNA as delivered by Control LNP C.
  • the retention rates of luciferase mRNA in the mice bone marrow during the 6-24 hour post-dosing period, as delivered by LNP101 were also superior to the retention rate of the mRNA as delivered by Control LNP C.
  • the amount of copies of luciferase mRNA in the mice bone marrow merely dropped for less than one order of magnitude in mice dosed with the inventive LNP formulation, as compared to a drop of greater than one order of magnitude within the same time window for mice dosed with Control LNP C.
  • Example 6 LNPs formulated with no helper lipid and a variety of ionzable lipids can result in increased expression in vivo and in vitro Stealth LNPs with no helper lipid were formulated using ionizable lipids with head or tail group variations of Ionizable Lipid 87.
  • LNPs containing mRNA cargo were administered in vitro to primary mouse hepatocytes. LNPs were uptaken uniformly, with no significative differences between the Ionizable Lipid 87 variants (e.g., Ionizable Lipids 113, 114 and 115). However, referring to FIG.9B LNP containing, Ionizable Lipid 114 and mRNA cargo showed about 2-fold increase in expression compared to Ionizable Lipid 87 containing LNP, while other ionizable lipids variants showed slightly lower expression compared to Ionizable Lipid 87.
  • Ionizable Lipid 87 variants e.g., Ionizable Lipids 113, 114 and 115.
  • FIG.9B LNP containing, Ionizable Lipid 114 and mRNA cargo showed about 2-fold increase in expression compared to Ionizable Lipid 87 containing LNP, while other ionizable lipids variants showed slightly lower expression compared to Ioniz
  • LNPs with Ionizable Lipid 87 of the present disclosure without or with GalNAc clearly showed stealth properties.
  • LNPs without GalNAc had low expression as they are long circulating and do not have a targeting agent to reach the target organ.
  • GalNAc was added as active targeting ligand, expression levels were increased notably, which indicates that the LNP was then capable of reaching the target organ due to the presence of the targeting ligand (i.e., GalNAc).
  • LNPs with Ionizable Lipid 114 of the present disclosure behaved similarly to LNPs with Ionizable Lipid 87 of the present disclosure.
  • Example 8 Smaller LNPs were formulated by adding alternative lipid-conjugated polymer to the base LNPs of the present disclosure Referring to FIG.10A, smaller ceDNA LNPs containing anchored polymer (e.g., DSG-PEG) were formulated through addition of dissociable polymer (e.g., 5% DMG-PEG or mono-C18-PEG having the structure . This study compares these smaller LNPs to control LNPs to observe if smaller particles lead to higher expression and improved tolerability. The LNPs were injected as an IV bolus injection via the tail vein of CD-1 mice.
  • anchored polymer e.g., DSG-PEG
  • dissociable polymer e.g., 5% DMG-PEG or mono-C18-PEG having the structure .
  • Expression was measured by IVIS on day 7 post-dose administration.
  • body weights were measured on days 1, 2, 3 and 7 post-dose administration.
  • LNP Formulation 623 two of the smaller formulations (LNP Formulations 620 and 623) had 0.5 to 1 log decrease in IVIS expression with additional PEG (5% DMG-PEG or 5% mono-C18-PEG, respectively) compared to control (LNP F 433), one of the formulations (LNP Formulation 621) showed comparable IVIS expression with the addition of 5% DMG-PEG.
  • Small single-stranded DNA LNPs were also formulated with Ionizable Lipid Z, as well as Ionizable Lipid 87, and the addition of 5% dissociable DMG-PEG.
  • test articles are compared against a control LNP Formulation 241 containing dissociable PEG, e.g., Ac- PEG, instead of anchored PEG, e.g., DSG-PEG, as its expression and tolerability profiles are well- documented and will confirm successful test article administration in mice.
  • dissociable PEG e.g., Ac- PEG
  • anchored PEG e.g., DSG-PEG
  • the LNPs of the present disclosure were confirmed to possess comparable physicochemical characteristics across processes based on particle size and polydispersity, while encapsulation efficiency was improved with New Process (NP).
  • NP New Process
  • in CD-1 mice there was no obvious difference observed in Luciferase expression, cytokine induction, or body weight loss across dose levels when comparing the LNPs of the present disclosure prepared by either OP or NP, or when prepared with or without a targeting ligand.
  • Expression of ceDNA luciferase cargo of the LNPs of the present disclosure was uniformly less than that of the control LNP Formulation 241, attributable to the control formulation’s incorporation of dissociable PEG-lipid and an ionizable lipid known to promote endosomal escape.
  • lipid nanoparticle formulation For Old Process (OP), individual lipid components were dissolved in tert-butanol at stock concentrations. Lipid mixtures were prepared according to described molar ratios of individual components, targeting a final molar concentration of 3.94 mM in tert-butanol.1.0 mg/mL ceDNA solution was prepared in water. Prior to formulation, the final lipid mixture was mixed at ratio of 10:1 (v/v) with ceDNA in water, resulting in a final solvent of 90.9% tert-butanol and 9.1% water. Nanoassemblr was used to prepare the lipid nanoparticle formulation.
  • the aqueous stream identity was 25 mM sodium acetate, pH 4, and the organic stream identity was the lipid/ceDNA mixture in 90.9% tert-butanol, a final lipid concentration of 3.59 mM, and a final ceDNA concentration of 0.091 mg/mL.
  • Mixing through the Nanoassemblr targeted a flow rate ratio of 3:1 (aqueous:organic).
  • Formulations were dialyzed against 1X DPBS for at least two hours at 4°C. Dialyzed formulations were concentrated via Amicon spin filters (100 kDa MWCO) spun at 2000 g via centrifuge. Concentrated formulations were sterile filtered through a 0.2 ⁇ m nylon filter.
  • NP New Process
  • EtOH/MeOH ethanol/methanol
  • t-BuOH tert-butanol
  • Individual lipid components were 135 ME146907648v.1 dissolved in ethanol at stock concentrations.
  • Lipid mixtures were prepared according to described molar ratios of individual components, targeting a final molar concentration of 3.94 mM in EtOH/MeOH.1.0 mg/mL ceDNA solution was prepared in water. Prior to formulation, the final lipid mixture was mixed at ratio of 10:1 (v/v) with ceDNA in water, resulting in a final solvent of 90.9% EtOH/MeOH and 9.1% water.
  • Nanoassemblr was used to prepare the lipid nanoparticle formulation.
  • the aqueous stream identity was 25 mM sodium acetate, pH 4, and the organic stream identity was the lipid/ceDNA mixture in 90.9% EtOH/MeOH, a final lipid concentration of 3.59 mM, and a final ceDNA concentration of 0.091 mg/mL.
  • Mixing through the Nanoassemblr targeted a flow rate ratio of 3:1 (aqueous:organic).
  • the LNP formulations were dialyzed against 1X DPBS for at least two hours at 4°C, performed a total of three times. Dialyzed formulations were concentrated via Amicon spin filters (100 kDa MWCO) spun at 2000 g via centrifuge.
  • FIG.11A shows CD-1 mouse body weight loss recorded on day 1 post-dose administration. Loss associated with 0.5 mpk dose groups was negligible when compared to PBS mice, while 2.0 mpk and 4.0 mpk dose groups had more variable impact on body weight loss. As shown in FIG.11B, mouse body weights trended toward full recovery by day 3 post-dose administration, with exception to LNP Formulation 117 that employed NP.
  • FIGS.12A and 12B show IVIS readouts performed at day 4 and day 7 post-dose administrating measuring the level of Luciferase protein expressed in CD-1 mouse liver tissue.
  • the LNPs of the present disclosure prepared without a targeting ligand resulted in near-baseline Luciferase expression in a dose-independent manner while the LNPs of the present disclosure containing a targeting ligand resulted in a modest boost in expression in a dose-independent manner.
  • FIGs.13A-13F show cytokine profile of test articles comparing 0.5 mpk and 2.0 mpk doses.
  • LNPs of the present 136 ME146907648v.1 disclosure prepared without a targeting ligand, and similarly for the liver-targeted LNP of the present disclousred prepared by Old Process (OP).
  • the liver-targeted LNP of the present disclosure prepared New Process (NP) induced control-like cytokines with exception to TNF-alpha at 2.0 mpk.
  • Example 10 PK profiles of the ceDNA cargo of the stealth LNP formulations of the present disclosure prepared by different processes This study aims to compare whole blood and plasma PK profiles of the stealth LNP formulations of the present disclosure prepared by Old Process (OP) or New Process (NP), with or without a GalNAc-based targeting ligand, as listed in Table 16 above, to better understand if formulation process impacts these properties of stealth LNPs. Test articles were compared against a control LNP prepared with dissociable DMG-PEG, serving as a non-stealthy LNP control formulation that rapidly distributes from the blood compartment toward tissue compartments.
  • OP Old Process
  • NP New Process
  • Test articles were compared against a control LNP prepared with dissociable DMG-PEG, serving as a non-stealthy LNP control formulation that rapidly distributes from the blood compartment toward tissue compartments.
  • PK of the test articles was determined by quantifying by qPCR the amount of genetic cargo present in blood and plasma samples drawn at 2 minutes, 60 minutes, 3 hours, and 6 hours post-dosing. Similarly prolonged blood and plasma PK profiles were observed for untargeted stealth LNP formulations of the present disclosure regardless of the LNP formulation process.
  • both stealth LNP formulations underwent faster clearance from the blood compartment compared to untargeted stealth LNP formulations.
  • the control LNP formulation containing dissociable PEG e.g., Ac-PEG
  • FIGs.14A and 14B show pharmacokinetics of LNP-encapsulated ceDNA in mouse whole blood and plasma.
  • ceDNA encapsulated in the control LNP formulation containing dissociable Ac- PEG was cleared from both compartments rapidly within the first hour post-administration, while ceDNA encapsulated within stealth LNP formulations lacking a targeting ligand underwent prolonged residence time in both blood and plasma over a period of six hours.
  • the same stealth formulations prepared with a GalNAc targeting ligand were cleared from both compartments at a faster rate.
  • Example 11 The stealth LNPs of the present disclosure formulated with higher amount of cholesterol, and without a targeting ligand, produced smaller particles that retained prolonged PK profiles
  • LNPs prepared with 2.9% DSG-PEG2000, 67.6% ionizable lipid, and 29.5% cholesterol, and lacking a helper lipid demonstrated prolonged blood residence time in the absence of a targeting ligand, and quick clearance from blood when containing a GalNAc targeting ligand.
  • these formulations 137 ME146907648v.1 were not optimized for particle size, which is believed to play a role in efficient delivery of the LNP to certain target cell types, in this case hepatocytes.
  • LNPs incorporating 2.9% DSG-PEG2000, 57.6% ionizable lipid, and 39.5% cholesterol produce 10-15 nm smaller particles, prepared by New Process (NP).
  • NP New Process
  • the purpose of this study is to confirm that untargeted LNPs prepared with this composition retain prolonged whole blood and plasma PK profiles, as well as the expected GalNAc-mediated blood clearance.
  • In-situ hybridization (ISH) analysis and payload concentration was carried out on extracted livers at 1 hour and 6 hours post-dosing to confirm that differences in blood PK are reflected in payload distribution to liver tissue.
  • ISH In-situ hybridization
  • Test articles were compared against a control LNP formulation prepared with dissociable DMG-PEG2000, serving as a non-stealthy LNP control formulation that rapidly distributes from the blood compartment toward tissue compartments.
  • PK of the test articles was determined by quantifying by qPCR the amount of genetic cargo present in blood and plasma samples drawn at 2 minutes, 60 minutes, 3 hours, and 6 hours post-dosing.
  • the untargeted LNP of the present disclosure was observed to have prolonged blood residence and cleared from blood quickly when containing a GalNAc targeting ligand, suggesting that 57.6% ionizable lipid and 39.5% cholesterol does not impact these stealth-like properties.
  • the dissociable PEG control LNP formulation containing dissociable Ac-PEG expectedly underwent rapid distribution out of the blood compartment. Differences in blood clearance at 1 and 6 hours did not translate to payload concentration in liver tissue, but were qualitatively observed by liver ISH. qPCR analysis of liver tissue suggested similar payload concentrations across all test groups with no statistical outliers. However, clear rank-ordering of LNP distribution to the liver could be established by ISH analysis of liver tissue. At both time points, the highest payload density in liver was observed with the dissociable Ac-PEG control LNP, followed by the liver-targeted stealth LNP of the present disclosure, while the lowest payload density was observed with the untargeted stealth LNP.
  • FIGs.15A and 15B show pharmacokinetics of LNP-encapsulated payload in mouse whole blood and plasma. Approximately 99% of payload encapsulated in the control LNP formulation containing dissociable Ac-PEG is cleared from the whole blood compartment within the first hour post-administration.
  • FIGs.16A-16C show ISH staining of mouse livers collected at 1 hour post-administration.
  • FIGs.16D-16F show SH staining of the mouse livers collected at 6 hours post-administration. ceDNA payload staining was less abundant in all test groups compared to the livers collected at 1 hour. The highest payload density was observed in the liver tissue dosed with control LNP formulation containining dissociable Ac-PEG, while the lowest payload density was observed in the liver tissue dosed with untargeted stealth LNPs.
  • Example 12 Smaller LNPs were formulated by increasing the cholesterol content Stealth-like properties, such as prolonged blood PK and low cytokine induction, have been demonstrated for LNP formulations containing ionizable lipid, cholesterol and DSG-PEG2000 at a molar ratio of 67.6/29.5/2.9. However, resulting LNP physicochemical properties were sub-optimal as particle sizes were often larger than 90 nm (e.g., ⁇ 100 nm) while encapsulation efficiency was often below 90%.
  • the purpose of this experiment was to observe murine-based in vivo Luciferase expression, body weight loss, and cytokine induction comparing this LNP formulation against LNP formulations containing higher cholesterol content, e.g., LNP formulations containing ionizable lipid, cholesterol and DSG-PEG2000 at a molar ratio of 57.6 : 39.5 : 2.9, which was demonstrated to produce LNPs less than 80 nm in size and greater than 90% encapsulated cargo. Comparable expression, body weight loss, and cytokine induction was observed between these two LNP formulations at both 0.5 and 2.0 mpk, implying that LNP potency and tolerability properties were not lost when modifying the lipid composition to improve physiochemical properties.
  • FIGs.17A and 17B show IVIS readouts performed at day 4 and day 7 post-dose administration measuring the level of Luciferase protein expressed in CD-1 mouse liver tissue. At 0.5 and 2.0 mpk, the LNPs of the present disclosure had comparable Luciferase expression at both time points. Expression data suggests that shifts from 29.5% to 39.5% cholesterol and from 67.6% to 57.6% ionizable lipid in the LNPs of the present disclosure did not significantly impact payload expression.
  • FIGs.18A and 18B show CD-1 mouse body weight loss at day 1 post-dose administration.
  • Mouse body weight loss at day 1 post-administration was generally consistent between PBS and 0.5 mpk groups.
  • the LNP Formulation 118 containing 29.5% cholesterol resulted in a small decrease in body weight compared to other groups, but values below 5% body weight loss may not be significant.
  • mice across all dose groups averaged 5-10% body weight loss.
  • FIGs.19A-19E show cytokine profiles of test articles comparing 0.5 and 2.0 mpk doses.
  • Example 13 Antibody conjugation of the LNPs of the present disclosure leads to significant increase in uptake and expression as compared to the parental LNPs, regardless of ionizable lipid
  • This example compares DiD uptake and mRNA luciferase expression in primary human hepatocytes of the LNPs of the present disclosure, with or without antibody conjugation (e.g., scFv, 140 ME146907648v.1 VHH, Fab, etc.), using the protocols as shown in FIG.20 (without an endocytosis inhibitor) and FIG. 23 (with an endocytosis inhibitor).
  • the aim of the study using the protocol as shown in FIG.23 is to ensure that uptake and expression of conjugated LNPs with varying ionizable lipids is clathrin- mediated by using an endocytosis inhibitor.
  • the media containing an endocytosis inhibitor is incomplete (no BSA or transferrin) media with 100 ⁇ M of DynGo 4a.
  • Table 19 Physicochemical properties of LNP formulations having mRNA-luciferase cargo Referring to FIGs.21A and 21B and Table 19, antibody (VHH) conjugation for targeting hepatic ASGPR1 protein led to significant increase in LNP uptake compared to the parental LNPs of the present disclosure without antibody conjugation, and regardless of the identity of the ionizable lipid.
  • antibody (VHH) conjugation for targeting hepatic ASGPR1 protein led to higher levels of expression of mLuc and rLuc luciferase cargo compared to the parental LNPs of the present disclosure without antibody conjugation, except for DOTAP.
  • DOTAP antibody conjugation for targeting hepatic ASGPR1 protein

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Abstract

Provided herein are lipid nanoparticles (LNPs) comprising a therapeutic nucleic acid (TNA) and uses thereof. The LNPs comprise an ionizable lipid; a structural lipid, e.g., a sterol; and one or more types of lipid-anchored polymers, and do not comprise a helper lipid.

Description

LIPID NANOPARTICLES AND USES THEREOF RELATED APPLICATIONS The instant application claims priority to U.S. Provisional Application No.63/429,226, filed on December 1, 2022; U.S. Provisional Application No.63/449,610, filed on March 3, 2023; and U.S. Provisional Application No.63/467,116, filed on May 17, 2023. The entire contents of each of the foregoing applications are expressly incorporated herein by reference in their entireties. BACKGROUND Lipid-based nanoparticles have played a pivotal role in the successes of COVID-19 vaccines and many other nanomedicines, such as Doxil® and Onpattro®, and have therefore been considered as a frontrunner among nanoscale drug delivery systems. However, effective targeted delivery of biologically active substances, such as therapeutic nucleic acids, represents a continuing medical challenge. This has severely limited broad applications of nucleic acids such as mRNA and DNA in non-viral gene replacement therapy, gene therapy, gene editing, and vaccination. Lack of effective methods and vehicles for non-viral delivery represents a major barrier to a broad use of nucleic acid therapeutics. Generally, non-viral delivery of the larger mRNA or DNA genetic cargoes is more challenging than that of small oligonucleotides, in part due to the fact that mRNA and DNA molecules (which typically range from 300 kDa to 5,000 kDa in size, or ∼1-15 kb) are significantly larger than other types of RNAs, such as small interfering RNAs or siRNA (which are typically ∼14 kDa) or antisense oligonucleotides or ASOs (which typically range from 4 kDa to 10 kDa). Furthermore, viral delivery of nucleic acid therapeutics to targeted cells is hindered greatly by the activation of the innate and/or adaptive immune responses. Whereas it is possible to avoid RNA sensing by myeloid dendritic cells (MDCs) by chemically modifying RNA cargo (e.g., with 1mΨ, 2’OMe, etc.), there are no known chemical modifications to a DNA cargo that can limit pattern recognition receptor (PRR) sensing and still maintain transcriptional activity. An alternative approach to gene therapy is the recombinant adeno-associated virus (rAAV) vector platform that packages heterologous DNA in a viral capsid. However, there are several major disadvantages to using rAAV vectors as a gene delivery vector. One major drawback associated with rAAV is its limited viral packaging capacity of about 4.5 kb of heterologous DNA. Another major drawback is capsid immunogenicity that prevents re-administration to patients. Thus, there remains a need for effective delivery vehicles that enable safe and effective non- viral delivery of nucleic acid therapeutics to desired cell populations. 1 ME146907648v.1 SUMMARY The present disclosure provides lipid nanoparticles (LNPs) and LNP compositions (e.g., pharmaceutical compositions) comprising a therapeutic nucleic acid (TNA), e.g., a gene expression vector such as closed-ended DNA (ceDNA), single-stranded DNA (ssDNA) vector, or messenger RNA (mRNA), wherein the structural LNP components comprise an ionizable lipid; a structural lipid, e.g., a sterol; and one or more types of lipid-anchored polymers, and wherein the entire structural LNP components do not comprise a helper lipid. The LNPs disclosed herein provide surprising and unexpected properties as compared to known LNPs comprising helper lipids. These properties include, e.g., improved tolerability and slower plasma clearance indicating an increased stealth property. Moreover, the disclosed LNPs and LNP compositions surprisingly reduce LNP related toxicity, as is evidenced by serum levels of immune response markers (see Examples herein). Further, the disclosed LNPs and LNP compositions comprising a certain molecular percentage of sterol (30%-45% molecular percentage of the total lipid) are characterized by a diameter of about 80 nm or less, making them particularly amenable and useful for therapeutic administration as most target organs require the size of LNP being smaller than 85 nm for efficient delivery. According to one aspect, the disclosure provides a lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; an ionizable lipid; a sterol; a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises: a polymer; a lipid moiety comprising at least one hydrophobic tail; and optionally a linker connecting the polymer to the lipid moiety; wherein the at least one hydrophobic tail comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; and wherein the LNP does not comprise a helper lipid. In some embodiments, the therapeutic nucleic acid (TNA) is about 100 to about 10,000 base pairs or about 100 to about 10,000 nucleotides in length, or about 250 to about 10,000 base pairs or about 250 to about 10,000 nucleotides in length, or about 500 to about 10,000 base pairs or about 500 to about 10,000 nucleotides in length, or about 1,000 to about 10,000 base pairs or about 1,000 to about 10,000 nucleotides in length, or about 2,500 to about 10,000 base pairs or about 2,500 to about 10,000 nucleotides in length, or about 3,000 to about 10,000 base pairs or about 3,000 to about 10,000 nucleotides in length, or about 4,000 to about 10,000 base pairs or about 4,000 to about 10,000 nucleotides in length. In some embodiments, the therapeutic nucleic acid (TNA) does not comprise short interfering RNA (siRNA). In some embodiments, the therapeutic nucleic acid (TNA) does not comprise microRNA (miRNA). In some embodiments, the therapeutic nucleic acid (TNA) does not comprise antisense oligonucleotide (ASO). 2 ME146907648v.1 In some embodiments, the LNP has an average particle size of about 50-100 nm in diameter. In some embodiments, the LNP has an average particle size of about 50-90 nm in diameter. In some embodiments, the LNP has an average particle size of about 50-80 nm in diameter. In some embodiments, the LNP has an average particle size of about 50-70 nm in diameter. In some embodiments, the LNP has an average particle size of about 50-60 nm in diameter. In some embodiments, the lipid moiety comprises at least two hydrophobic tails. In some embodiments, the two hydrophobic tails each independently comprise 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 16, 17, 18, 19, 20, or 21 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 16, 17, 18, 19 or 20 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 16, 17, 18, or 19 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprises 16, 17, or 18 carbon atoms. In some embodiments, the two hydrophobic tails each comprise 18, 19 or 20 carbon atoms. In some embodiments, the two hydrophobic tails each comprise 16 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 18 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 20 carbon atoms. In some embodiments, the at least one or two hydrophobic tails are each a fatty acid. In some embodiments, the at least one or two hydrophobic tails are each independently selected from the group consisting of octadecane, octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and a derivative thereof. In some embodiments, the at least two hydrophobic tails are each independently selected from the group consisting of lauric acid, myristic acid, myristoleic acid, and a derivative thereof. In some embodiments, the lipid moiety comprises two hydrophobic tails. In some embodiments, the two hydrophobic tails are each a fatty acid. In some embodiments, the two hydrophobic tails are each independently selected from the group consisting of octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and a derivative thereof. In some embodiments, the two hydrophobic tails are each independently selected from the group consisting of lauric acid, myristic acid, myristoleic acid, and a derivative thereof. In some embodiments, the first lipid-anchored polymer comprises a glycerolipid. In some embodiments, the first lipid-anchored polymer comprises a phospholipid. In some embodiments, the first lipid-anchored polymer comprises a linker-lipid moiety selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn- 3 ME146907648v.1 glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn- phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn- glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), a derivative thereof, and a combination of any of the foregoing. In some embodiments, the first lipid- anchored polymer comprises a linker-lipid moiety selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, a derivative thereof, and a combination of any of the foregoing. In some embodiments, the polymer in the first lipid-anchored polymer is selected from the group consisting of polyethylene (PE), polypropylene (PP), polyethylene glycol (PEG), polyglycerol (PG), polyvinyl alcohol (PVOH), polysarcosine (pSar), and a combination thereof. In some embodiments, the polymer is selected from the group consisting of polyethylene glycol (PEG), polyglycerol (PG), polysarcosine (pSar), and a combination thereof. In some embodiments, the polymer has a molecular weight of between about 1000 Da and about 5000 Da. In some embodiments, the polymer has a molecular weight of between about 2000 Da and about 5000 Da. In some embodiments, the polymer has a molecular weight of about 2000 Da. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, the sterol is selected from the group consisting of cholesterol, beta- sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and a derivative of thereof, and a combination of any of the foregoing. In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is beta-sitosterol. In some embodiments, the ionizable lipid is a lipid represented by: a) Formula (A):
Figure imgf000005_0001
Formula (A), or a pharmaceutically acceptable salt thereof, wherein: R1 and R1’ are each independently optionally substituted linear or branched C1-3 alkylene; 4 ME146907648v.1 R2 and R2’ are each independently optionally substituted linear or branched C1-6 alkylene; R3 and R3’ are each independently optionally substituted linear or branched C1-6 alkyl; or alternatively, when R2 is optionally substituted branched C1-6 alkylene, R2 and R3, taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; or alternatively, when R2’ is optionally substituted branched C1-6 alkylene, R2’ and R3', taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; R4 and R4’ are each independently –CRa, –C(Ra)2CRa, or –[C(Ra)2]2CRa; Ra, for each occurrence, is independently H or C1-3 alkyl; or alternatively, when R4 is –C(Ra)2CRa, or –[C(Ra)2]2CRa and when Ra is C1-3 alkyl, R3 and R4, taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; or alternatively, when R4’ is –C(Ra)2CRa, or –[C(Ra)2]2CRa and when Ra is C1-3 alkyl, R3’ and R4’, taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; R5 and R5’ are each independently hydrogen, C1-20 alkylene or C2-20 alkenylene; R6 and R6’, for each occurrence, are independently C1-20 alkylene, C3-20 cycloalkylene, or C2-20 alkenylene; and m and n are each independently an integer selected from 1, 2, 3, 4, and 5; or b) Formula (B):
Figure imgf000006_0001
Formula (B); or a pharmaceutically acceptable salt thereof, wherein: a is an integer ranging from 1 to 20; b is an integer ranging from 2 to 10; R1 is absent or is selected from (C2-C20)alkenyl, -C(O)O(C2-C20)alkyl, and cyclopropyl substituted with (C2-C20)alkyl; and R2 is (C2-C20)alkyl; or c) Formula (C):
Figure imgf000006_0002
Formula (C); or a pharmaceutically acceptable salt thereof, wherein: 5 ME146907648v.1 R1 and R1’ are each independently (C1-C6)alkylene optionally substituted with one or more groups selected from Ra; R2 and R2’ are each independently (C1-C2)alkylene; R3 and R3’ are each independently (C1-C6)alkyl optionally substituted with one or more groups selected from Rb; or alternatively, R2 and R3 and/or R2’ and R3’ are taken together with their intervening N atom to form a 4- to 7-membered heterocyclyl; R4 and R4’ are each a (C2-C6)alkylene interrupted by –C(O)O-; R5 and R5’ are each independently a (C2-C30)alkyl or (C2-C30)alkenyl, each of which are optionally interrupted with –C(O)O- or (C3-C6)cycloalkyl; and Ra and Rb are each halo or cyano; or d) Formula (D):
Figure imgf000007_0001
Formula (D), or a pharmaceutically acceptable salt thereof, wherein: R’ is absent, hydrogen, or C1-C6 alkyl; provided that when R’ is hydrogen or C1-C6 alkyl, the nitrogen atom to which R’, R1, and R2 are allpositively charged; R1 and R2 are each independently hydrogen, C1-C6 alkyl, or C2-C6 alkenyl; R3 is C1-C12 alkylene or C2-C12 alkenylene; R4 is C1-C18 unbranched alkyl, C2-C18 unbranched alkenyl, or
Figure imgf000007_0002
wherein: R4a and R4b are each independently C1-C16 unbranched alkyl or C2-C16 unbranched alkenyl; R5 is absent, C1-C8 alkylene, or C2-C8 alkenylene; R6a and R6b are each independently C7-C16 alkyl or C7-C16 alkenyl; provided that the total number of carbon atoms in R6a and R6b as combined is greater than 15; X1 and X2 are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(Ra)=N-, -N=C(Ra)-, -C(Ra)=NO-, -O-N=C(Ra)-, -C(=O)NRa-, -NRaC(=O)-, -NRaC(=O)NRa-, -OC(=O)O-, -OSi(Ra)2O-, -C(=O)(CRa2)C(=O)O-, or OC(=O)(CRa2)C(=O)-; wherein: Ra, for each occurrence, is independently hydrogen or C1-C6 alkyl; and 6 ME146907648v.1 n is an integer selected from 1, 2, 3, 4, 5, and 6; wherein, in some embodiments, R4 is C1-C16 unbranched alkyl, C2-C16 unbranched alkenyl, or
Figure imgf000008_0001
R4a and R4b are as defined above; or e) Formula (E):
Figure imgf000008_0002
Formula (E), or a pharmaceutically acceptable salt thereof, wherein: R’ is absent, hydrogen, or C1-C3 alkyl; provided that when R’ is hydrogen or C1-C3 alkyl, the nitrogen atom to which R’, R1, and R2 are all attached ispositively charged; R1 and R2 are each independently hydrogen or C1-C3 alkyl; R3 is C3-C10 alkylene or C3-C10 alkenylene; R4 is C1-C16 unbranched alkyl, C2-C16 unbranched alkenyl, or
Figure imgf000008_0003
wherein: R4a and R4b are each independently C1-C16 unbranched alkyl or C2-C16 unbranched alkenyl; R5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; R6a and R6b are each independently C7-C14 alkyl or C7-C14 alkenyl; X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(Ra)=N-, -N=C(Ra)-, -C(Ra)=NO-, -O-N=C(Ra)-, -C(=O)NRa-, -NRaC(=O)-, -NRaC(=O)NRa-, -OC(=O)O-, -OSi(Ra)2O-, -C(=O)(CRa2)C(=O)O-, or OC(=O)(CRa2)C(=O)-; wherein: Ra, for each occurrence, is independently hydrogen or C1-C6 alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6; or f) an ionizable lipid selected from any of the ionizable lipids in Table 1, 4, 5, 6 or 7. In some embodiments, the LNP further comprises a targeting moiety. In some embodiments, the LNP comprises a second lipid-anchored polymer and the targeting moiety is conjugated to the second lipid-anchored polymer. In some embodiments, the second lipid- anchored polymer comprises a linker-lipid moiety selected from the group consisting of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- 7 ME146907648v.1 phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3- phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); and dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), or a derivative thereof. In some embodiments, the second lipid-anchored polymer comprises a linker-lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, a derivative thereof, and a combination of any of the foregoing. In some embodiments, the first and the second lipid-anchored polymers are different lipid- anchored polymers; and the first and the second lipid-anchored polymers comprise one of the following combinations: DSG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DSPE (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); and DMG (the first lipid-anchored polymer) and DPG (the second lipid-anchored polymer). In some embodiments, the first and the second lipid-anchored polymers are the same lipid- anchored polymers; and wherein the first and the second lipid-anchored polymers comprise one of the following combinations: DSG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DSPE (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); and DPG (the first lipid-anchored polymer) and DPG (the second lipid-anchored polymer). In some embodiments, the targeting moiety is conjugated to a DSPE-anchored polymer. In some embodiments, the DSPE-anchored polymer is DSPE-PEG or a derivative thereof. In some embodiments, the targeting moiety is conjugated to a DSG-anchored polymer. In some embodiments, the DSG-anchored polymer is DSG-PEG or a derivative thereof. 8 ME146907648v.1 In some embodiments, the targeting moiety is capable of binding to a liver cell. In some embodiments, the liver cell is a hepatocyte. In some embodiments, the targeting moiety is N-acetyl galactosamine (GalNAc) or a GalNAc derivative. In some embodiments, the targeting moiety is a tri- antennary GalNAc conjugate or a tetra-antennary GalNAc conjugate. In some embodiments, the targeting moiety is an ApoE protein, an ApoE polypeptide, an ApoB protein, an ApoB polypeptide, a fragment thereof, or a derivative of any of the foregoing. In some embodiments, the targeting moiety is capable of binding to ASGPR1 protein. In some embodiments, the targeting moiety is an antibody, antibody fragment, or an antibody derivative. In some embodiments, the antibody, antibody fragment, or antibody derivative is selected from the group consisting of a full-length antibody, an Fab, an Fab’, a single-domain antibody, a single-chain antibody, a nanobody, and a VHH. In some embodiments, the LNP further comprises a dissociable lipid-conjugated polymer; wherein the dissociable lipid-conjugated polymer comprises: a polymer; a lipid moiety comprising at least one hydrophobic tail; and optionally a linker connecting the polymer to the lipid moiety; wherein the at least one hydrophobic tail comprises 10-15 carbon atoms in a single aliphatic chain backbone. In some embodiments, the at least one hydrophobic tail comprises 14 carbon atoms in a single aliphatic chain backbone. In some embodiments, the dissociable lipid-conjugated polymer is 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol (DMG-PEG) or mono-C18-PEG. In some embodiments, the mono-C18-PEG is mono-C18-PEG2000. In some embodiments, the dissociable lipid-conjugated polymer is present in the LNP in an amount of about 5%. In one embodiment, the ionizable lipid in an LNP of the present disclosure in accordance with any of the foregoing embodiments is Ionizable Lipid 87:
Figure imgf000010_0001
, or a pharmaceutically acceptable salt thereof. In some embodiments, the ionizable lipid is present in the LNP in an amount of about 35 mol% to about 60 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of no less than about 30 mol% (i.e., ≥ 30 mol%) of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 20 mol% to about 45 mol% of the total lipid present in the 9 ME146907648v.1 LNP. In some embodiments, the sterol is present in the LNP in an amount of about 35 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 35 mol% to about 40 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 40 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 35 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 40 mol% of the total lipid present in the LNP. In some embodiments, the sterol is present in the LNP in an amount of about 45 mol% of the total lipid present in the LNP. In some embodiments, the first lipid-anchored polymer is present in the LNP in an amount of about 0.005 mol% to about 5 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.05 mol% to about 2 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.1 mol% to about 1 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.5 mol% of the total lipid present in the LNP. In some embodiments, the first lipid-anchored polymer and the second lipid anchored polymer are present in the LNP in an amount of about 2.5 mol% and 0.5 mol%, respectively, of the total lipid present in the LNP. In some embodiments, the LNP is suitable for intravenous administration. In some embodiments, the LNP is less immunogenic than a reference LNP that comprises a helper lipid; and/or does not comprise the first lipid-anchored polymer and comprises a reference lipid-anchored polymer comprising one or more hydrophobic tails that each independently comprises less than 16 carbon atoms in a single aliphatic chain backbone. In some embodiments, the LNP elicits lower pro-inflammatory cytokine response than the reference LNP. In some embodiments, the reference lipid-anchored polymer is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) or methoxypolyethyleneglycoloxy-N,N-ditetradecylacetamide (Ac-PEG). In some embodiments, the LNP elicits lower pro-inflammatory cytokine response than the reference LNP. In some embodiments, the LNP results in a lower uptake level of TNA by a blood cell than that of the reference LNP. In some embodiments, the blood cell is selected from the group consisting of a red blood cell, a macrophage, and a peripheral blood mononuclear cell. In some embodiments, the therapeutic nucleic acid (TNA) is selected from the group consisting of a minigene, a plasmid, a minicircle, a small interfering RNA (siRNA), a microRNA (miRNA), a guide RNA (gRNA) an antisense oligonucleotide (ASO), a ribozyme, a closed-ended DNA (ceDNA), single-stranded DNA (ssDNA), a ministring, a doggybone™, a protelomere closed ended DNA, a dumbbell linear DNA, synthetic single stranded AAV vector, a dicer-substrate dsRNA, a small hairpin RNA (shRNA), an asymmetrical interfering RNA (aiRNA), mRNA, tRNA, rRNA, a DNA viral vector, a viral RNA vector, a non-viral vector and any combination thereof. 10 ME146907648v.1 In some embodiments, the TNA is greater than about 200 bp or greater than about 200 nt in length. In some embodiments, the TNA is greater than about 500 bp or greater than about 500 nt in length. In some embodiments, the TNA is greater than about 1000 bp or greater than about 1000 nt in length. In some embodiments, the TNA is greater than about 4000 bp or greater than about 4000 nt in length. In some embodiments, the TNA is a closed-ended DNA (ceDNA). In some embodiments, the TNA is a messenger RNA (mRNA). In one embodiment, the TNA is a single- stranded DNA (ssDNA). In some embodiments, the TNA is a single-stranded (ss) nucleic acid. In some embodiments, the TNA is a double-stranded nucleic acid. In some aspects, the present disclosure also provides a pharmaceutical composition comprising the LNP of the present disclosure and a pharmaceutically acceptable carrier. In some aspects, the present disclosure also provides a method of treating a genetic disorder in a subject, the method comprising administering to the subject an effective amount of the LNP of the present disclosure or the pharmaceutical composition of the present disclosure. In some embodiments, the subject is a human. In some embodiments, the genetic disorder is selected from the group consisting of sickle cell anemia; melanoma; hemophilia A (clotting factor VIII (FVIII) deficiency); hemophilia B (clotting factor IX (FIX) deficiency); cystic fibrosis (CFTR); familial hypercholesterolemia (LDL receptor defect); hepatoblastoma; Wilson disease; phenylketonuria (PKU); congenital hepatic porphyria; an inherited disorder of hepatic metabolism; Lesch Nyhan syndrome; a thalassaemia; xeroderma pigmentosum; Fanconi’s anemia; retinitis pigmentosa; ataxia telangiectasia; Bloom’s syndrome; retinoblastoma; a mucopolysaccharide storage disease; a Niemann-Pick Disease; Fabry disease; Schindler disease; GM2-gangliosidosis Type II (Sandhoff Disease); Tay-Sachs disease; Metachromatic Leukodystrophy; Krabbe disease; a mucolipidosis (ML); Sialidosis Type II, a glycogen storage disease (GSD); Gaucher disease; cystinosis; Batten disease; Aspartylglucosaminuria; Salla disease; Danon disease (LAMP-2 deficiency); Lysosomal Acid Lipase (LAL) deficiency; a neuronal ceroid lipofuscinoses (NCL); a sphingolipidoses, galactosialidosis; amyotrophic lateral sclerosis (ALS); Parkinson’s disease; Alzheimer’s disease; Huntington’s disease; spinocerebellar ataxia; spinal muscular atrophy (SMA); Friedreich’s ataxia; Duchenne muscular dystrophy (DMD); a Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB); ectonucleotide pyrophosphatase 1 deficiency; generalized arterial calcification of infancy (GACI); Leber Congenital Amaurosis; Stargardt disease; wet macular degeneration (wet AMD); ornithine transcarbamylase (OTC) deficiency; Usher syndrome; alpha-1 antitrypsin deficiency; a progressive familial intrahepatic cholestasis (PFIC); and Cathepsin A deficiency. In some embodiments, the genetic disorder is phenylketonuria (PKU). In some embodiments, the genetic disorder is hemophilia A (Factor VIII deficiency). In some embodiments, the genetic disorder is Wilson disease. In some embodiments, the genetic disorder is Gaucher disease. In some embodiments, the genetic disorder is Gaucher disease Type I, Gaucher disease Type II or Gaucher 11 ME146907648v.1 disease type III. In some embodiments, the genetic disorder is Leber congenital amaurosis (LCA). In some embodiments, the LCA is LCA10. In some embodiments, the genetic disorder is Stargardt disease. In some embodiments, the genetic disorder is wet macular degeneration (wet AMD). In some aspects, the present disclosure also provides a method of providing anti-tumor immunity in a subject, the method comprising administering to the subject an effective amount of the LNP of the present disclosure or the pharmaceutical composition of the present disclosure. In some aspects, the present disclosure also provides a method of treating a subject having a disease, disorder or condition associated with an elevated expression of a tumor antigen, the method comprising administering to the subject an effective amount of the LNP of the present disclosure or the pharmaceutical composition of the present disclosure. In some embodiments, the subject is a human. In some embodiments, the TNA is retained in the spleen for at least about 6 hours, or at least about 9 hours, or at least about 12 hours, or at least about 15 hours, or at least about 18 hours, or at least about 21 hours, or at least about 24 hours, or at least about 27 hours, or at least about 30 hours, or at least about 33 hours, or at least about 36 hours after dosing. In some embodiments, the concentration of the TNA at the start of a 12, 18, or 24-hour time window post-dosing and the concentration of the TNA at the end of the time window are within the same order of magnitude. In one embodiment, the TNA is a messenger RNA (mRNA). In one embodiment, the TNA is a single-stranded DNA (ssDNA). In some aspects, the present disclosure further provides a method of treating a blood disease, disorder or condition in a subject, the method comprising administering to the subject an effective amount of the LNP of the present disclosure or the pharmaceutical composition of the present disclosure. In some embodiments, the blood disease, disorder or condition is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Hodgkin lymphoma (HL), multiple myeloma, a myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), adrenoleukodystrophy (ALD), Hurler syndrome, Krabbe disease (Globoid-cell leukodystrophy or GLD), metachromatic leukodystrophy (MLD), severe aplastic anemia (SAA), severe combined immunodeficiency (SCID), sickle cell disease (SCD), thalassemia, Wiskott-Aldrich syndrome, Diamond-Blackfan anemia, essential thrombocytosis, Fanconi anemia, hemophagocytic lymphohistiscytosis (HLH), juvenile myelomonocytic leukemia (JMML), myelofibrosis, polycythemia vera, and a combination thereof. In one embodiment, the TNA is a messenger RNA (mRNA). In one embodiment, the TNA is a single-stranded DNA (ssDNA). In some aspects, the present disclosure also provides a lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; wherein the TNA is a deoxyribonucleic acid (DNA) or a messenger ribonucleic acid (mRNA); an ionizable lipid; a sterol; 12 ME146907648v.1 a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises: a polymer; a lipid moiety comprising at least one or two hydrophobic tails; and optionally a linker connecting to the polymer to the lipid moiety; wherein each of the at least two hydrophobic tails independently comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; and wherein the LNP does not comprise a helper lipid. In some aspects, the present disclosure also provides a lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; an ionizable lipid; a sterol; a first lipid-anchored polymer and a second lipid-anchored polymer; wherein the first lipid- anchored polymer and the second lipid-anchored polymer each comprise: a polymer; a lipid moiety comprising at least one or two hydrophobic tails; wherein each of the at least two hydrophobic tails independently comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; and a linker connecting the polymer to the lipid moiety; wherein the second lipid-anchored polymer is conjugated to a targeting moiety; and wherein the LNP does not comprise a helper lipid. In some aspects, the present disclosure also provides a lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; an ionizable lipid; a sterol; a first lipid-anchored polymer and a second lipid-anchored polymer; wherein the first lipid- anchored polymer and the second lipid-anchored polymer each comprise: a polymer; a lipid moiety comprising at least one or two hydrophobic tails; wherein each of the at least two hydrophobic tails independently comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; and optionally a linker connecting the polymer to the lipid moiety; wherein the second lipid-anchored polymer is conjugated to a targeting moiety; and 13 ME146907648v.1 wherein the LNP does not comprise a helper lipid, and wherein the composition is prepared by a method comprising the following steps: a) adding the TNA to a first solution comprising one or more low molecular weight alcohols to result in a TNA solution having an alcohol content of >80%; b) adding the TNA solution to a second solution comprising the ionizable lipid, the sterol, the first lipid-anchored polymer and the second lipid-anchored polymer in one or more low molecular weight alcohols to result in a TNA/lipid solution having an alcohol content of between about 80% and about 95%; c) mixing the TNA/lipid solution with an acidic aqueous buffer to form a treated TNA/lipid solution; and d) subjecting the treated TNA/lipid solution to buffer exchange with a neutral-pH aqueous buffer to produce the LNP. In some aspects, the present disclosure also provides a lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; an ionizable lipid; a sterol; a first lipid-anchored polymer and a second lipid-anchored polymer; wherein the first lipid- anchored polymer and the second lipid-anchored polymer each comprise: a polymer; a lipid moiety comprising at least one or two hydrophobic tails; wherein each of the at least two hydrophobic tails independently comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; and optionally a linker connecting the polymer to the lipid moiety; wherein the second lipid-anchored polymer is conjugated to a targeting moiety; and wherein the LNP does not comprise a helper lipid, and wherein the composition is prepared by a method comprising the following steps: a) adding the TNA cargo to a solution comprising the ionizable lipid, the sterol, the first lipid- anchored polymer and the second lipid-anchored polymer in one or more low molecular weight alcohols to result in a TNA/lipid solution having an alcohol content of between about 80% and about 95%; b) mixing the TNA/lipid solution with an acidic aqueous buffer to form a treated TNA/lipid solution; and c) subjecting the treated TNA/lipid solution to buffer exchange with a neutral-pH aqueous buffer to thereby produce the LNP. 14 ME146907648v.1 According to some embodiments, the targeting moiety is a tissue- and/or cell-type specific targeting moiety. According to some embodiments, the targeting moiety is selected from the group consisting of a protein, a nucleic acid, and a sugar. According to some embodiments, the targeting moiety is an antibody, antibody fragment, or antibody derivative. According to some embodiments, the antibody, antibody fragment, or antibody derivative is selected from the group consisting of a full- length antibody, a Fab, a Fab’, a single-domain antibody, a single-chain antibody, and a VHH. According to some embodiments, the antibody, antibody fragment, or antibody derivative is an scFv. According to some embodiments, the antibody, antibody fragment, or antibody derivative is a VHH. According to further embodiments, the VHH is a nanobody. According to some embodiments, the targeting moiety is located on the exterior of the LNP. According to some embodiments, the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. According to some embodiments, the targeting moiety is an aptamer. According to some embodiments, the targeting moiety binds specifically to a T cell antigen. According to some embodiments, the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, PD-1, and TCR. According to some embodiments, the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD5, CD6, and CD7. According to some embodiments, the LNP further comprises a linker between the second lipid-anchored polymer and the targeting moiety. According to some embodiments, the first lipid-linker and the second lipid-linker are each independently selected from the group consisting of a non-ester-containing linker and an ester-containing linker. According to some embodiments, the ester-containing linker is selected from the group consisting of an amide linker and a carbamate linker. According to some embodiments, the targeting moiety is conjugated to the second lipid-anchored polymer via maleimide conjugation. According to some embodiments, the targeting moiety is conjugated to the second lipid-anchored polymer via click chemistry. BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the present disclosure, briefly summarized above and discussed in greater detail below, can be understood by reference to the illustrative embodiments of the disclosure depicted in the appended drawings. However, the appended drawings illustrate only typical embodiments of the disclosure and are therefore not to be considered limiting of scope, for the disclosure may admit to other equally effective embodiments. FIG.1 shows the percent change in body weight of mice at Day 1 following administration of inventive LNP formulations LNP4, LNP5, LNP6, LNP7 (e.g., having no helper lipid) and Control LNP B formulation that contains the helper lipid distearoylphosphatidylcholine (DSPC)). FIGS.2A-2F depict blood serum levels of the cytokines IFN-alpha (FIG.2A), IL-6 (FIG. 2B), IFN-gamma (FIG.2C), TNF-alpha (FIG.2D), IL-18 (FIG.2E), and IP-10 (FIG.2F) measured in CD-1 mice 6 hours after dosing of LNP formulations LNP4, LNP6, LNP7 (e.g., having no helper 15 ME146907648v.1 lipid) and the Control LNP B formulation that contains the helper lipid distearoylphosphatidylcholine (DSPC)). FIG.3 shows the whole blood and plasma levels of the ceDNA cargo of Control LNP B formulation and inventive LNP formulation LNP4 in CD-1 mice at 1 hour, 3 hours, and 6 hours post- dosing. FIG.4A shows the in vitro expression of luciferase in primary mouse hepatocytes that were treated with inventive LNP formulations LNP8, LNP9, LNP10, LNP11 (e.g., having no helper lipid) that each carried an mRNA luciferase cargo. FIG.4B shows the DiD signals that indicate the uptake of these inventive LNP formulations into the primary mouse hepatocytes. FIG.5 shows and compares the 24-hour total IVIS fluorescence in the liver of CD-1 mice groups dosed with LNP101, LNP102, LNP103, LNP104, and Control LNP C, all of which carry luciferase mRNA as a nucleic acid cargo. FIG.6A is a curve quantifying, via qPCR, concentrations of luciferase mRNA (µg/mL) in whole blood at 2 minutes, 1 hour, 6 hours, and 24 hours after dosing for CD-1 mice groups dosed with LNP101, LNP102, LNP103, LNP104, and Control LNP C. FIG.6B is a curve quantifying, via qPCR, copies of luciferase mRNA in the liver at 6 hours and 24 hours after dosing for CD-1 mice groups dosed with LNP101, LNP102, LNP103, LNP104, and Control LNP C. FIG.6C is a curve quantifying, via qPCR, copies of luciferase mRNA) in the spleen at 6 hours and 24 hours after dosing for CD-1 mice groups dosed with LNP101, LNP102, LNP103, LNP104, and Control LNP C. FIG.6D is a curve quantifying, via qPCR, copies of luciferase mRNA in the bone marrow at 6 hours and 24 hours after dosing for CD-1 mice groups dosed with LNP101, LNP102, LNP103, LNP104, and Control LNP C. FIG.7A shows and compares the 7-day total IVIS fluorescence in the liver of CD-1 mice groups dosed with the LNPs of the present disclosure, as well as Control LNP 010, all of which carry ceDNA luciferase as a nucleic acid cargo. FIG.7B shows and compares percent change in body weight at Day 1 of CD-1 mice groups dosed with the LNPs of the present disclosure, as well as Control LNP 010, all of which carry ceDNA luciferase as a nucleic acid cargo. FIG.8A shows the IVIS expression data collected on Day 7 in the liver of CD-1 mice (5 per group) dosed at 1.0 mg/kg with the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different variants of Ionizable Lipid 87, and with or without a GalNAc-based targeting ligand. 16 ME146907648v.1 FIG.8B shows the percent change in body weight of CD-1 mice (5 per group) at Day 1 following administration of the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different variants of Ionizable Lipid 87, and with or without a GalNAc-based targeting ligand. FIG.9A shows the DiD signals that indicate the uptake of these inventive LNP formulations (e.g., having no helper lipid) into the primary mouse hepatocytes. FIG.9B shows the in vitro expression of luciferase in primary mouse hepatocytes that were treated with inventive LNP formulations (e.g., having no helper lipid) that each carried an mRNA luciferase cargo. FIG.10A schematically shows the formulation approach of adding/subtracting alternative polymer (e.g., 5% DMG-PEG or mono-C18-PEG) to/from total lipid content of the base LNP compositions (e.g., LNP Formulations 617, 618 and 619) of the present disclosure (e.g., having no helper lipid). FIG.10B shows the IVIS expression data collected on Day 7 in the liver of CD-1 mice (5 per group) dosed at 2.0 mg/kg with the LNPs of the present disclosure (e.g., having no helper lipid) formulated according to the scheme as shown in FIG.10A. FIG.10C shows the percent change in body weight of CD-1 mice (5 per group) at Day 1 following administration of the LNPs of the present disclosure (e.g., having no helper lipid) formulated according to the scheme as shown in FIG.10A. FIGs.11A and 11B show the percent change in body weight of CD-1 mice (5 per group) at Day 1, and longitudinal body weight loss over the course of the study, following administration of the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different processes, and with or without a GalNAc-based targeting ligand. FIGs.12A and 12B show the IVIS expression data collected on Days 4 and 7, respectively, in the liver of CD-1 mice (5 per group) dosed at 0.5, 2.0 and 4.0 mg/kg with the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different processes, and with or without a GalNAc-based targeting ligand. FIGs.13A-13F depict the blood serum levels of the cytokines IFN-alpha (FIG.13A), IFN- gamma (FIG.13B), IL-18 (FIG.13C), IL-6 (FIG.13D), IP-10 (FIG.13E), and TNF-alpha (FIG. 13F) measured in CD-1 mice (5 per group) dosed at 0.5 and 2.0 mg/kg with the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different processes, and with or without a GalNAc-based targeting ligand. FIGs.14A and 14B show the whole blood and plasma PK profiles of the ceDNA cargo of the stealth LNP formulations of the present disclosure prepared by Old Process (OP) or New Process (NP), with or without a GalNAc-based targeting ligand. Test articles were compared against a control LNP prepared with dissociable C14 tailed DMG-PEG2000. 17 ME146907648v.1 FIGs.15A and 15B show the whole blood and plasma PK profiles of the ceDNA cargo of the stealth LNP formulations of the present disclosure with higher amount of cholesterol, and with or without a GalNAc-based targeting ligand. Test articles were compared against a control LNP prepared with dissociable Ac-PEG2000. FIGs.16A-16C show ISH staining of mouse livers collected at 1-hour post-administration of of the stealth LNP formulations of the present disclosure with higher amount of cholesterol, and with or without a GalNAc-based targeting ligand. Test articles were compared against a control LNP prepared with dissociable Ac-PEG. FIGs.16D-16F show ISH staining of mouse livers collected at 6 hours post-administration of of the stealth LNP formulations of the present disclosure with higher amount of cholesterol, and with or without a GalNAc-based targeting ligand. Test articles were compared against a control LNP prepared with dissociable Ac-PEG. FIGs.17A and 17B show the IVIS expression data collected on Days 4 and 7 in the liver of CD-1 mice (5 per group) dosed at 0.5 and 2.0 mg/kg with the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different amounts of cholesterol. FIGs.18A and 18B show the percent change in body weight of CD-1 mice (5 per group) at Day 1, and longitudinal body weight loss over the course of the study, following administration of the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different amounts of cholesterol. FIGs.19A-19E depict the blood serum levels of the cytokines IFN-alpha (FIG.19A), IFN- gamma (FIG.19B), IL-18 (FIG.19C), TNF-alpha (FIG.19D), and IL-6 (FIG.19E) measured in CD-1 mice (5 per group) dosed at 0.5 and 2.0 mg/kg with the LNPs of the present disclosure (e.g., having no helper lipid) formulated with different amounts of cholesterol. FIG.20 depicts a workflow for using primary human hepatocytes to screen and compare various LNP formulations for the ability to enter cells, without an endocytosis inhibitor. FIGs.21A and 21B show the results of a screening study of the LNP formulations of the present disclosure with antibody (VHH; “A05”) conjugation for targeting hepatic ASGPR1 protein, for their relative ability to gain entry into primary human hepatocytes after 24 hours, according to the workflow as depicted in FIG.20. FIGs.22A and 22B show the results of a screening study of the LNP formulations of the present disclosure with antibody (VHH; “A05”) conjugation for targeting hepatic ASGPR1 protein, for their relative ability to express mLuc and rLuc cargo, according to the workflow as depicted in FIG.20. FIG.23 depicts a workflow for using primary human hepatocytes to screen and compare various LNP formulations for their relative ability to enter cells, with an endocytosis inhibitor. FIGs.24A and 24B show the results of a screening study of the LNP formulations of the present disclosure with antibody (VHH (“A05”) and scFv) conjugation for targeting hepatic ASGPR1 18 ME146907648v.1 protein, for their relative ability to gain entry into primary human hepatocytes after 24 hours, according to the workflow as depicted in FIG.23. FIGs.25A and 25B show the results of a screening study of the LNP formulations of the present disclosure with antibody (VHH (“A05”) and scVc) conjugation for targeting hepatic ASGPR1 protein, for their relative ability to express mLuc cargo, with varying inhibition conditions, according to the workflow as depicted in FIG.23. FIGs.26A and 26B show the results of a screening study of the LNP formulations of the present disclosure with antibody (VHH; “A05”) conjugation for targeting hepatic ASGPR1 protein, for their relative ability to gain entry into primary human hepatocytes after 24 hours, according to the workflow as depicted in FIG.23. FIGs.27A and 27B show the results of a screening study of the LNP formulations of the present disclosure with antibody (VHH; “A05”) conjugation for targeting hepatic ASGPR1 protein, for their relative ability to express mLuc and rLuc cargo, according to the workflow as depicted in FIG.23. DETAILED DESCRIPTION The present disclosure provides lipid nanoparticles (LNPs) and LNP compositions (e.g., pharmaceutical compositions), wherein the LNP comprises a therapeutic nucleic acid (TNA), an ionizable lipid; a structural lipid, e.g., a sterol; and one or more types of lipid-anchored polymers, wherein the LNP does not comprise a helper lipid. I. Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0- 911910-19-3); Robert S. Porter et al. (eds.), Fields Virology, 6th Edition, published by Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013), Knipe, D.M. and Howley, P.M. (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1- 56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 19 ME146907648v.1 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al. Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. The abbreviation, “e.g.” is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the term “about,” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. As used herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. As used herein, “comprise,” “comprising,” and “comprises” are meant to be synonymous with “include”, “including”, “includes” or “contain”, “containing”, “contains” and are inclusive or open- ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein. The term “consisting of” refers to compositions, methods, processes, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the disclosure. 20 ME146907648v.1 As used herein, the terms “such as”, “for example” and the like are intended to refer to exemplary embodiments and not to limit the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, preferred materials and methods are described herein. As used herein the terms, “administration,” “administering” and variants thereof refers to introducing a composition or agent (e.g., nucleic acids, in particular ceDNA) into a subject and includes concurrent and sequential introduction of one or more compositions or agents. “Administration” can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. “Administration” also encompasses in vitro and ex vivo treatments. The introduction of a composition or agent into a subject is by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intratumorally, or topically. Administration includes self-administration and the administration by another. Administration can be carried out by any suitable route. A suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject. The term “immunogenicity of an LNP” or “immunogenicity of a composition comprising an LNP”, as used herein, refers to the ability of a composition comprising LNPs of the present disclosure to stimulate an undesired immune response in a subject after the LNPs of the disclosure or a composition comprising the LNPs of the disclosure are administered to the subject. In some embodiments, the immune response, e.g., before and after administration of a composition comprising LNPs of the present disclosure, may be measured by measuring levels of one or more pro- inflammatory cytokines. Exemplary pro-inflammatory cytokines that may be used to determine immunogenicity of LNPs of the present disclosure or a composition comprising LNPs of the present disclosure include, but are not limited to, granulocyte colony stimulating factor (G-CSF), interleukin 1 alpha (IL-1α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8 or CXCL8), interleukin 11 (IL-11), interleukin 17 (IL-17), interleukin 18 (IL-18), interferon α (IFN-α), interferon β (IFN-β), interferon γ (IFN-γ), C-X-C motif chemokine ligand 10 (CXCL10 or IP-10), monocyte chemoattractant protein 1 (MCP-1), CD40L, CCL2, CCL3, CCL4, CCL5, CCL11, tumor necrosis factor α (TNF-α), and combinations thereof. The term “off-target delivery”, as used herein, refers to delivery of LNPs of the disclosure to non-target cells. For example, an LNP of the disclosure comprising GalNAc targets delivery of the LNP to hepatocytes, and off-target delivery of the LNP refers to the delivery of the LNP to random, non-target cells that are not, for example, hepatocytes. In some embodiments, the non-target cell may be a blood cell, or macrophage,. In some embodiments, the non-target cell is a macrophage. In some 21 ME146907648v.1 embodiments, the non-target cell may be a liver sinusoidal endothelial cell (LSEC cell), a spleen cell or a Kupffer cell. After administration to a subject, an LNP may be delivered to a non-target cell, e.g., one or more of blood cells listed above, and may result in expression of a therapeutic nucleic acid (TNA) in the non-target cell, or may be degraded once engulfed by, e.g., a macrophage. In some embodiments, a reference LNP that comprises a helper lipid; and /or does not comprise the first lipid-anchored polymer and comprises a reference lipid-anchored polymer comprising one or more hydrophobic tails that each independently comprise less than 16 carbon atoms in a single aliphatic chain backbone., may be characterized by a higher rate of delivery to a non-target cell, e.g., one or more of blood cells listed above, as compared to an LNP of the present disclosure. In some embodiments, an LNP of the present disclosure results in an uptake level of TNA (e.g., ceDNA, ssDNA, or mRNA) in a blood cell that is lower than that of a reference LNP. In some embodiments, the reference LNP is an LNP that comprises a helper lipid; and /or does not comprise the first lipid-anchored polymer and comprises a reference lipid-anchored polymer comprising one or more hydrophobic tails that each independently comprise less than 16 carbon atoms in a single aliphatic chain backbone.. In some embodiments, the blood cell is a cell selected from the group consisting of a red blood cell, a macrophage, and a peripheral blood mononuclear cell. As used herein, the term “aqueous solution” refers to a composition comprising in whole, or in part, water. As used herein, the term “bases” includes purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides. As used herein, the terms “carrier” and “excipient” are meant to include any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically- acceptable” refers to molecular entities and compositions that do not produce a toxic, an allergic, or similar untoward reaction when administered to a host. As used herein, the term “ceDNA” refers to capsid-free closed-ended linear double stranded (ds) duplex DNA for non-viral gene transfer, synthetic or otherwise. According to some embodiments, the ceDNA is a closed-ended linear duplex (CELiD) CELiD DNA. According to some embodiments, the ceDNA is a DNA-based minicircle. According to some embodiments, the ceDNA is a minimalistic immunological-defined gene expression (MIDGE)-vector. According to some embodiments, the ceDNA is a ministering DNA. According to some embodiments, the ceDNA is a 22 ME146907648v.1 dumbbell shaped linear duplex closed-ended DNA comprising two hairpin structures of ITRs in the 5’ and 3’ ends of an expression cassette. According to some embodiments, the ceDNA is a doggybone™ DNA. Detailed description of ceDNA is described in International Patent Application No. PCT/US2017/020828, filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Certain methods for the production of ceDNA comprising various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Patent Application Nos. PCT/US18/49996, filed September 7, 2018, and PCT/US2018/064242, filed December 6, 2018, each of which is incorporated herein in its entirety by reference. Certain methods for the production of synthetic ceDNA vectors comprising various ITR sequences and configurations are described, e.g., in International application PCT/US2019/14122, filed January 18, 2019, the entire content of which is incorporated herein by reference. As used herein, the term “closed-ended DNA vector” refers to a capsid-free DNA vector with at least one covalently closed end and where at least part of the vector has an intramolecular duplex structure. As used herein, the terms “ceDNA vector” and “ceDNA” are used interchangeably and refer to a closed-ended DNA vector comprising at least one terminal palindrome. In some embodiments, the ceDNA comprises two covalently-closed ends. As used herein, the term “ceDNA-bacmid” refers to an infectious baculovirus genome comprising a ceDNA genome as an intermolecular duplex that is capable of propagating in E. coli as a plasmid, and so can operate as a shuttle vector for baculovirus. As used herein, the term “ceDNA-baculovirus” refers to a baculovirus that comprises a ceDNA genome as an intermolecular duplex within the baculovirus genome. As used herein, the terms “ceDNA-baculovirus infected insect cell” and “ceDNA-BIIC” are used interchangeably, and refer to an invertebrate host cell (including, but not limited to an insect cell (e.g., an Sf9 cell)) infected with a ceDNA-baculovirus. As used herein, the term “ceDNA genome” refers to an expression cassette that further incorporates at least one inverted terminal repeat (ITR) region. A ceDNA genome may further comprise one or more spacer regions. In some embodiments the ceDNA genome is incorporated as an intermolecular duplex polynucleotide of DNA into a plasmid or viral genome. As used herein, the terms “DNA regulatory sequences,” “control elements,” and “regulatory elements,” are used interchangeably herein, and refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., DNA-targeting RNA) or a coding sequence (e.g., site-directed modifying polypeptide, or Cas9/Csn1 polypeptide) and/or regulate translation of an encoded polypeptide. As used herein, the term “terminal repeat” or “TR” includes any viral or non-viral terminal repeat or synthetic sequence that comprises at least one minimal required origin of replication and a 23 ME146907648v.1 region comprising a palindromic hairpin structure. A Rep-binding sequence (“RBS” or also referred to as Rep-binding element (RBE)) and a terminal resolution site (“TRS”) together constitute a “minimal required origin of replication” for an AAV and thus the TR comprises at least one RBS and at least one TRS. TRs that are the inverse complement of one another within a given stretch of polynucleotide sequence are typically each referred to as an “inverted terminal repeat” or “ITR”. In the context of a virus, ITRs plays a critical role in mediating replication, viral particle and DNA packaging, DNA integration and genome and provirus rescue. TRs that are not inverse complement (palindromic) across their full length can still perform the traditional functions of ITRs, and thus, the term ITR is used to refer to a TR in a viral or non-viral AAV vector that is capable of mediating replication of in the host cell. It will be understood by one of ordinary skill in the art that in a complex AAV vector configurations more than two ITRs or asymmetric ITR pairs may be present. The “ITR” can be artificially synthesized using a set of oligonucleotides comprising one or more desirable functional sequences (e.g., palindromic sequence, RBS). The ITR sequence can be an AAV ITR, an artificial non-AAV ITR, or an ITR physically derived from a viral AAV ITR (e.g., ITR fragments removed from a viral genome). For example, the ITR can be derived from the family Parvoviridae, which encompasses parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin that serves as the origin of SV40 replication can be used as an ITR, which can further be modified by truncation, substitution, deletion, insertion and/or addition. Parvoviridae family viruses consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the viral family of the adeno-associated viruses (AAV) which are capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine and ovine species. Typically, ITR sequences can be derived not only from AAV, but also from Parvovirus, lentivirus, goose virus, B19, in the configurations of wildtype, “doggy bone” and “dumbbell shape”, symmetrical or even asymmetrical ITR orientation. Although the ITRs are typically present in both 5’ and 3’ ends of an AAV vector, ITR can be present in only one of end of the linear vector. For example, the ITR can be present on the 5’ end only. Some other cases, the ITR can be present on the 3’ end only in synthetic AAV vector. For convenience herein, an ITR located 5’ to (“upstream of”) an expression cassette in a synthetic AAV vector is referred to as a “5’ ITR” or a “left ITR”, and an ITR located 3’ to (“downstream of”) an expression cassette in a vector or synthetic AAV is referred to as a “3’ ITR” or a “right ITR”. As used herein, a “wild-type ITR” or “WT-ITR” refers to the sequence of a naturally occurring ITR sequence in an AAV genome or other dependovirus that remains, e.g., Rep binding activity and Rep nicking ability. The nucleotide sequence of a WT-ITR from any AAV serotype may slightly vary from the canonical naturally occurring sequence due to degeneracy of the genetic code or drift, and therefore WT-ITR sequences encompasses for use herein include WT-ITR sequences as result of naturally occurring changes (e.g., a replication error). 24 ME146907648v.1 As used herein, the term “substantially symmetrical WT-ITRs” or a “substantially symmetrical WT-ITR pair” refers to a pair of WT-ITRs within a synthetic AAV vector that are both wild type ITRs that have an inverse complement sequence across their entire length. For example, an ITR can be considered to be a wild-type sequence, even if it has one or more nucleotides that deviate from the canonical naturally occurring canonical sequence, so long as the changes do not affect the physical and functional properties and overall three-dimensional structure of the sequence (secondary and tertiary structures). In some aspects, the deviating nucleotides represent conservative sequence changes. As one non-limiting example, a sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical sequence (as measured, e.g., using BLAST at default settings), and also has a symmetrical three-dimensional spatial organization to the other WT-ITR such that their 3D structures are the same shape in geometrical space. The substantially symmetrical WT-ITR has the same A, C-C’ and B-B’ loops in 3D space. A substantially symmetrical WT-ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE’) and terminal resolution site (trs) that pairs with the appropriate Rep protein. One can optionally test other functions, including transgene expression under permissive conditions. As used herein, the phrases of “modified ITR” or “mod-ITR” or “mutant ITR” are used interchangeably and refer to an ITR with a mutation in at least one or more nucleotides as compared to the WT-ITR from the same serotype. The mutation can result in a change in one or more of A, C, C’, B, B’ regions in the ITR, and can result in a change in the three-dimensional spatial organization (i.e. its 3D structure in geometric space) as compared to the 3D spatial organization of a WT-ITR of the same serotype. As used herein, the term “asymmetric ITRs” also referred to as “asymmetric ITR pairs” refers to a pair of ITRs within a single synthetic AAV genome that are not inverse complements across their full length. As one non-limiting example, an asymmetric ITR pair does not have a symmetrical three- dimensional spatial organization to their cognate ITR such that their 3D structures are different shapes in geometrical space. Stated differently, an asymmetrical ITR pair have the different overall geometric structure, i.e., they have different organization of their A, C-C’ and B-B’ loops in 3D space (e.g., one ITR may have a short C-C’ arm and/or short B-B’ arm as compared to the cognate ITR). The difference in sequence between the two ITRs may be due to one or more nucleotide addition, deletion, truncation, or point mutation. In one embodiment, one ITR of the asymmetric ITR pair may be a wild-type AAV ITR sequence and the other ITR a modified ITR as defined herein (e.g., a non-wild- type or synthetic ITR sequence). In another embodiment, neither ITRs of the asymmetric ITR pair is a wild-type AAV sequence and the two ITRs are modified ITRs that have different shapes in geometrical space (i.e., a different overall geometric structure). In some embodiments, one mod-ITRs of an asymmetric ITR pair can have a short C-C’ arm and the other ITR can have a different modification (e.g., a single arm, or a short B-B’ arm etc.) such that they have different three- dimensional spatial organization as compared to the cognate asymmetric mod-ITR. 25 ME146907648v.1 As used herein, the term “symmetric ITRs” refers to a pair of ITRs within a single stranded AAV genome that are wild-type or mutated (e.g., modified relative to wild-type) dependoviral ITR sequences and are inverse complements across their full length. In one non-limiting example, both ITRs are wild type ITRs sequences from AAV2. In another example, neither ITRs are wild type ITR AAV2 sequences (i.e., they are a modified ITR, also referred to as a mutant ITR), and can have a difference in sequence from the wild type ITR due to nucleotide addition, deletion, substitution, truncation, or point mutation. For convenience herein, an ITR located 5’ to (upstream of) an expression cassette in a synthetic AAV vector is referred to as a “5’ ITR” or a “left ITR”, and an ITR located 3’ to (downstream of) an expression cassette in a synthetic AAV vector is referred to as a “3’ ITR” or a “right ITR”. As used herein, the terms “substantially symmetrical modified-ITRs” or a “substantially symmetrical mod-ITR pair” refers to a pair of modified-ITRs within a synthetic AAV that are both that have an inverse complement sequence across their entire length. For example, the modified ITR can be considered substantially symmetrical, even if it has some nucleotide sequences that deviate from the inverse complement sequence so long as the changes do not affect the properties and overall shape. As one non-limiting example, a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical sequence (as measured using BLAST at default settings), and also has a symmetrical three-dimensional spatial organization to their cognate modified ITR such that their 3D structures are the same shape in geometrical space. Stated differently, a substantially symmetrical modified-ITR pair have the same A, C-C’ and B-B’ loops organized in 3D space. In some embodiments, the ITRs from a mod-ITR pair may have different reverse complement nucleotide sequences but still have the same symmetrical three-dimensional spatial organization – that is both ITRs have mutations that result in the same overall 3D shape. For example, one ITR (e.g., 5’ ITR) in a mod-ITR pair can be from one serotype, and the other ITR (e.g., 3’ ITR) can be from a different serotype, however, both can have the same corresponding mutation (e.g., if the 5’ITR has a deletion in the C region, the cognate modified 3’ITR from a different serotype has a deletion at the corresponding position in the C’ region), such that the modified ITR pair has the same symmetrical three- dimensional spatial organization. In such embodiments, each ITR in a modified ITR pair can be from different serotypes (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) such as the combination of AAV2 and AAV6, with the modification in one ITR reflected in the corresponding position in the cognate ITR from a different serotype. In one embodiment, a substantially symmetrical modified ITR pair refers to a pair of modified ITRs (mod-ITRs) so long as the difference in nucleotide sequences between the ITRs does not affect the properties or overall shape and they have substantially the same shape in 3D space. As a non-limiting example, a mod-ITR that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the canonical mod-ITR as determined by standard means well known in the art such as BLAST (Basic Local Alignment Search Tool), or BLASTN at default settings, and also has a symmetrical three-dimensional spatial organization such that their 3D structure is the same 26 ME146907648v.1 shape in geometric space. A substantially symmetrical mod-ITR pair has the same A, C-C’ and B-B’ loops in 3D space, e.g., if a modified ITR in a substantially symmetrical mod-ITR pair has a deletion of a C-C’ arm, then the cognate mod-ITR has the corresponding deletion of the C-C’ loop and also has a similar 3D structure of the remaining A and B-B’ loops in the same shape in geometric space of its cognate mod-ITR. As used herein, the phrase an “effective amount” or “therapeutically effective amount” of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to produce the desired effect, e.g., inhibition of expression of a target sequence in comparison to the expression level detected in the absence of a therapeutic nucleic acid. Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art. As used herein, the term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. As used herein, the phrase “expression products” include RNA transcribed from a gene (e.g., transgene), and polypeptides obtained by translation of mRNA transcribed from a gene. As used herein, the term “expression vector” refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the host cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. The expression vector may be a recombinant vector. As used herein, the term “flanking” refers to a relative position of one nucleic acid sequence with respect to another nucleic acid sequence. Generally, in the sequence ABC, B is flanked by A and C. The same is true for the arrangement AxBxC. Thus, a flanking sequence precedes or follows a flanked sequence but need not be contiguous with, or immediately adjacent to the flanked sequence. As used herein, the term “spacer region” refers to an intervening sequence that separates functional elements in a vector or genome. In some embodiments, spacer regions keep two functional elements at a desired distance for optimal functionality. In some embodiments, the spacer regions provide or add to the genetic stability of the vector or genome. In some embodiments, spacer regions facilitate ready genetic manipulation of the genome by providing a convenient location for cloning sites and a gap of design number of base pair. As used herein, the terms “expression cassette” and “expression unit” are used interchangeably, and refer to a heterologous DNA sequence that is operably linked to a promoter or other DNA regulatory sequence sufficient to direct transcription of a transgene of a DNA vector, e.g., 27 ME146907648v.1 synthetic AAV vector. Suitable promoters include, for example, tissue specific promoters. Promoters can also be of AAV origin. As used herein, the phrase “genetic disease” or “genetic disorder” refers to a disease, partially or completely, directly or indirectly, caused by one or more abnormalities in the genome, including and especially a condition that is present from birth. The abnormality may be a mutation, an insertion or a deletion in a gene. The abnormality may affect the coding sequence of the gene or its regulatory sequence. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. According to some embodiments, a polypeptide of the disclosure is an ApoE or an ApoB polypeptide. According to some embodiments, the ApoE polypeptide is a functional fragment (or a functional portion) of the full length ApoE polypeptide. According to some embodiments, the ApoE polypeptide is a functional fragment (or a functional portion) of the full length ApoB polypeptide. According to some embodiments, the ApoE polypeptide is 30 amino acids in length or less. According to some embodiments, the ApoB polypeptide is 30 amino acids in length or less. As used herein, the term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols, and β-acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipids described above can be mixed with other lipids including triglycerides and sterols. As used herein, the term “helper lipid” refers to a non-cationic lipid comprising at least one non-polar chain. Exemplary helper lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 28 ME146907648v.1 distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16- O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid,cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is to be understood that a reference LNP used herein contains a helper lipid and the reference LNP was compared with an LNP of the invention which contains no helper lipid. The helper lipid as referred to herein is not conjugated to a polymer (e.g., PEG or PG). The molecular structures of the representative helper lipids DSPC and DOPE having C18 hydrophobic tails are shown below.
Figure imgf000030_0001
DOPE. As used herein, the term “lipid-anchored polymer”, which may be used herein interchangeably with the term “lipid conjugate” or “lipid-conjugated polymer”, refers to a molecule comprising a lipid moiety covalently attached to a polymer, e.g., optionally via a linker. Without wishing to be bound by a specific theory, it is believed that a lipid-anchored polymer can inhibit aggregation of LNPs and provide steric stabilization. Exemplary lipid-anchored polymers include, but are not limited to, PEGylated lipids such as, e.g., PEG coupled to dialkyloxypropyls (e.g., PEG- DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides (see, e.g., U.S. Patent No.5,885,613), cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ- DAA conjugates; see, e.g., U.S. Provisional Application No.61/294,828, filed Jan.13, 2010, and U.S. 29 ME146907648v.1 Provisional Application No.61/295,140, filed Jan.14, 2010), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. Additional examples of POZ-lipid conjugates are described in International Patent Application Publication No. WO 2010/006282. PEG or POZ can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG or the POZ to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties. In certain preferred embodiments, non-ester containing linker moieties, such as amides or carbamates, are used. The disclosures of each of the above patent documents are herein incorporated by reference in their entirety for all purposes. As used herein, the term “dissociable lipid-conjugated polymer” refers to a lipid-conjugated polymer that comprises a lipid moiety comprising at least one or two hydrophobic tails, wherein the at least two hydrophobic tails each comprise 10-15 carbons atoms, e.g., 14 carbon atoms, and wherein the at least one hydrophobic tail comprises 16-22 carbon atoms, e.g., 18 carbon atoms, in a single aliphatic chain backbone. An example of a dissociable lipid-conjugated polymer comprising at least two hydrophobic tails is DMG-PEG2000 having the structure
Figure imgf000031_0001
dissociable lipid-conjugated polymer comprising at least one hydrophobic tail is mono-C18-PEG2000 having the structure
Figure imgf000031_0002
As used herein, the term “lipid encapsulated” refers to a lipid particle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., a ceDNA, ssDNA, mRNA), with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is fully encapsulated in the lipid particle (e.g., to form a nucleic acid containing lipid particle). As used herein, the term “lipid particle” or “lipid nanoparticle” (LNP) refer to a lipid formulation that can be used to deliver a therapeutic agent, such as therapeutic nucleic acid, to a target site of interest (e.g., cell, tissue, organ, and the like). In some embodiments, the lipid nanoparticle of the disclosure is typically formed from an ionizable lipid (e.g., a pH-sensitive cationic lipid), sterol (e.g., cholesterol), a conjugated lipid (e.g., lipid-anchored polymer) that prevents aggregation of the particle. In some embodiments, the lipid particle of the disclosure is a nucleic acid containing lipid particle, which is typically formed from a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle. In other preferred embodiments, a therapeutic agent such as a therapeutic nucleic acid may be encapsulated in the lipid portion of the 30 ME146907648v.1 particle, thereby protecting it from enzymatic degradation. In one embodiment, the lipid particle comprises a nucleic acid (e.g., ceDNA, ssDNA, mRNA) and a lipid comprising one or more - tertiary amino groups, one or more phenyl ester bonds and a disulfide bond. According to some embodiments, lipid particles of the disclosure typically have a mean diameter of from about 20 nm to about 75 nm, about 20 nm to about 70 nm, about 25 nm to about 75 nm, about 25 nm to about 70 nm, from about 30 nm to about 75 nm, from about 30 nm to about 70 nm, from about 35 nm to about 75 nm, from about 35 nm to about 70 nm, from about 40 nm to about 75 nm, from about 40 nm to about 70 nm, from about 45 nm to about 75 nm, from about 50 nm to about 75 nm, from about 50 nm to about 70 nm, from about 60 nm to about 75 nm, from about 60 nm to about 70 nm, from about 65 nm to about 75 nm, from about 65 nm to about 70 nm, or about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 51 nm, about 52 nm, about 53 nm, about 54 nm, about 55 nm, about 56 nm, about 57 nm, about 58 nm, about 59 nm about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm, about 65 nm, about 66 nm, about 67 nm, about 68 nm, about 69 nm, about 70 nm, about 71 nm, about 72 nm, about 73 nm, about 74 nm, or about 75 nm (± 3 nm) in size. Generally, the LNPs of the disclosure have a mean diameter selected to provide an intended therapeutic effect. For example, the LNPs of the disclosure have a mean diameter that is compatible with a target organ, such that the LNPs of the disclosure are able to diffuse through the fenestrations of a target organ (e.g., liver) or a target cell subpopulation (e.g., hepatocytes). According to some embodiments, the lipid particles of the disclosure typically have a mean diameter of less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, less than about 20 nm in size. As used herein, the term “cationic lipid” refers to any lipid that is positively charged at physiological pH. The cationic lipid in the lipid particles may comprise, e.g., one or more cationic lipids such as 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ- DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), “SS-cleavable lipid”, or a mixture thereof. In some embodiments, a cationic lipid can also be an ionizable lipid, i.e., an ionizable cationic lipid. i.e. The term “cationic lipids” also encompasses lipids that are positively charged at any pH, e.g., lipids comprising quaternary amine groups, i.e., quarternary lipids. Any cationic lipid described herein comprising a primary, secondary or tertiary amine group may be converted to a corresponding quaternary lipid, for example, by treatment with chloromethane (CH3Cl) in acetonitrile (CH3CN) and chloroform (CHCl3). As used herein, the term “ionizable lipid” refers to a lipid, e.g., cationic lipid, having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below 31 ME146907648v.1 physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7. In some embodiments, ionizable lipid may include “cleavable lipid” or “SS- cleavable lipid”. As used herein, the term “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols. As used herein, the term “non-cationic lipid” refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid that can be used as a helper lipid in LNP formulation. As used herein, the term “cleavable lipid” or “SS-cleavable lipid” refers to an ionizable lipid comprising a disulfide bond cleavable unit. Cleavable lipids may include cleavable disulfide bond (“ss”) containing lipid-like materials that comprise a pH-sensitive amine, e.g., a tertiary amine, and self-degradable phenyl ester. For example, a SS-cleavable lipid can be an ss-OP lipid (COATSOME® SS-OP), an ss-M lipid (COATSOME® SS-M), an ss-E lipid (COATSOME® SS-E), an ss-EC lipid (COATSOME® SS-EC), an ss-LC lipid (COATSOME® SS-LC), an ss-OC lipid (COATSOME® SS- OC), and an ss-PalmE lipid (see, for example, Formulae I-IV), or a lipid described by Togashi et al., (2018) Journal of Controlled Release “A hepatic pDNA delivery system based on an intracellular environment sensitive vitamin E -scaffold lipid-like material with the aid of an anti-inflammatory drug” 279:262-270. Additional examples of cleavable lipids are described in US Patent No. 9,708,628, and US Patent No.10,385,030, the entire contents of which are incorporated herein by reference. In one embodiment, cleavable lipids comprise a tertiary amine, which responds to an acidic compartment, e.g., an endosome or lysosome for membrane destabilization and a disulfide bond that can be cleaved in a reducing environment, such as the cytoplasm. In one embodiment, a cleavable lipid is a cationic lipid. In one embodiment, a cleavable lipid is an ionizable cationic lipid. Cleavable lipids are described in more detail herein. As used herein, the term “organic lipid solution” refers to a composition comprising in whole, or in part, an organic solvent having a lipid. As used herein, the term “liposome” refers to lipid molecules assembled in a spherical configuration encapsulating an interior aqueous volume that is segregated from an aqueous exterior. Liposomes are vesicles that possess at least one lipid bilayer. Liposomes are typical used as carriers for drug/ therapeutic delivery in the context of pharmaceutical development. They work by fusing with a cellular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical ingredient. Liposome compositions for such delivery are typically composed of 32 ME146907648v.1 phospholipids, especially compounds having a phosphatidylcholine group, however these compositions may also include other lipids. As used herein, the term “local delivery” refers to delivery of an active agent such as an interfering RNA (e.g., siRNA) directly to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site such as a tumor or other target site such as a site of inflammation or a target organ such as the liver, heart, pancreas, kidney, and the like. As used herein, the term “nucleic acid,” refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. DNA may be in the form of minicircle, plasmid, bacmid, minigene, ministring DNA (linear covalently closed DNA vector), closed-ended linear duplex DNA (CELiD or ceDNA), single-stranded DNA (ssDNA), doggybone™ DNA, dumbbell shaped DNA, minimalistic immunological-defined gene expression (MIDGE)-vector, viral vector or nonviral vectors. RNA may be in the form of small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, gRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs and/or modified residues include, without limitation, phosphorothioates, phosphorodiamidate morpholino oligomer (morpholino), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, locked nucleic acid (LNA™), and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. As used herein, the phrases “nucleic acid therapeutic”, “therapeutic nucleic acid” and “TNA” are used interchangeably and refer to any modality of therapeutic using nucleic acids as an active component of therapeutic agent to treat a disease or disorder. As used herein, these phrases refer to RNA-based therapeutics and DNA-based therapeutics. Non-limiting examples of RNA-based therapeutics include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNAs (RNAi), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigene, viral DNA (e.g., Lentiviral or AAV genome) or non-viral synthetic DNA 33 ME146907648v.1 vectors, closed-ended linear duplex DNA (ceDNA / CELiD), single-stranded DNA (ssDNA), plasmids, bacmids, DOGGYBONE™ DNA vectors, minimalistic immunological-defined gene expression (MIDGE)-vector, nonviral ministring DNA vector (linear-covalently closed DNA vector), or dumbbell-shaped DNA minimal vector (“dumbbell DNA”). As used herein, “nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound. By “receptor” is meant a polypeptide, or portion thereof, present on a cell membrane that selectively binds one or more ligands. The term “receptor” as used herein is intended to encompass the entire receptor or ligand-binding portions thereof. These portions of the receptor particularly include those regions sufficient for specific binding of the ligand to occur. As used herein, the term “subject” refers to a human or animal, to whom treatment, including prophylactic treatment, with the therapeutic nucleic acid according to the present disclosure, is provided. Usually, the animal is a vertebrate such as, but not limited to a primate, rodent, domestic animal or game animal. Primates include but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, but are not limited to, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate or a human. A subject can be male or female. Additionally, a subject can be an infant or a child. In some embodiments, the subject can be a neonate or an unborn subject, e.g., the subject is in utero. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of diseases and disorders. In addition, the methods and compositions described herein can be used for domesticated animals and/or pets. A human subject can be of any age, gender, race or ethnic group, e.g., Caucasian (white), Asian, African, black, African American, African European, Hispanic, Mideastern, etc. In some embodiments, the subject can be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment. In some embodiments, the subject is an embryo, a fetus, neonate, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, human neonate, human infant, human child, human 34 ME146907648v.1 adolescent, or human adult. In some embodiments, the subject is an animal embryo, or non-human embryo or non-human primate embryo. In some embodiments, the subject is a human embryo. As used herein, the phrase “subject in need” refers to a subject that (i) will be administered a ceDNA, mRNA or ssDNA-containing lipid particle (or pharmaceutical composition comprising a ceDNA lipid particle) according to the described disclosure, (ii) is receiving a ceDNA, mRNA, or ssDNA-containing lipid particle (or pharmaceutical composition comprising aceDNA lipid particle) according to the described disclosure; or (iii) has received a ceDNA, mRNA, ssDNA-containing lipid particle (or pharmaceutical composition comprising a ceDNA, mRNA, or ssDNA-containing lipid particle) according to the described disclosure, unless the context and usage of the phrase indicates otherwise. As used herein, the term “suppress,” “decrease,” “interfere,” “inhibit” and/or “reduce” (and like terms) generally refers to the act of reducing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition. As used herein, the term “systemic delivery” refers to delivery of lipid particles that leads to a broad biodistribution of an active agent such as an interfering RNA (e.g., siRNA) within an organism. Some techniques of administration can lead to the systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body. To obtain broad biodistribution generally requires a blood lifetime such that the agent is not rapidly degraded or cleared (such as by first pass organs (liver, lung, etc.) or by rapid, nonspecific cell binding) before reaching a disease site distal to the site of administration. Systemic delivery of LNPs can be by any means known in the art including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of LNPs is by intravenous delivery. As used herein, the terms “effective amount”, which may be used interchangeably with the terms “therapeutic amount”, “therapeutically effective amount”, an “amount effective”, or “pharmaceutically effective amount” of an active agent (e.g., a ceDNA, ssDNA, mRNA as described herein), refers to an amount that is sufficient to provide the intended benefit of treatment or effect, e.g., expression or inhibition of expression of a target sequence in comparison to the expression level detected in the absence of a therapeutic nucleic acid. Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art. Dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular active agent employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. Additionally, the terms “effective amount”, 35 ME146907648v.1 “therapeutic amount”, “therapeutically effective amounts” and “pharmaceutically effective amounts” include prophylactic or preventative amounts of the compositions of the described invention. In prophylactic or preventative applications of the described invention, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and/or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes presenting during development of the disease, disorder or condition. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to some medical judgment. The terms “dose” and “dosage” are used interchangeably herein. In one aspect of any of the aspects or embodiments herein, “therapeutic amount”, “therapeutically effective amounts” and “pharmaceutically effective amounts” refer to non-prophylactic or non-preventative applications. As used herein the term “therapeutic effect” refers to a consequence of treatment, the results of which are judged to be desirable and beneficial. A therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation. A therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation. For any therapeutic agent described herein therapeutically effective amount may be initially determined from preliminary in vitro studies and/or animal models. A therapeutically effective dose may also be determined from human data. The applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan. General principles for determining therapeutic effectiveness, which may be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below. Pharmacokinetic principles provide a basis for modifying a dosage regimen to obtain a desired degree of therapeutic efficacy with a minimum of unacceptable adverse effects. In situations where the drug's plasma concentration can be measured and related to therapeutic window, additional guidance for dosage modification can be obtained. As used herein, the terms “treat,” “treating,” and/or “treatment” include abrogating, inhibiting, slowing or reversing the progression of a condition, ameliorating clinical symptoms of a condition, or preventing the appearance of clinical symptoms of a condition, obtaining beneficial or desired clinical results. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); 36 ME146907648v.1 and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s). In one aspect of any of the aspects or embodiments herein, the terms “treat,” “treating,” and/or “treatment” include abrogating, inhibiting, slowing or reversing the progression of a condition, or ameliorating clinical symptoms of a condition. Beneficial or desired clinical results, such as pharmacologic and/or physiologic effects include, but are not limited to, preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization (i.e., not worsening) of the disease, disorder or condition, preventing spread of the disease, disorder or condition, delaying or slowing of the disease, disorder or condition progression, amelioration or palliation of the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment. As used herein, the term “combination therapy” refers to treatment regimens for a clinical indication that comprise two or more therapeutic agents. Thus, the term refers to a therapeutic regimen in which a first therapy comprising a first composition (e.g., active ingredient) is administered in conjunction with a second therapy comprising a second composition (active ingredient) to a patient, intended to treat the same or overlapping disease or clinical condition. The first and second compositions may both act on the same cellular target, or discrete cellular targets. The phrase “in conjunction with,” in the context of combination therapies, means that therapeutic effects of a first therapy overlaps temporarily and/or spatially with therapeutic effects of a second therapy in the subject receiving the combination therapy. Thus, the combination therapies may be formulated as a single formulation for concurrent administration, or as separate formulations, for sequential administration of the therapies. As used herein, the term “alkyl” refers to a saturated monovalent hydrocarbon radical of 1 to 20 carbon atoms (i.e., C1-20 alkyl). “Monovalent” means that alkyl has one point of attachment to the remainder of the molecule. In one embodiment, the alkyl has 1 to 12 carbon atoms (i.e., C1-12 alkyl) or 1 to 10 carbon atoms (i.e., C1-10 alkyl). In one embodiment, the alkyl has 1 to 8 carbon atoms (i.e., C1- 8 alkyl), 1 to 7 carbon atoms (i.e., C1-7 alkyl), 1 to 6 carbon atoms (i.e., C1-6 alkyl), 1 to 4 carbon atoms (i.e., C1-4 alkyl), or 1 to 3 carbon atoms (i.e., C1-3 alkyl). Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2- pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2- hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2- methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like. A linear or branched alkyl, such as a “linear or branched C1-6 alkyl,” “linear or branched C1-4 alkyl,” or “linear or branched C1-3 alkyl” means that the saturated monovalent hydrocarbon radical is a linear or 37 ME146907648v.1 branched chain. As used herein, the term “linear” as referring to aliphatic hydrocarbon chains means that the chain is unbranched. The term “alkylene” as used herein refers to a saturated divalent hydrocarbon radical of 1 to 20 carbon atoms (i.e., C1-20 alkylene), examples of which include, but are not limited to, those having the same core structures of the alkyl groups as exemplified above. “Divalent” means that the alkylene has two points of attachment to the remainder of the molecule. In one embodiment, the alkylene has 1 to 12 carbon atoms (i.e., C1-12 alkylene) or 1 to 10 carbon atoms (i.e., C1-10 alkylene). In one embodiment, the alkylene has 1 to 8 carbon atoms (i.e., C1-8 alkylene), 1 to 7 carbon atoms (i.e., C1-7 alkylene), 1 to 6 carbon atoms (i.e., C1-6 alkylene), 1 to 4 carbon atoms (i.e., C1-4 alkylene), 1 to 3 carbon atoms (i.e., C1-3 alkylene), ethylene, or methylene. A linear or branched alkylene, such as a “linear or branched C1-6 alkylene,” “linear or branched C1-4 alkylene,” or “linear or branched C1-3 alkylene” means that the saturated divalent hydrocarbon radical is a linear or branched chain. The term “alkenyl” refers to straight or branched aliphatic hydrocarbon radical with one or more (e.g., one or two) carbon-carbon double bonds, wherein the alkenyl radical includes radicals having “cis” and “trans” orientations, or by an alternative nomenclature, “E” and “Z” orientations. “Alkenylene” as used herein refers to aliphatic divalent hydrocarbon radical of 2 to 20 carbon atoms (i.e., C2-20 alkenylene) with one or two carbon-carbon double bonds, wherein the alkenylene radical includes radicals having “cis” and “trans” orientations, or by an alternative nomenclature, “E” and “Z” orientations. “Divalent” means that alkenylene has two points of attachment to the remainder of the molecule. In one embodiment, the alkenylene has 2 to 12 carbon atoms (i.e., C2-16 alkenylene), 2 to 10 carbon atoms (i.e., C2-10 alkenylene). In one embodiment, the alkenylene has 2 to four carbon atoms (C2-4). Examples include, but are not limited to, ethylenylene or vinylene (-CH=CH-), allyl (- CH2CH=CH-), and the like. A linear or branched alkenylene, such as a “linear or branched C2-6 alkenylene,” “linear or branched C2-4 alkenylene,” or “linear or branched C2-3 alkenylene” means that the unsaturated divalent hydrocarbon radical is a linear or branched chain. “Cycloalkylene” as used herein refers to a divalent saturated carbocyclic ring radical having 3 to 12 carbon atoms as a monocyclic ring, or 7 to 12 carbon atoms as a bicyclic ring. “Divalent” means that the cycloalkylene has two points of attachment to the remainder of the molecule. In one embodiment, the cycloalkylene is a 3- to 7-membered monocyclic or 3- to 6-membered monocyclic. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, cyclononylene, cyclodecylene, cycloundecylene, cyclododecylene, and the like. In one embodiment, the cycloalkylene is cyclopropylene. The terms “heterocycle,” “heterocyclyl,” heterocyclic and “heterocyclic ring” are used interchangeably herein and refer to a cyclic group which contains at least one N atom has a heteroatom and optionally 1-3 additional heteroatoms selected from N and S, and are non-aromatic (i.e., partially or fully saturated). It can be monocyclic or bicyclic (bridged or fused). Examples of 38 ME146907648v.1 heterocyclic rings include, but are not limited to, aziridinyl, diaziridinyl, thiaziridinyl, azetidinyl, diazetidinyl, triazetidinyl, thiadiazetidinyl, thiazetidinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, isothiazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, azepanyl, azocanyl, and the like. The heterocycle contains 1 to 4 heteroatoms, which may be the same or different, selected from N and S. In one embodiment, the heterocycle contains 1 to 3 N atoms. In another embodiment, the heterocycle contains 1 or 2 N atoms. In another embodiment, the heterocycle contains 1 N atom. A “4- to 8-membered heterocyclyl” means a radical having from 4 to 8 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. A “5- or 6-membered heterocyclyl” means a radical having from 5 or 6 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. The term “heterocycle” is intended to include all the possible isomeric forms. Heterocycles are described in Paquette, Leo A., Principles of Modern Heterocyclic Chemistry (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. The heterocyclyl groups may be carbon (carbon-linked) or nitrogen (nitrogen-linked) attached to the rest of the molecule where such is possible. If a group is described as being “optionally substituted,” the group may be either (1) not substituted, or (2) substituted. If a carbon of a group is described as being optionally substituted with one or more of a list of substituents, one or more of the hydrogen atoms on the carbon (to the extent there are any) may separately and/or together be replaced with an independently selected optional substituent. Suitable substituents for an alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl, are those which do not significantly adversely affect the biological activity of the molecule. Unless otherwise specified, exemplary substituents for these groups include linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, aryl, heteroaryl, heterocyclyl, halogen, guanidinium [-NH(C=NH)NH2], -OR100, NR101R102, -NO2, -NR101COR102, -SR100, a sulfoxide represented by -SOR101, a sulfone represented by -SO2R101, a sulfonate -SO3M, a sulfate -OSO3M, a sulfonamide represented by -SO2NR101R102, cyano, an azido, -COR101, -OCOR101, -OCONR101R102 and a polyethylene glycol unit (-OCH2CH2)nR101 wherein M is H or a cation (such as Na+ or K+); R101, R102 and R103 are each independently selected from H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH2CH2)n-R104, wherein n is an integer from 1 to 24, an aryl having from 6 to 10 carbon atoms, a heterocyclic ring having from 3 to 10 carbon atoms and a heteroaryl having 5 to 10 carbon atoms; and R104 is H or a linear or branched alkyl having 1 to 4 carbon atoms, wherein the alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl in the groups represented by R100, R101, R102, R103 and R104 are optionally substituted with one or more (e.g., 2, 3, 4, 5, 6 or more) substituents independently selected from halogen, -OH, -CN, -NO2, and 39 ME146907648v.1 unsubstituted linear or branched alkyl having 1 to 4 carbon atoms. Preferably, the substituent for the optionally substituted alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl described above is selected from the group consisting of halogen, -CN, -NR101R102, -CF3, -OR100, aryl, heteroaryl, heterocyclyl, -SR101, -SOR101, -SO2R101, and -SO3M. Alternatively, the suitable substituent is selected from the group consisting of halogen, -OH, -NO2, -CN, C1-4 alkyl, -OR100, NR101R102, -NR101COR102, - SR100, -SO2R101, -SO2NR101R102, -COR101, -OCOR101, and -OCONR101R102, wherein R100, R101, and R102 are each independently -H or C1-4 alkyl. “Halogen” as used herein refers to F, Cl, Br or I. “Cyano” is –CN. “Amine” or “amino” as used herein interchangeably refers to a functional group that contains a basic nitrogen atom with a lone pair. The term “pharmaceutically acceptable salt” as used herein refers to pharmaceutically acceptable organic or inorganic salts of an ionizable lipid of the disclosure. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate “mesylate,” ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1’-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion. The counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counter ion. Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. In some embodiments of any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes. 40 ME146907648v.1 Other terms are defined herein within the description of the various aspects of the disclosure. All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims. Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure. The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. It should be understood that this disclosure is not limited in any manner to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. 41 ME146907648v.1 II. Lipid Nanoparticles (LNPs) Provided herein are lipid nanoparticles (LNPs) comprising a therapeutic nucleic acid (TNA); an ionizable lipid; a structural lipid (e.g., a sterol); and one or more lipid-anchored polymers, e.g., a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the LNP does not comprise a helper lipid. Also provided herein are LNPs consisting essentially of a therapeutic nucleic acid (TNA); an ionizable lipid; a structural lipid (e.g., a sterol); and one or more lipid-anchored polymers, e.g., a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the LNP does not comprise a helper lipid. Also provided herein are LNPs consisting of a therapeutic nucleic acid (TNA); an ionizable lipid; a structural lipid (e.g., a sterol); and one or more lipid-anchored polymers, e.g., a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the LNP does not comprise a helper lipid. The helper lipids as referred to herein are not conjugated to a polymer. An LNP of the present disclosure does not comprise a helper lipid, such as distearoylphosphatidylcholine (DSPC). In some embodiments, an LNP of the present disclosure does not comprise 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, an LNP of the present disclosure does not comprise a phosphatidylcholine that is not conjugated to a polymer. In some embodiments, an LNP of the present disclosure does not comprise 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE). A. Ionizable Lipids In some embodiments, the ionizable lipid is present in the LNP provided by the present disclosure in an amount of about 20 mol% to about 70 mol%, about 20 mol% to about 65 mol%, about 20 mol% to about 60 mol%, about 20 mol% to about 55 mol%, about 20 mol% to about 50 mol%, about 25 mol% to about 70 mol%, about 25 mol% to about 65 mol%, about 25 mol% to about 60 mol%, about 25 mol% to about 55 mol%, about 25 mol% to about 50 mol%, about 30 mol% to about 70 mol%, about 30 mol% to about 65 mol%, about 30 mol% to about 60 mol%, about 30 mol% to about 55 mol%, about 30 mol% to about 50 mol%, about 35 mol% to about 70 mol%, about 35 mol% to about 65 mol%, about 35 mol% to about 60 mol%, about 35 mol% to about 55 mol%, about 35 mol% to about 50 mol%, 40 mol% to about 70 mol%, about 40 mol% to about 65 mol%, about 40 mol% to about 60 mol%, about 40 mol% to about 55 mol%, or about 40 mol% to about 50 mol%, of the total lipid present in the LNP. In some embodiments, the LNPs provided by the present disclosure comprise an ionizable lipid. Exemplary ionizable lipids in the LNPs of the present disclosure are described in International Patent Application Publication Nos. WO2015/095340, WO2015/199952, WO2018/011633, WO2017/049245, WO2015/061467, WO2012/040184, WO2012/000104, WO2015/074085, WO2016/081029, WO2017/004143, WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541, WO2013/116126, WO2011/153120, WO2012/044638, WO2012/054365, 42 ME146907648v.1 WO2011/090965, WO2013/016058, WO2012/162210, WO2008/042973, WO2010/129709, WO2010/144740 , WO2012/099755, WO2013/049328, WO2013/086322, WO2013/086373, WO2011/071860, WO2009/132131, WO2010/048536, WO2010/088537, WO2010/054401, WO2010/054406 , WO2010/054405, WO2010/054384, WO2012/016184, WO2009/086558, WO2010/042877, WO2011/000106, WO2011/000107, WO2005/120152, WO2011/141705, WO2013/126803, WO2006/007712, WO2011/038160, WO2005/121348, WO2011/066651, WO2009/127060, WO2011/141704, WO2006/069782, WO2012/031043, WO2013/006825, WO2013/033563, WO2013/089151, WO2017/099823, WO2015/095346, and WO2013/086354, and US Patent Application Publication Nos. US2016/0311759, US2015/0376115, US2016/0151284, US2017/0210697, US2015/0140070, US2013/0178541, US2013/0303587, US2015/0141678, US2015/0239926, US2016/0376224, US2017/0119904, US2012/0149894, US2015/0057373, US2013/0090372, US2013/0274523, US2013/0274504, US2013/0274504, US2009/0023673, US2012/0128760, US2010/0324120, US2014/0200257, US2015/0203446, US2018/0005363, US2014/0308304, US2013/0338210, US2012/0101148, US2012/0027796, US2012/0058144, US2013/0323269, US2011/0117125, US2011/0256175, US2012/0202871, US2011/0076335, US2006/0083780, US2013/0123338, US2015/0064242, US2006/0051405, US2013/0065939, US2006/0008910, US2003/0022649, US2010/0130588, US2013/0116307, US2010/0062967, US2013/0202684, US2014/0141070, US2014/0255472, US2014/0039032, US2018/0028664, US2016/0317458, and US2013/0195920, the contents of all of which are incorporated herein by reference in their entirety. Formula (A) In some embodiments, the ionizable lipid in the LNPs of the present disclosure is represented by Formula (A):
Figure imgf000044_0001
or a pharmaceutically acceptable salt thereof, wherein: R1 and R1’ are each independently C1-3 alkylene; R2 and R2’ are each independently linear or branched C1-6 alkylene, or C3-6 cycloalkylene; 43 ME146907648v.1 R3 and R3’ are each independently optionally substituted C1-6 alkyl or optionally substituted C3-6 cycloalkyl; or alternatively, when R2 is branched C1-6 alkylene and when R3 is C1-6 alkyl, R2 and R3, taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; or alternatively, when R2’ is branched C1-6 alkylene and when R3’ is C1-6 alkyl, R2’ and R3', taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; R4 and R4’ are each independently –CH, –CH2CH, or –(CH2)2CH; R5 and R5’ are each independently hydrogen, C1-20 alkylene or C2-20 alkenylene; R6 and R6’, for each occurrence, are independently C1-20 alkylene, C3-20 cycloalkylene, or C2-20 alkenylene; and m and n are each independently an integer selected from 1, 2, 3, 4, and 5. In some embodiments, R2 and R2’ are each independently C1-3 alkylene. In some embodiments, the linear or branched C1-3 alkylene represented by R1 or R1’, the linear or branched C1-6 alkylene represented by R2 or R2’, and the optionally substituted linear or branched C1-6 alkyl are each optionally substituted with one or more halo and cyano groups. In some embodiments, R1 and R2 taken together are C1-3 alkylene and R1’ and R2’ taken together are C1-3 alkylene, e.g., ethylene. In some embodiments, R3 and R3’ are each independently optionally substituted C1-3 alkyl, e.g., methyl. In some embodiments, R4 and R4’ are each –CH. In some embodiments, R2 is optionally substituted branched C1-6 alkylene; and R2 and R3, taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl. In some embodiments, R2’ is optionally substituted branched C1-6 alkylene; and R2’ and R3’, taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl, such as pyrrolidinyl or piperidinyl. In some embodiments, R4 is –C(Ra)2CRa, or –[C(Ra)2]2CRa and Ra is C1-3 alkyl; and R3 and R4, taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl. In some embodiments, R4’ is –C(Ra)2CRa, or –[C(Ra)2]2CRa and Ra is C1-3 alkyl; and R3’ and R4’, taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl, such as pyrrolidinyl or piperidinyl. In some embodiments, R5 and R5’ are each independently C1-10 alkylene or C2-10 alkenylene. In one embodiment, R5 and R5’ are each independently C1-8 alkylene or C1-6 alkylene. In some embodiments, R6 and R6’, for each occurrence, are independently C1-10 alkylene, C3-10 cycloalkylene, or C2-10 alkenylene. In one embodiment, C1-6 alkylene, C3-6 cycloalkylene, or C2-6 alkenylene. In one embodiment the C3-10 cycloalkylene or the C3-6 cycloalkylene is cyclopropylene. In some embodiments, m and n are each 3. 44 ME146907648v.1 In some embodiments, the ionizable lipid in the LNPs of the present disclosure may be selected from any one of the lipids listed in Table 1 below, or a pharmaceutically acceptable salt thereof: Table 1. Exemplary ionizable lipids of Formula (A)
Figure imgf000046_0001
45 ME146907648v.1
Figure imgf000047_0001
46 ME146907648v.1
Figure imgf000048_0001
47 ME146907648v.1
Figure imgf000049_0001
48 ME146907648v.1
Figure imgf000050_0001
49 ME146907648v.1
Figure imgf000051_0001
50 ME146907648v.1
Figure imgf000052_0001
51 ME146907648v.1
Figure imgf000053_0001
52 ME146907648v.1
Figure imgf000054_0001
53 ME146907648v.1
Figure imgf000055_0001
54 ME146907648v.1
Figure imgf000056_0002
Formula (B) In some embodiments, the ionizable lipid in the LNPs of the present disclosure is represented by Formula (B):
Figure imgf000056_0001
or a pharmaceutically acceptable salt thereof, wherein: a is an integer ranging from 1 to 20 (e.g., a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); b is an integer ranging from 2 to 10 (e.g., b is 2, 3, 4, 5, 6, 7, 8, 9, or 10); 55 ME146907648v.1 R1 is absent or is selected from (C2-C20)alkenyl, -C(O)O(C2-C20)alkyl, and cyclopropyl substituted with (C2-C20)alkyl; and R2 is (C2-C20)alkyl. In a second embodiment of Formula (B), the ionizable lipid of Formula (B) is represented by Formula (B-1):
Figure imgf000057_0001
or a pharmaceutically acceptable salt thereof, wherein c and d are each independently integers ranging from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and wherein the remaining variables are as described for Formula (B). In a third embodiment of Formula (B), c and d in Formula (B-1) are each independently integers ranging from 2 to 8, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 8, 4 to 7, 4 to 6, 5 to 8, 5 to 7, or 6 to 8, wherein the remaining variables are as described for Formula (B-1). In a fourth embodiment of Formula (B), c in Formula (B-1) is 2, 3, 4, 5, 6, 7, or 8, wherein the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B). Alternatively, c and d in Formula (B-1) are each independently 1, 3, 5, or 7, wherein the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B). In a fifth embodiment of Formula (B), d in the cationic lipid of Formula (B-1) is 2, 3, 4, 5, 6, 7, or 8, wherein the remaining variables are as described for Formula (B), or the second, third or fourth embodiments of Formula (B). Alternatively, at least one of c and d in Formula (B-1) is 7, wherein the remaining variables are as described for Formula (B), or the second, third or fourth embodiments of Formula (B). In a sixth embodiment of Formula (B), the ionizable lipid of Formula (B) or Formula (B-1) is represented by Formula (B-2):
Figure imgf000057_0002
(B-2); or a pharmaceutically acceptable salt thereof, wherein the remaining variables are as described for Formula (B) or Formula (B-1). 56 ME146907648v.1 In a seventh embodiment of Formula (B), b in Formula (B), (B-1), or (B-2) is an integer ranging from 3 to 9, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth or sixth embodiments of Formula (B). Alternatively, b in Formula (B), (B-1), or (B-2) is an integer ranging from 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 9, 5 to 8, 5 to 7, 6 to 9, 6 to 8, or 7 to 9, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth or sixth embodiments of Formula (B). Alternatively, b in Formula (B), (B-1), or (B-2) is 3, 4, 5, 6, 7, 8, or 9, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth or sixth embodiments of Formula (B). In an eighth embodiment of Formula (B), a in Formula (B), (B-1), or (B-2) is an integer ranging from 2 to 18, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth or seventh embodiment of Formula (B). Alternatively, a in Formula (B), (B- 1), or (B-2) is an integer ranging from 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 25 to 8, 5 to 7, 6 to 18, 6 to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 8 to 18, 8 to 17, 8 to 16, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 9 to 18, 9 to 17, 9 to 16, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 10 to 18, 10 to 17, 10 to 16, 10 to 15, 10 to 14, 10 to 13, 11 to 18, 11 to 17, 11 to 16, 11 to 15, 11 to 14, 11 to 13, 12 to 18, 12 to 17, 12 to 16, 12 to 15, 12 to 14, 13 to 18, 13 to 17, 13 to 16, 13 to 15, 14 to 18, 14 to 17, 14 to 16, 15 to 18, 15 to 17, or 16 to 18, , wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth or seventh embodiment of Formula (B). Alternatively, a in Formula (B), (B-1), or (B-2) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, , wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth or seventh embodiment of Formula (B). In a ninth embodiment of Formula (B), R1 in Formula (B), Formula (B-1), or Formula (B-2) is absent or is selected from (C5-C15)alkenyl, -C(O)O(C4-C18)alkyl, and cyclopropyl substituted with (C4-C16)alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B). Alternatively, R1 in Formula (B), Formula (B-1), or Formula (B-2) is absent or is selected from (C5-C15)alkenyl, -C(O)O(C4-C16)alkyl, and cyclopropyl substituted with (C4-C16)alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B). Alternatively, R1 in Formula (B), Formula (B-1), or Formula (B-2) is absent or is selected from (C5- C12)alkenyl, -C(O)O(C4-C12)alkyl, and cyclopropyl substituted with (C4-C12)alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B). In another alternative, R1 in the cationic lipid of Formula (B), 57 ME146907648v.1 Formula (B-1), or Formula (B-2) is absent or is selected from (C5-C10)alkenyl, -C(O)O(C4-C10)alkyl, and cyclopropyl substituted with (C4-C10)alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B). In a tenth embodiment of Formula (B), R1 is C10 alkenyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B). In an eleventh embodiment of Formula (B), the alkyl in C(O)O(C2-C20)alkyl, -C(O)O(C4- C18)alkyl, -C(O)O(C4-C12)alkyl, or -C(O)O(C4-C10)alkyl of R1 in Formula (B), Formula (B-1), or Formula (B-2) is an unbranched alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, eighth or ninth embodiments of Formula (B). In one embodiment, R1 is -C(O)O(C9 alkyl). Alternatively, the alkyl in -C(O)O(C4-C18)alkyl, - C(O)O(C4-C12)alkyl, or -C(O)O(C4-C10)alkyl of R1 in Formula (B), Formula (B-1), or Formula (B-2) is a branched alkyl, wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth or ninth embodiments of Formula (B). In one embodiment, R1 is -C(O)O(C17 alkyl), wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth or ninth embodiments of Formula (B). In a twelfth embodiment of Formula (B), R1 in Formula (B), Formula (B-1), or Formula (B-2) is selected from any group listed in Table 2 below, wherein the wavy bond in each of the groups indicates the point of attachment of the group to the rest of the ionizable lipid molecule, and wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B). The present disclosure further contemplates the combination of any one of the R1 groups in Table 2 with any one of the R2 groups in Table 3 in Formula (B), wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B). Table 2. Exemplary R1 groups in Formula (B), Formula (B-1), or Formula (B-2)
Figure imgf000059_0001
58 ME146907648v.1 In a thirteenth embodiment, R2 in Formula (B) or a pharmaceutically acceptable salt thereof is selected from any group listed in Table 3 below, wherein the wavy bond in each of the groups indicates the point of attachment of the group to the rest of the ionizable lipid molecule, and wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh or eighth, ninth, tenth, eleventh or twelfth embodiments of Formula (B). Table 3 Exemplary R2 groups in Formula (B)
Figure imgf000060_0001
Table 4 below provides specific examples of ionizable lipids of Formula (B). Pharmaceutically acceptable salts as well as ionized and neutral forms are also included. Table 4. Exemplary ionizable lipids of Formula (B), (B-1), or (B-2)
Figure imgf000060_0002
59 ME146907648v.1
Figure imgf000061_0001
60 ME146907648v.1
Figure imgf000062_0001
61 ME146907648v.1
Figure imgf000063_0001
62 ME146907648v.1
Figure imgf000064_0001
63 ME146907648v.1
Figure imgf000065_0002
Formula (C) In some embodiments, the ionizable lipid in the LNPs of the present disclosure are represented by Formula (C):
Figure imgf000065_0001
or a pharmaceutically acceptable salt thereof, wherein: R1 and R1’ are each independently (C1-C6)alkylene optionally substituted with one or more groups selected from Ra; R2 and R2’ are each independently (C1-C2)alkylene; R3 and R3’ are each independently (C1-C6)alkyl optionally substituted with one or more groups selected from Rb; or alternatively, R2 and R3 and/or R2’ and R3’ are taken together with their intervening N atom to form a 4- to 7-membered heterocyclyl; R4 and R4’ are each a (C2-C6)alkylene interrupted by –C(O)O-; R5 and R5’ are each independently a (C2-C30)alkyl or (C2-C30)alkenyl, each of which are optionally interrupted with –C(O)O- or (C3-C6)cycloalkyl; and Ra and Rb are each halo or cyano. 64 ME146907648v.1 In a second embodiment of Formula (C), R1 and R1 are each independently (C1-C6)alkylene, wherein the remaining variables are as described above for Formula (C). Alternatively, R1 and R1’ are each independently (C1-C3)alkylene, wherein the remaining variables are as described above for Formula (C). In a third embodiment of Formula (C), the ionizable lipid of the Formula (C) is represented by Formula (C-1):
Figure imgf000066_0001
or a pharmaceutically acceptable salt thereof, wherein R2 and R2’, R3 and R3’, R4 and R4’ and R5 and R5’ are as described above for Formula (C) or the second embodiment of Formula (C). In a fourth embodiment, the ionizable lipid of Formula (C) is represented by Formula (C-2) or Formula
Figure imgf000066_0002
or a pharmaceutically acceptable salt thereof, wherein R4 and R4’ and R5 and R5’ are as described above for Formula (C). In a fifth embodiment of Formula (C), the ionizable lipid of Formula (C) is represented by Formula (C-4) or (C-5):
Figure imgf000066_0003
or a pharmaceutically acceptable salt thereof, wherein R5 and R5’ are as described above for Formula (C). In a sixth embodiment of Formula (C), the ionizable lipid of Formula (C) is represented by Formula (C-6), (C-7), (C-8), or (C-9): 65 ME146907648v.1 or a pharmaceutically acceptable salt thereof, wherein R5 and R5’ are as described above for Formula (XV). In a seventh embodiment of Formula (C), at least one of R5 and R5’ in Formula (C), (C-1), (C- 2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, one of R5 and R5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). In an eighth embodiment of Formula (C), R5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C6-C26)alkyl or (C6-C26)alkenyl, each of which are optionally interrupted with –C(O)O- or (C3-C6)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R5 in Formula (C), (C-1), (C- 2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C6-C26)alkyl or (C6-C26)alkenyl, each of which are optionally interrupted with –C(O)O- or (C3-C5)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C7-C26)alkyl or (C7- C26)alkenyl, each of which are optionally interrupted with –C(O)O- or (C3-C5)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C8-C26)alkyl or (C8-C26)alkenyl, each of which are optionally interrupted with –C(O)O- or (C3- C5)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C- 6), (C-7), (C-8), or (C-9) is a (C6-C24)alkyl or (C6-C24)alkenyl, each of which are optionally 66 ME146907648v.1 interrupted with –C(O)O- or cyclopropyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R5 in Formula (C), (C-1), (C- 2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C8-C24)alkyl or (C8-C24)alkenyl, wherein said (C8-C24)alkyl is optionally interrupted with –C(O)O- or cyclopropyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C8-C10)alkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C14-C16)alkyl interrupted with cyclopropyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C10-C24)alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C16-C18)alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R5 in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is –(CH2)3C(O)O(CH2)8CH3, –(CH2)5C(O)O(CH2)8CH3, – (CH2)7C(O)O(CH2)8CH3, –(CH2)7C(O)OCH[(CH2)7CH3]2, –(CH2)7-C3H6-(CH2)7CH3, –(CH2)7CH3, – (CH2)9CH3, –(CH2)16CH3, –(CH2)7CH=CH(CH2)7CH3, or –(CH2)7CH=CHCH2CH=CH( CH2)4CH3, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). In a ninth embodiment, R5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C- 8), or (C-9) is a (C15-C28)alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). Alternatively, R5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C17-C28)alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). Alternatively, R5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C19-C28)alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). Alternatively, R5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C17-C26)alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). Alternatively, R5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C19-C26)alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). Alternatively, R5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a (C20-C26)alkyl interrupted with –C(O)O-, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). Alternatively, R5’ is a (C22-C24)alkyl interrupted with –C(O)O-, and the remaining 67 ME146907648v.1 variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). Alternatively, R5’ is –(CH2)5C(O)OCH[(CH2)7CH3]2, –(CH2)7C(O)OCH[(CH2)7CH3]2, – (CH2)5C(O)OCH(CH2)2[(CH2)7CH3]2, or –(CH2)7C(O)OCH(CH2)2[(CH2)7CH3]2, and the remaining variables are as described above for Formula (C) or the second or eighth embodiments of Formula (C). In some embodiments, the ionizable lipid of Formula (C), (C-1), (C-3), (C-3), (C-4), (C-5), (C-7), (C-8), or (C-9) may be selected from any of the lipids listed in Table 5 below, or pharmaceutically acceptable salts thereof. Table 5. Exemplary ionizable lipids of Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9)
Figure imgf000069_0001
68 ME146907648v.1
Figure imgf000070_0002
Formula (D) In some embodiments, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (D):
Figure imgf000070_0001
or a pharmaceutically acceptable salt thereof, wherein: R’ is absent, hydrogen, or C1-C6 alkyl; provided that when R’ is hydrogen or C1-C6 alkyl, the nitrogen atom to which R’, R1, and R2 are all attached is positively charged; R1 and R2 are each independently hydrogen, C1-C6 alkyl, or C2-C6 alkenyl; R3 is C1-C12 alkylene or C2-C12 alkenylene; R4 is C1-C18 unbranched alkyl, C2-C18 unbranched alkenyl, or ; wherein: R4a and R4b are each independently C1-C16 unbranched alkyl or C2-C16 unbranched alkenyl; R5 is absent, C1-C8 alkylene, or C2-C8 alkenylene; 69 ME146907648v.1 R6a and R6b are each independently C7-C16 alkyl or C7-C16 alkenyl; provided that the total number of carbon atoms in R6a and R6b as combined is greater than 15; X1 and X2 are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(Ra)=N-, -N=C(Ra)-, -C(Ra)=NO-, -O-N=C(Ra)-, -C(=O)NRa-, -NRaC(=O)-, -NRaC(=O)NRa-, -OC(=O)O-, -OSi(Ra)2O-, -C(=O)(CRa2)C(=O)O-, or OC(=O)(CRa2)C(=O)-; wherein: Ra, for each occurrence, is independently hydrogen or C1-C6 alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6. In a second embodiment of Formula (D), X1 and X2 are the same; and all other remaining variables are as described for Formula (C). In a third embodiment of Formula (D), X1 and X2 are each independently -OC(=O)-, - SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -S-S-; or X1 and X2 are each independently -C(=O)O-, - C(=O)S-, or -S-S-; or X1 and X2 are each independently -C(=O)O- or -S-S-; and all other remaining variables are as described for Formula (D) or the second embodiment of Formula (D). In a fourth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure, is represented by Formula (D-1):
Figure imgf000071_0001
(D-1) or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, 3, and 4; and all other remaining variables are as described for Formula (D) or the second or third embodiments of Formula (D). In a fifth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure, is represented by Formula (D-2):
Figure imgf000071_0002
(D-2) or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, and 3; and all other remaining variables are as described for Formula (D) or the second or third embodiments of Formula (D). 70 ME146907648v.1 In a sixth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (D-3):
Figure imgf000072_0001
or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (D) or the second or third embodiments of Formula (D). In a seventh embodiment of Formula (D), in the ionizable lipid, e.g., cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or the second or third embodiments of Formula (D), R1 and R2 are each independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl, or C1-C5 alkyl or C2-C5 alkenyl, or C1-C4 alkyl or C2-C4 alkenyl, or C6 alkyl, or C5 alkyl, or C4 alkyl, or C3 alkyl, or C2 alkyl, or C1 alkyl, or C6 alkenyl, or C5 alkenyl, or C4 alkenyl, or C3 alkenyl, or C2 alkenyl; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3) or the second or third embodiments of Formula (D). In an eighth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (D-4):
Figure imgf000072_0002
or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3) or the second, third or seventh embodiments of Formula (D). In a ninth embodiment of Formula (D), in the ionizable lipid, e.g., cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third or seventh embodiments of Formula (D), R3 is C1-C9 alkylene or C2-C9 alkenylene, C1-C7 alkylene or C2- C7 alkenylene, C1-C5 alkylene or C2-C5 alkenylene, or C2-C8 alkylene or C2-C8 alkenylene, or C3-C7 alkylene or C3-C7 alkenylene, or C5-C7 alkylene or C5-C7 alkenylene; or R3 is C12 alkylene, C11 alkylene, C10 alkylene, C9 alkylene, or C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C2 alkylene, or C1 alkylene, or C12 alkenylene, C11 alkenylene, C10 alkenylene, C9 alkenylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, or C2 alkenylene; and all other remaining variables are as described 71 ME146907648v.1 for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third or seventh embodiments of Formula (D). In a tenth embodiment of Formula (D), in the ionizable lipid, e.g., cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third or seventh embodiments of Formula (D), R5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; or R5 is absent, C1-C4 alkylene, or C2-C4 alkenylene; or R5 is absent; or R5 is C8 alkylene, C7 alkylene, C6 alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, C1 alkylene, C8 alkenylene, C7 alkenylene, C6 alkenylene, C5 alkenylene, C4 alkenylene, C3 alkenylene, or C2 alkenylene; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third, seventh, or ninth embodiments of Formula (D). In an eleventh embodiment of Formula (D), in the ionizable lipid, e.g., cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third, seventh, ninth or tenth embodiments of Formula (D), R4 is C1-C14 unbranched alkyl, C2- C14 unbranched alkenyl, or
Figure imgf000073_0001
R4a and R4b are each independently C1-C12 unbranched alkyl or C2-C12 unbranched alkenyl; or R4 is C2-C12 unbranched alkyl or C2-C12 unbranched alkenyl; or R4 is C5-C7 unbranched alkyl or C5-C7 unbranched alkenyl; or R4 is C16 unbranched alkyl, C15 unbranched alkyl, C14 unbranched alkyl, C13 unbranched alkyl, C12 unbranched alkyl, C11 unbranched alkyl, C10 unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, C1 unbranched alkyl, C16 unbranched alkenyl, C15 unbranched alkenyl, C14 unbranched alkenyl, C13 unbranched alkenyl, C12 unbranched alkenyl, C11 unbranched alkenyl, C10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl; or R4 is
Figure imgf000073_0002
R4a and R4b are each independently C2-C10 unbranched alkyl or C2-C10 unbranched alkenyl; or R4 is , wherein R4a and R4b are each independently C16 unbranched alkyl, C15 unbranched alkyl, C14 unbranched alkyl, C13 unbranched alkyl, C12 unbranched alkyl, C11 unbranched alkyl, C10 unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C16 unbranched alkenyl, C15 unbranched alkenyl, C14 unbranched alkenyl, C13 unbranched alkenyl, C12 unbranched alkenyl, C11 unbranched alkenyl, C10 unbranched alkenyl, C9 72 ME146907648v.1 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third, seventh, ninth or tenth embodiments of Formula (D). In a twelfth embodiment, in the ionizable lipid, e.g., cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth or eleventh embodiments of Formula (D), R6a and R6b are each independently C6-C14 alkyl or C6- C14 alkenyl; or R6a and R6b are each independently C8-C12 alkyl or C8-C12 alkenyl; or R6a and R6b are each independently C16 alkyl, C15 alkyl, C14 alkyl, C13 alkyl, C12 alkyl, C11 alkyl, C10 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C16 alkenyl, C15 alkenyl, C14 alkenyl, C13 alkenyl, C12 alkenyl, C11 alkenyl, C10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl; provided that the total number of carbon atoms in R6a and R6b as combined is greater than 15; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4) , or the second, third, seventh, ninth, tenth or eleventh embodiments of Formula (D). In a thirteenth embodiment of Formula (D), in the ionizable lipid, e.g., cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh or twelfth embodiments of Formula (D), or a pharmaceutically acceptable salt thereof, R6a and R6b contain an equal number of carbon atoms with each other; or R6a and R6b are the same; or R6a and R6b are both C16 alkyl, C15 alkyl, C14 alkyl, C13 alkyl, C12 alkyl, C11 alkyl, C10 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C16 alkenyl, C15 alkenyl, C14 alkenyl, C13 alkenyl, C12 alkenyl, C11 alkenyl, C10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl; provided that the total number of carbon atoms in R6a and R6b as combined is greater than 15; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third, seventh, ninth, tenth, eleventh or twelfth embodiments of Formula (D). In a fourteenth embodiment of Formula (D), in the ionizable lipid, e.g., cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh, twelfth or thirteenth embodiments of Formula (D), R6a and R6b as defined in any one of the preceding embodiments each contain a different number of carbon atoms with each other; or the number of carbon atoms R6a and R6b differs by one or two carbon atoms; or the number of carbon atoms R6a and R6b differs by one carbon atom; or R6a is C7 alkyl and R6a is C8 alkyl, R6a is C8 alkyl and R6a is C7 alkyl, R6a is C8 alkyl and R6a is C9 alkyl, R6a is C9 alkyl and R6a is C8 alkyl, R6a is C9 alkyl and R6a is C10 alkyl, R6a is C10 alkyl and R6a is C9 alkyl, R6a is C10 alkyl and R6a is C11 alkyl, R6a is C11 alkyl and R6a is C10 alkyl, R6a is C11 alkyl and R6a is C12 alkyl, R6a is C12 alkyl and R6a is C11 alkyl, R6a is C7 alkyl and R6a is C9 alkyl, R6a is C9 alkyl and R6a is C7 alkyl, R6a is C8 alkyl and R6a is C10 alkyl, R6a is C10 alkyl and R6a is C8 alkyl, R6a is C9 alkyl and R6a is C11 alkyl, R6a is C11 alkyl and R6a is C9 alkyl, R6a is C10 alkyl and R6a is C12 alkyl, R6a is C12 alkyl and R6a is C10 alkyl, R6a is C11 alkyl and R6a is C13 alkyl, or R6a is C13 alkyl and R6a is C11 alkyl, etc.; and all other remaining 73 ME146907648v.1 variables are as described for Formula I, Formula II, Formula III, Formula IV, Formula V , or the second, third, seventh, ninth, tenth, eleventh, twelfth or thirteenth embodiments of Formula (D). In a fifteenth embodiment of Formula (D), R4 is C1-C16 unbranched alkyl, C2-C16 unbranched alkenyl,
Figure imgf000075_0001
, wherein R4a and R4b are as described above for the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth or fourteenth embodiments of Formula (D). In one embodiment, the ionizable lipid, e.g., cationic lipid, of the present disclosure or the ionizable lipid of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or Formula (D-4) is any one lipid selected from the lipids listed in Table 6 below, or a pharmaceutically acceptable salt thereof: Table 6. Exemplary lipids of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3) or Formula (D-4)
Figure imgf000075_0002
74 ME146907648v.1
Figure imgf000076_0001
75 ME146907648v.1
Figure imgf000077_0001
76 ME146907648v.1
Figure imgf000078_0001
77 ME146907648v.1
Figure imgf000079_0003
In one embodiment, the ionizable lipid in the LNPs of the present disclosure comprises Lipid No.87:
Figure imgf000079_0001
or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof. Formula (E) In some embodiments, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E):
Figure imgf000079_0002
78 ME146907648v.1 (E) or a pharmaceutically acceptable salt thereof, wherein: R’ is absent, hydrogen, or C1-C3 alkyl; provided that when R’ is hydrogen or C1-C3 alkyl, the nitrogen atom to which R’, R1, and R2 are all attached is positively charged; R1 and R2 are each independently hydrogen or C1-C3 alkyl; R3 is C3-C10 alkylene or C3-C10 alkenylene; R4 is C1-C16 unbranched alkyl, C2-C16 unbranched alkenyl, or ; wherein: R4a and R4b are each independently C1-C16 unbranched alkyl or C2-C16 unbranched alkenyl; R5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; R6a and R6b are each independently C7-C14 alkyl or C7-C14 alkenyl; X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(Ra)=N-, -N=C(Ra)-, -C(Ra)=NO-, -O-N=C(Ra)-, -C(=O)NRa-, -NRaC(=O)-, -NRaC(=O)NRa-, -OC(=O)O-, -OSi(Ra)2O-, -C(=O)(CRa2)C(=O)O-, or OC(=O)(CRa2)C(=O)-; wherein: Ra, for each occurrence, is independently hydrogen or C1-C6 alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6. In a second embodiment of Formula (E), in the ionizable lipid, e.g., cationic lipid, according to the first embodiment, or a pharmaceutically acceptable salt thereof, X is -OC(=O)-, -SC(=O)-, - OC(=S)-, -C(=O)O-, -C(=O)S-, or -S-S-; and all other remaining variables are as described for Formula I or the first embodiment. In a third embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E-1):
Figure imgf000080_0001
(E-1) or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, 3, and 4; and all other remaining variables are as described for Formula (E) or the second embodiment of Formula (E). Alternatively, n is an integer selected from 1, 2, and 3; and all other remaining variables are as described for Formula (E) or the second embodiment of Formula (E). In a fourth embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E-2): 79 ME146907648v.1 (E-2) or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-1) or the second embodiment of Formula (E). In a fifth embodiment of Formula (E), in the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure, R1 and R2 are each independently hydrogen or C1-C2 alkyl, or C2-C3 alkenyl; or R’, R1, and R2 are each independently hydrogen, C1-C2 alkyl; and all other remaining variables are as described for Formula (E), Formula (E-1) or the second embodiment of Formula (E). In a sixth embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E-3):
Figure imgf000081_0001
(E-3) or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2) or the second or fifth embodiments of Formula (E). In a seventh embodiment of Formula (E), in the ionizable lipid, e.g., cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3) or the second or firth embodiments of Formula (E), R5 is absent or C1-C8 alkylene; or R5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; or R5 is absent, C1-C4 alkylene, or C2-C4 alkenylene; or R5 is absent; or R5 is C8 alkylene, C7 alkylene, C6 alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, C1 alkylene, C8 alkenylene, C7 alkenylene, C6 alkenylene, C5 alkenylene, C4 alkenylene, C3 alkenylene, or C2 alkenylene; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3) or the second or fifth embodiments of Formula (E). In an eighth embodiment of Formula (E), he ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E-4): 80 ME146907648v.1 (E-4) or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3) or the second, fifth or seventh embodiments of Formula (E). In a ninth embodiment, in the ionizable lipid, e.g., cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth or seventh embodiments of Formula (E), or a pharmaceutically acceptable salt thereof, R4 is C1-C14 unbranched alkyl, C2-C14 unbranched alkenyl, or
Figure imgf000082_0001
, wherein R4a and R4b are each independently C1-C12 unbranched alkyl or C2-C12 unbranched alkenyl; or R4 is C2-C12 unbranched alkyl or C2-C12 unbranched alkenyl; or R4 is C5-C12 unbranched alkyl or C5-C12 unbranched alkenyl; or R4 is C16 unbranched alkyl, C15 unbranched alkyl, C14 unbranched alkyl, C13 unbranched alkyl, C12 unbranched alkyl, C11 unbranched alkyl, C10 unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, C1 unbranched alkyl, C16 unbranched alkenyl, C15 unbranched alkenyl, C14 unbranched alkenyl, C13 unbranched alkenyl, C12 unbranched alkenyl, C11 unbranched alkenyl, C10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl; or R4 is
Figure imgf000082_0002
, wherein R4a and R4b are each independently C2-C10 unbranched alkyl or C2-C10 unbranched alkenyl; or R4 is
Figure imgf000082_0003
, wherein R4a and R4b are each independently C16 unbranched alkyl, C15 unbranched alkyl, C14 unbranched alkyl, C13 unbranched alkyl, C12 unbranched alkyl, C11 unbranched alkyl, C10 unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C16 unbranched alkenyl, C15 unbranched alkenyl, C14 unbranched alkenyl, C13 unbranched alkenyl, C12 unbranched alkenyl, C11 unbranched alkenyl, C10 unbranched alkenyl, C9 81 ME146907648v.1 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth or seventh embodiments of Formula (E). In a tenth embodiment, in the ionizable lipid, e.g., cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh or ninth embodiments of Formula (E), R3 is C3-C8 alkylene or C3-C8 alkenylene, C3-C7 alkylene or C3-C7 alkenylene, or C3-C5 alkylene or C3-C5 alkenylene,; or R3 is C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C1 alkylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh or ninth embodiments of Formula (E). In an eleventh embodiment, in the ionizable lipid, e.g., cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth or tenth embodiments of Formula (E), R6a and R6b are each independently C7-C12 alkyl or C7-C12 alkenyl; or R6a and R6b are each independently C8-C10 alkyl or C8-C10 alkenyl; or R6a and R6b are each independently C12 alkyl, C11 alkyl, C10 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C12 alkenyl, C11 alkenyl, C10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth or tenth embodiments of Formula (E). In a twelfth embodiment, in the ionizable lipid, e.g., cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth or eleventh embodiments of Formula (E), R6a and R6b contain an equal number of carbon atoms with each other; or R6a and R6b are the same; or R6a and R6b are both C12 alkyl, C11 alkyl, C10 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C12 alkenyl, C11 alkenyl, C10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth or eleventh embodiments of Formula (E). In a thirteenth embodiment, in the ionizable lipid, e.g., cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), R6a and R6b as defined in any one of the preceding embodiments each contain a different number of carbon atoms with each other; or the number of carbon atoms R6a and R6b differs by one or two carbon atoms; or the number of carbon atoms R6a and R6b differs by one carbon atom; or R6a is C7 alkyl and R6a is C8 alkyl, R6a is C8 alkyl and R6a is C7 alkyl, R6a is C8 alkyl and R6a is C9 alkyl, R6a is C9 alkyl and R6a is C8 alkyl, R6a is C9 alkyl and R6a is C10 alkyl, R6a is C10 alkyl and R6a is C9 alkyl, R6a is C10 alkyl and R6a is C11 alkyl, R6a is C11 alkyl and R6a is C10 alkyl, R6a is C11 alkyl and R6a is C12 alkyl, R6a is C12 alkyl and R6a is C11 alkyl, R6a is C7 alkyl and R6a is C9 alkyl, R6a is C9 alkyl and R6a is C7 alkyl, R6a is C8 alkyl and R6a is C10 alkyl, R6a is 82 ME146907648v.1 C10 alkyl and R6a is C8 alkyl, R6a is C9 alkyl and R6a is C11 alkyl, R6a is C11 alkyl and R6a is C9 alkyl, R6a is C10 alkyl and R6a is C12 alkyl, R6a is C12 alkyl and R6a is C10 alkyl, etc.; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E- 4) or the second, fifth, seventh, ninth, tenth, eleventh or twelfth embodiments of Formula (E). In a fourteenth embodiment, in the ionizable lipid, e.g., cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth, eleventh, twelfth or thirteenth embodiments of Formula (E), R’ is absent; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth, eleventh, twelfth or thirteenth embodiments of Formula (E). In one embodiment, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure or the cationic lipid of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) is any one lipid selected from the lipids in Table 7 or a pharmaceutically acceptable salt thereof: Table 7. Exemplary lipids of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E- 4)
Figure imgf000084_0001
83 ME146907648v.1
Figure imgf000085_0001
84 ME146907648v.1
Figure imgf000086_0001
Specific examples are provided in the exemplification section below and are included as part of the cationic or ionizable lipids described herein. Pharmaceutically acceptable salts as well as neutral forms are also included. Cleavable Lipids In some embodiments, the LNPs provided by the present disclosure comprise an ionizable lipid that is also a cleavable lipid. As used herein, the term “cleavable lipid”, which may be used interchangeably with the term “SS-cleavable lipid” refers to an ionizable lipid comprising a disulfide bond (“SS”). The SS in the cleavable lipid is a cleavable unit. In one embodiment, a cleavable lipid comprises an amine, e.g., a tertiary amine, e.g.and a disulfide bond. In this cleavable lipid, an amine can become protonated in an acidic compartment (e.g., an endosome or a lysosome), leading to LNP destabilization, and the cleavable lipid can become cleaved in a reductive environment (e.g., the cytoplasm). Cleavable lipids also include pH-activated lipid-like materials, such as ss-OP lipids, ssPalm lipids, ss-M lipids, ss-E lipids, ss-EC lipids, ss-LC lipids and ss-OC lipids, etc. According to some embodiments, SS-cleavable lipids are described in International Patent Application Publication No. WO2019188867, incorporated by reference in its entirety herein. In one embodiment, a cleavable lipid may comprise three components: an amine head group, a linker group, and a hydrophobic tail(s). In one embodiment, the cleavable lipid comprises one or more phenyl ester bonds, one of more tertiary amino groups, and a disulfide bond. The tertiary amine groups provide pH responsiveness and induce endosomal escape, the phenyl ester bonds enhance the degradability of the structure (self- degradability) and the disulfide bond becomes cleaved in a reductive environment. 85 ME146907648v.1 In one embodiment, the cleavable lipid is an ss-OP lipid. In one embodiment, an ss-OP lipid comprises the structure of Lipid A shown below: Lipid A
Figure imgf000087_0001
. In one embodiment, the SS-cleavable lipid is an SS-cleavable and pH-activated lipid-like material (ssPalm). ssPalm lipids are well known in the art. For example, see Togashi et al., Journal of Controlled Release, 279 (2018) 262-270, the entire contents of which are incorporated herein by reference. In one embodiment, the ssPalm is an ssPalmM lipid comprising the structure of Lipid B shown below: Lipid B
Figure imgf000087_0002
. In one embodiment, the ssPalmE lipid is a ssPalmE-P4-C2 lipid comprising the structure of Lipid C below: Lipid C
Figure imgf000087_0003
. In one embodiment, the ssPalmE lipid is a ssPalmE-Paz4-C2 lipid, comprising the structure of Lipid D below: Lipid D
Figure imgf000087_0004
86 ME146907648v.1 In one embodiment, the cleavable lipid is an ss-M lipid. In one embodiment, an ss-M lipid comprises the structure shown in Lipid E below: Lipid E
Figure imgf000088_0001
In one embodiment, the cleavable lipid is an ss-E lipid. In one embodiment, an ss-E lipid comprises the structure shown in Lipid F below: Lipid F
Figure imgf000088_0002
. In one embodiment, the cleavable lipid is an ss-EC lipid. In one embodiment, an ss-EC lipid comprises the structure shown for Lipid G below: Lipid G
Figure imgf000088_0003
In one embodiment, the cleavable lipid is an ss-LC lipid. In one embodiment, an ss-LC lipid comprises the structure shown for Lipid H below: Lipid H
Figure imgf000088_0004
87 ME146907648v.1 In one embodiment, the cleavable lipid is an ss-OC lipid. In one embodiment, an ss-OC lipid comprises the structure shown for Lipid J below: Lipid J
Figure imgf000089_0001
. Other Lipids In some embodiments, the ionizable lipid in the LNPs of the present disclosure is selected from the group consisting of N-[1-(2,3-dioleyloxy)propyll-N,N,N-trimethylammonium chloride (DOTMA); N-[1-(2,3-dioleoyloxy)propyll-N,N,N-trimethylammonium chloride (DOTAP); 1,2- dioleoyl-sn-glycero -3-ethylphosphocholine (DOEPC); 1,2-dilauroyl-sn-glycero-3- ethylphosphocholine (DLEPC); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2- dimyristoleoyl- sn-glycero-3-ethylphosphocholine (14:1), N1- [2-((1S)-1-[(3-aminopropyl)amino]-4- [di(3-amino-propyl) aminolbutylc arboxamidoiethy11-3 ,4 -di[oleyloxy]-benzamide(MVL5); Dioctadecylamido-glycylspermine (DOGS); 3b-[N-(N’,N’-dimethylaminoethyl)carb amoyl] cholesterol (DC-Chol); Dioctadecyldimethylammonium Bromide (DDAB); a Saint lipid (e.g., SAINT-2, N-methy1-4-(dioleyl)methylpyridinium); 1,2-dimyristyloxypropy1-3- dimethylhydroxyethylammonium bromide (DMRIE); 1,2-dioleoy1-3-dimethyl-hydroxyethyl ammonium bromide (DORIE); 1,2-dioleoyloxypropy1-3-dimethylhydroxyethyl ammonium chloride (DORI); Di-alkylated Amino Acid (DILA2) (e.g., C18 :1 -norArg -C16); Dioleyldimethylammonium chloride (DODAC); 1-palmitoy1-2-oleoyl-sn-glycero-3 -ethylpho sphocholine (POEPC); and 1,2 - dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC). In some variations, the condensing agent, e.g. a cationic lipid, is a lipid such as, e.g., Dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoley1-4-(2dimethylaminoethyl)- [1,31-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19- yl-4- (dimethylamino)butanoate (DLin-MC3-DMA), 1,2-Dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-Dioleyloxy-3-dimethylaminopropane (DODMA), Morpholinocholesterol (Mo-CHOL), (R)-5- (dimethylamino)pentane-1,2-diy1 dioleate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane- 1,2-diy1 dioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethy1-4,5-bis(oleoyloxy)pentan-1- aminium chloride(DOTAPen). In some embodiments, the ionizable lipid in the LNP of the present disclosure is represented by the following structure: 88 ME146907648v.1 5 , 89 ME146907648v.1 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof. B. Structural Lipids In some embodiments, the LNPs provided by the present disclosure comprise a structural lipid. Without wishing to be bound by a specific theory, it is believed that a structural lipid, when present in an LNP, contributes to membrane integrity and stability of the LNP. In some embodiments, the structural lipid is a sterol, e.g., cholesterol, or a derivative thereof. In one embodiment, the structural lipid is cholesterol. In another embodiment, the structural lipid is a derivative of cholesterol. Non-limiting examples of cholesterol derivatives include polar analogues such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2’-hydroxy)-ethyl ether, cholesteryl-(4’- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4’-hydroxy)- butyl ether. In some embodiments, cholesterol derivative is cholestryl hemisuccinate (CHEMS). 90 ME146907648v.1 Exemplary cholesterol derivatives are described in International Patent Application Publication No. WO2009/127060 and U.S. Patent Application Publication No. US2010/0130588, contents of both of which are incorporated herein by reference in their entirety. In some embodiments, the sterol in the LNPs of the present disclosure is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and derivatives thereof, and any combination thereof. In one embodiment, the sterol is cholesterol. In another embodiment, the sterol is beta-sitosterol. In some embodiments, the structural lipid constitutes about 20 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid constitutes about 25 mol% to about 45 mol% of the total lipid content of the LNP. In some embodiments, the structural lipid constitutes about 30 to about 45% of the total lipid present in the LNP. In some embodiments, the structural lipid constitutes about 30 mol% to about 40 mol% of the total lipid present in the LNP. In some embodiments, such a component is about 40 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid, e.g., a sterol, constitutes about 20 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid, e.g., a sterol, constitutes about 30 mol% to about 40 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 30 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 35 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 40 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 40 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 40 mol% to about 45 mol% of the total lipid present in the LNP, wherein the encapsulation efficiency (“Enc. Eff.”) of TNA is greater than 95% and/or the average size of the LNP ranges about 70 nm to 90 nm in diameter. Table 8 in Example 1 shows the effect of cholesterol on the average LNP particle size. As the molar percentage of cholesterol increases from 25% up to 45% of the total lipid percentage, the average LNP particle size (measured as particle diameter) gradually decreases from 110 nm to 82 nm in the formulation disclosed herein. This suggests that a structural lipid like cholesterol plays an essenstial role in achieving desirable LNP attributes like the particle size that are useful for delivery of therapeutic molecules. C. Lipid-Anchored Polymers In some embodiments, the LNPs provided by the present disclosure comprise at least one type of lipid-anchored polymer, i.e., a first lipid-anchored polymer. As used herein, the term “lipid- anchored polymer” refers to a molecule comprising a lipid moiety covalently attached to a polymer, 91 ME146907648v.1 e.g., via a linker. Without wishing to be bound by a specific theory, it is believed that a lipid-anchored polymer can inhibit aggregation of LNPs and provide steric stabilization. In some embodiments, the LNPs provided by the present disclosure comprise two lipid-anchored polymers, i.e., a first lipid- anchored polymer and a second lipid-anchored polymer. Lipid moieties in lipid-anchored polymers More specifically, in one embodiment, a lipid-anchored polymer, e.g., a first lipid-anchored polymer, in accordance with the present disclosure comprises: (i) a polymer; (ii) a lipid moiety comprising at least two hydrophobic tails (which may be linear or branched); and (iii) a linker connecting the polymer to the lipid moiety; wherein the at least two hydrophobic tails (which may be linear or branched) comprise 16 to 22 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, 21, or 22 carbon atoms in a single aliphatic chain backbone. In one embodiment, the at least two hydrophobic tails are each a fatty acid. In one embodiment, the lipid-anchored polymer, e.g., a first lipid-anchored polymer, comprises a lipid moiety comprising two hydrophobic tails, wherein the two hydrophobic tails each independently comprise 16 to 22 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, 21, or 22 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 to 21 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, or 21 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 to 20 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, or 20 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 to 19 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, or 19 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 to 18 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, or 18 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 or 18 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 or 20 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 18 or 20 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 16 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 17 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 18 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 19 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 20 carbon atoms in a single aliphatic chain backbone. 92 ME146907648v.1 The term “linker-lipid moiety”, as used herein, refers to a lipid moiety comprising at least two hydrophobic tails, e.g., two hydrophobic tails, covalently attached to a linker. In some embodiments, the linker-lipid moiety may be a part of a lipid-anchored polymer. The term “derivative,” when used herein in reference to hydrophobic tails in a lipid-anchored polymer, refers to a hydrophobic tail that has been modified as compared to the original or native hydrophobic tail. In some embodiments, the derivative contains one or more of the following modifications as compared to the original or native hydrophobic tail: a) carboxylate group has been replaced with an amine group, an amide group, an ether group, or a carbonate group; b) one or more points of saturation, e.g., double bonds, have been introduced into (e.g., via dehydrogenation) the hydrophobic tail; c) one or more points of saturation, e.g., double bonds, have been removed from (e.g., via hydrogenation) the hydrophobic tail; and d) configuration of one or more double bonds, if present, has been changed, e.g., from a cis configuration to a trans configuration, or from a trans configuration to a cis configuration. The derivative contains the same number of carbon atoms as its original or native hydrophobic tail. As used herein the term “a single aliphatic chain backbone” when referring to a hydrophobic tail in a lipid-anchored polymer refers to the main linear aliphatic chain or carbon chain, i.e., the longest continuous linear aliphatic chain or carbon chain. For example, the alkyl chain below that has several branchings contains 18 carbon atoms in a single aliphatic chain backbone, i.e., the longest continuous linear alkyl chain contains 18 carbon atoms. Note that the one or two carbon atoms (all indicated with *) in the several branching points are not included in the carbon atom count in the single aliphatic chain backbone.
Figure imgf000094_0001
Linkers in lipid-anchored polymers In some embodiments, in a lipid-anchored polymer of the present disclosure, a lipid moiety is covalently attached to a polymer via a linker. In some embodiments, the linker in the lipid-anchored polymer of the present disclosure is a glycerol linker, a phosphate linker, an ether linker, an amide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, or any combination thereof. In some embodiments, the linker in the lipid-anchored polymer in the LNPs of the present disclosure a glycerol linker. Accordingly, in some embodiments, the lipid-anchored polymer in the LNPs of the present disclosure is a glycerolipid, wherein the glycerolipid comprises glycerol as a linker and one or more lipid moieties as described above, e.g., distearoyl-rac-glycerol (DSG). 93 ME146907648v.1 In some embodiments, the linker in the lipid-anchored polymer in the LNPs of the present disclosure is a phosphate linker. Accordingly, in some embodiments, the lipid-anchored polymer in the LNPs of the present disclosure is a phospholipid, wherein the phospholipid comprises a phosphate group as a linker and one or more lipid moieties as described above. In some embodiments, the lipid-anchored polymer in an LNP of the present disclosure is both a glycerolipid and a phospholipid, such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). In some embodiments, the first lipid-anchored polymer comprises a linker-lipid moiety (i.e., with one or more hydrophobic tails containing 16 to 22 carbon atoms in a single aliphatic chain) selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1- palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn- glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); and dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), a derivative thereof, and a combination any of the foregoing. As used herein, the term “derivative” when used in reference to a linker-lipid moiety, means a linker-lipid moiety containing one or more of the following modifications: a) a phosphatidylethanolamine (PE) head group, if present, is modified to convert an amino group into a methylamino group or a dimethylamino group; b) the modified linker-lipid moiety comprises one or more additional functional groups or moieties, such as -OH, -OCH3, -NH2, a maleimide, an azide or a cyclooctyne such as dibonzeocyclooctyne (DBCO). In one embodiment, the first lipid-anchored polymer comprises a linker-lipid moiety (i.e., with one or more hydrophobic tails containing 16 to 22 carbon atoms in a single aliphatic chain) selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, a derivative thereof, and a combination of any of the foregoing. Polymers in lipid-anchored polymers In some embodiments, the polymer in the lipid-anchored polymer is selected from the group consisting of polyethylene (PE), polypropylene (PP), polyethylene glycol (PEG), polyglycerol (PG), polyvinyl alcohol (PVOH), polysarcosine (pSar), and a combination thereof. In one embodiment, the polymer is selected from the group consisting of polyethylene glycol (PEG), polyglycerol (PG), polysarcosine (pSar), and a combination thereof. In one embodiment, the polymer is polyethyelene glycol (PEG). In another embodiment, the polymer is polyglycerol (PG). 94 ME146907648v.1 In some embodiments, the polymer in the lipid-anchored polymer has a molecular weight of about 5000 Da or less, e.g., about 4500 Da or less, about 4000 Da or less, about 3500 Da or less, about 3200 Da or less, about 3000 Da or less, about 2500 Da or less, about 2000 Da or less, about 1500 Da or less, about 1000 Da or less, about 500 Da or less, about 100 Da or less or about 50 Da or less. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 20 Da to about 100 Da, about 50 Da to about 500 Da, about 500 Da to about 2000 Da, about 1000 Da to about 5000 Da, e.g., about 2000 Da to about 5000 Da, about 1000 Da to about 3000 Da, about 1500 Da to about 2500 Da, about 2000 Da to about 4000 Da or about 2000 Da to about 5000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 1000 Da, about 1500 Da, about 2000 Da, about 2500 Da, about 3000 Da, about 3200 Da, about 3300 Da, about 3350 Da, about 3400 Da, about 3500 Da, about 4000 Da, about 4500 Da or about 5000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 2000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 2000 Da. In some embodiments, the polymer in the lipid- anchored polymer has an average molecular weight of about 3200 Da to about 3500 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 3300 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 3350 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 3400 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 3500 Da. Targeting moiety and second lipid-anchored polymer In some embodiments, an LNP of the present disclosure further comprises one or more targeting moieties. The targeting moiety targets the LNP for delivery to a specific site or a tissue in a subject, e.g., liver. In some embodiments, the targeting moiety is capable of binding to specific liver cells, such as hepatocytes. In one embodiment, the targeting moiety is capable of binding to the asialoglycoprotein receptor (ASGPR), e.g., hepatocyte-specific ASGPR. In one embodiment, the targeting moiety comprises an N-acetylgalactosamine molecule (GalNAc) or a GalNAc derivative thereof. As used herein, a “GalNAc derivative” refers to a modified GalNAc molecule or a conjugate of one or more GalNAc molecules (modified or unmodified) covalently linked to, for example, a lipid-anchored polymer as defined herein. In one embodiment, the targeting moiety is a tri-antennary or tri-valent GalNAc conjugate (i.e., GalNAc3) which is a ligand conjugate having three GalNAc molecules or three GalNAc derivatives. In one embodiment, the targeting moiety is a tri-antennary GalNAc represented by the following structural formula: 95 ME146907648v.1
In one embodiment, the targeting moiety is a tetra-antennary GalNAc conjugate. In one embodiment, the targeting moiety is a tetra-antennary or tetra-valent GalNAc conjugate (i.e., GalNAc4) which is a ligand having four GalNAc molecules or four GalNAc derivatives. In one embodiment, the targeting moiety is capable of binding to low-density lipoprotein receptors (LDLRs), e.g., hepatocyte-specific LDLRs. In one embodiment, the targeting moiety comprises an apoliprotein E (ApoE) protein, an ApoE polypeptide (or peptide), an apoliprotein B (ApoB) protein, an ApoB polypeptide (or peptide), a fragment of any of the foregoing, or a derivative of any of the foregoing. In one embodiment, the ApoE polypeptide, ApoB polypeptide, or a fragment thereof is a ApoE polypeptide, ApoB polypeptide, or a fragment thereof as disclosed in International Patent Application Publication No. WO2022/261101, which is incorporated herein by reference in its entirety. In one embodiment, the ApoE protein is a modified ApoE protein and the ApoB protein is a modified ApoB protein. In one embodiment, the ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98, or at least about 99% sequence identity to the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQE LRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYR GEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRER LGPLVEQGRVR (SEQ ID NO: 1). In one embodiment, the ApoE protein comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98, or at least about 99% sequence identity to the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQE LRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYR 96 ME146907648v.1 GEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRER LGPLVEQGRVRHHHHHH (SEQ ID NO: 2). In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98, or at least about 99% sequence identity to the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQE LRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYR GEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRER LGPLVEQGRVR (SEQ ID NO: 3). In one embodiment, the ApoE protein comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98, or at least about 99% sequence indentity to the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQE LRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYR GEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRER LGPLVEQGRVRHHHHHHGGSSGSGC (SEQ ID NO: 4). In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO: 4. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO: 4. As used herein, the term “sequence identity” refers to the ratio of the number of identical amino acids between the 2 aligned sequences over the aligned length, expressed as a percentage. In some embodiments, the 2 aligned sequences are identical in length, i.e., have the same number of amino acids. In one embodiment, the targeting moiety in an LNP of the present disclosure is an ApoE protein conjugate in an ApoB protein conjugate, which is a conjugate of one or more ApoE and/or ApoB protein molecules (native or modified) or a fragment thereof covalently linked to, for example, a lipid-anchored polymer as defined herein. In one embodiment, the targeting moiety in an LNP of the present disclosure is an ApoE polypeptide conjugate in an ApoB polypeptide conjugate, which is a conjugate of one or more ApoE and/or ApoB polypeptide molecules or a fragment thereof covalently linked to, for example, a lipid-anchored polymer as defined herein. 97 ME146907648v.1 Accordingly, one key embodiment of an LNP of the present disclosure is that the LNP comprises a second lipid-anchored polymer and the targeting moiety as defined herein (and including GalNAc, ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide) is conjugated to the second lipid-anchored polymer. The second lipid-anchored polymer is structurally similar to the first lipid-anchored polymer as described herein in that the second lipid-anchored polymer also contains a lipid moiety covalently attached to a polymer via a linker. In one embodiment, the second lipid- anchored polymer comprises a linker-lipid moiety selected from the group consisting of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn- glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn- phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn- glycero-3-phosphoethanolamine (DPHyPE); and dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), a derivative thereof, and a combination any of the foregoing. In one embodiment, the second lipid-anchored polymer comprises a linker-lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and a derivative of thereof, and a combination of any of the foregoing. In one embodiment, the ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, or a fragment thereof, is covalently linked to a lipid-anchored polymer (e.g., second lipid-anchored polymer) or to an LNP of the present disclosure via strain promoted alkyne-azide cycloaddition (SPAAC) chemistry, such as via an azide-modified lipid-anchored polymer (e.g., DSG-PEG2000- azide, DSPE-PEG2000-azide, DSG-PEG3400-azide, DSPE-PEG3400-azide, DSG-PEG5000-azide, DSPE-PEG5000-azide) and a dibenzocyclooctyne (DBCO)-functionalized ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, or a fragment thereof. In some embodiments, the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer. For example, the LNPs of the present disclosure may comprise a first lipid-anchored polymer that does not comprise a targeting moiety, and a second type of lipid-anchored polymer that comprises a targeting moiety, such as GalNAc. For example, the LNPs of the present disclosure may comprise DSG-PEG2000 modified to comprise an additional OCH3 group (DSG-PEG2000-OMe) as a first lipid-anchored polymer and DSPE-PEG2000-GalNAc3 as a second lipid-anchored polymer. In some embodiments, the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety. In some embodiments, the second lipid-anchored polymer comprses a lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and a derivative of 98 ME146907648v.1 thereof. In some embodiments, the first lipid-anchored polymer is any lipid-anchored polymer as described hereinabove. In some embodiments, the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety, and the first lipid-anchored polymer and the second lipid-anchored polymer are different lipid-anchored polymers and are selected from one of the following combinations: DSG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DSPE (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); and DMG (the first lipid-anchored polymer) and DPG (the second lipid-anchored polymer). In some embodiments, the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety, and the first lipid-anchored polymer and the second lipid-anchored polymer are the same lipid-anchored polymers and are selected from one of the following combinations: DSG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DSPE (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); and DPG (the first lipid-anchored polymer) and DPG (the second lipid-anchored polymer). In some embodiments, the targeting moiety is conjugated to a DSPE-anchored polymer. In some embodiments, the DSPE-anchored polymer is DSPE-PEG or a derivative thereof. In some embodiments, the targeting moiety is conjugated to a DSG-anchored polymer. In some embodiments, the DSG-anchored polymer is DSG-PEG or a derivative thereof. In some embodiments, the lipid-anchored polymers (first and second lipid-anchored polymers in combination) constitute about 0.1 mol% to about 20 mol% of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 0.5 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 1 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 2 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute more than about 2 mol% (e.g., 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, 2.5 mol%, 2.6 99 ME146907648v.1 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%) to about 10 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 3 mol% to about 8 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 3 mol% to about 7 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 3 mol% to about 5 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 2 mol% to about 4 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 2% to about 3% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 2 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 2.5 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 3 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 3.5 mol% present in the LNP. In some embodiments, the lipid-anchored polymers constitute about 4 mol% present in the LNP. In some embodiments, the first lipid-anchored polymer is present in about 0.1 mol% to about 10 mol% of the total lipid present in the LNP, or about 0.2 mol% to about 8 mol%, or about 0.2 mol% to about 7 mol%, or about 0.2% mol% to about 5 mol%, or about 0.3 mol to about 4 mol%, or about 0.4 mol% to about 4 mol%, or about 0.5 mol% to about 5 mol%, or about 0.5 mol% to about 4 mol%, or about 0.5 mol% to about 3.5 mol%, or about 0.5 mol% to about 3 mol%, or about 0.7 mol% to about 5 mol%, or about 0.7 mol% to about 4 mol%, or about 0.7 mol% to about 3.5 mol%, or about 0.7 mol% to about 3 mol%, or about 1 mol% to about 5 mol%, or about 1 mol% to about 4 mol%, or about 1 mol% to about 3.5 mol%, or about 1 mol% to about 3 mol%, or about 1.5 mol% to about 5 mol%, or about 1.5 mol% to about 4 mol%, or about 1.5 mol% to about 3.5 mol%, or about 1.5 mol% to about 3 mol%, or about 2 mol% to about 5 mol%, or about 2 mol% to about 4 mol%, or about 2 mol% to about 3.5 mol%, or about 2 mol% to about 3 mol%, or about 2.5 mol% to about 5 mol%, or about 2.5 mol% to about 4 mol%, or about 2.5 mol% to about 3.5 mol%, or about 2.5 mol% to about 3 mol%, or about 3 mol% to about 5 mol%, or about 3 mol% to about 4.5 mol% or about 3 mol% to about 4 mol%, or about 3 mol% to about 3.5 mol%, or about 3.5 mol% to about 5 mol%, or about 3.5 mol% to about 4.5 mol% or about 3.5 mol% to about 4 mol%. In some embodiments, the second lipid-anchored polymer, if present, is present in about 0.005 mol% to about 5 mol% of the total lipid present in the LNP, or about 0.005 mol% to about 3 mol%, or about 0.005 mol% to about 2 mol%, or about 0.005 mol% to about 1 mol%, or about 0.005 mol% to about 0.5 mol%, or about 0.01 mol% to about 3 mol%, or about 0.01 mol% to about 2 mol%, or about 0.01 mol% to about 1 mol%, or about 0.01 mol% to about 0.5 mol%, or about 0.025 mol% to about 3 mol%, or about 0.025 mol% to about 2 mol%, or about 0.025 mol% to about 1 mol%, or about 0.025 mol% to about 0.5 mol%, or about 0.05 mol% to about 3 mol%, or about 0.05 mol% to about 2 mol%, or about 0.05 mol% to about 1 mol%, or about 0.05 mol% to about 0.5 mol%, or about 0.01 mol% to about 0.4 mol%, or about 0.01 mol% to about 0.3 mol%, or about 0.01 mol% to about 0.25 mol%, or about 0.01 mol% to about 0.2 mol%, or about 0.01 mol% to about 0.1 mol%, or about 100 ME146907648v.1 0.025 mol% to about 0.4 mol%, or about 0.025 mol% to about 0.3 mol%, or about 0.025 mol% to about 0.25 mol%, or about 0.025 mol% to about 0.2 mol%, or about 0.025 mol% to about 0.1 mol%, or about 0.05 mol% to about 0.4 mol%, or about 0.05 mol% to about 0.3 mol%, or about 0.05 mol% to about 0.25 mol%, or about 0.05 mol% to about 0.2 mol%, or about 0.05 mol% to about 0.1 mol%. Lipid nanoparticles (LNPs) comprising ceDNA are disclosed in International Patent Application No. PCT/US2018/050042, filed on September 7, 2018, which is incorporated herein in its entirety and envisioned for use in the methods and compositions as disclosed herein. The size of LNPs can be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK). In some embodiments, LNPs of the present disclosure have a mean diameter as determined by light scattering of less than about 90 nm, e.g., less than about 80 nm or less than about 75 nm. According to some embodiments, LNPs of the present disclosure have a mean diameter as determined by light scattering of between about 50 nm and about 75 nm or between about 50 nm and about 70 nm. The pKa of formulated cationic lipids can be correlated with the effectiveness of the LNPs for delivery of nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010), both of which are incorporated by reference in their entireties). In one embodiment, the pKa of each cationic lipid is determined in lipid nanoparticles using an assay based on fluorescence of 2-(p-toluidino)-6- napthalene sulfonic acid (TNS). LNPs in PBS at a concentration of 0.4 mM total lipid can be prepared using the in-line process as described herein and elsewhere. TNS can be prepared as a 100 mM stock solution in distilled water. Vesicles can be diluted to 24 mM lipid in 2 mL of buffered solutions containing, 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where the pH ranges from 2.5 to 11. An aliquot of the TNS solution can be added to give a final concentration of 1 mM and following vortex mixing fluorescence intensity is measured at room temperature in a SLM Aminco Series 2 Luminescence Spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best fit analysis can be applied to the fluorescence data and the pKa is measured as the pH giving rise to half-maximal fluorescence intensity. In one embodiment, relative activity can be determined by measuring luciferase expression in the liver 4 hours following administration via tail vein injection. The activity is compared at a dose of 0.3 and 1.0 mg ceDNA/kg and expressed as ng luciferase/g liver measured 4 hours after administration. Without limitations, LNP of the present disclosure includes a lipid formulation that can be used to deliver a capsid-free, non-viral DNA vector to a target site of interest (e.g., cell, tissue, organ, and the like). Generally, the LNP comprises capsid-free, non-viral DNA vector and a cationic lipid or a salt thereof. 101 ME146907648v.1 Further exemplary lipid-anchored polymers include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide -lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEGylated lipid, for example, a (methoxy polyethylene glycol)- conjugated lipid. PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG)), PEG- dialkyloxypropyl (DAA), PEG-phospholipid, a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0- (2’,3’-di(tetradecanoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypoly ethylene glycol 2000)-l,2-distearoyl-sn- glycero-3-phosphoethanolamine sodium salt, or a mixture thereof. Additional exemplary PEG-lipid conjugates are described, for example, in International Patent Application Publication Nos. WO 1996/010392, WO1998/051278, W02002/087541, W02005/026372, WO2008/147438, W02009/086558, W02012/000104, WO2017/117528, WO2017/099823, WO2015/199952, W02017/004143, WO2015/095346, WO2012/000104, WO2012/000104, and WO2010/006282, U.S. Patent Application Publication Nos. US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2013/0303587, US2018/0028664, US2015/0376115, US2016/0376224, US2016/0317458, US2013/0303587, US2013/0303587, and US20110123453, and U.S. Patent Nos. US5,885,613, US6,287,591, US6,320,017, and US6,586,559, the contents of all of which are incorporated herein by reference in their entireties. Additional examples of PEG-DAA PEGylated lipids include, for example, PEG- dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG- disterylglycamide, PEG-cholesterol (l-[8’-(Cholest-5-en-3[beta]- oxy)carboxamido-3’,6’-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-Ditetradecoxylbenzyl-[omega]- methyl-poly(ethylene glycol) ether), and l,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In one embodiment, the PEG-lipid can be selected from the group consisting of PEG-DMG, l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000],
Figure imgf000103_0001
, 102 ME146907648v.1 . Yet further exemplary lipid-anchored polymers include N-(Carbonyl- methoxypo1yethy1eneg1yco1n)-1,2-dimyristoyl-sn-glycero-3 -phosphoethanolamine (DMPE-PEGn, where n is 350, 500, 750, 1000 or 2000), N-(Carbonyl-methoxypolyethyleneglycoln)-1,2-distearoyl- sn-glycero-3-phosphoethanolamine (DSPE-PEGn, where n is 350, 500, 750, 1000 or 2000), DSPE- polyglycelin-cyclohexyl-carboxylic acid, DSPE-polyglycelin-2-methylglutar-carboxylic acid, 1,2- Distearoyl-sn-Glycero-3-Phosphoethanolamine (DSPE) conjugated Polyethylene Glycol (DSPE-PEG- OH), polyethylene glycol-dimyristolglycerol (PEG-DMG), or polyethylene glycol-distearoyl glycerol (PEG-DSG). In some examples of DMPE-PEGn, where n is 350, 500, 750, 1000 or 2000, the PEG- lipid is N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE-PEG 2,000). In some examples of DSPE-PEGn. where n is 350, 500, 750, 1000 or 2000, the PEG-lipid is N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl- sn-glycero-3-phosphoethanolamine (DSPE-PEG 2,000). In some embodiments, the PEG-lipid is DSPE-PEG-OH. In some embodiments, the PEG-lipid is PEG-DMG having two C14 hydrophobic tails and PEG2000. D. Therapeutic Nucleic Acids The LNPs provided by the present disclosure also comprise a therapeutic nucleic acid (TNA). According to embodiments, also provided are pharmaceutical compositions comprising the LNPs of the disclosure. Illustrative therapeutic nucleic acids in the LNPs of the present disclosure can include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, closed ended double stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA (“ministring”), single-stranded DNA (ssDNA), doggybone™, protelomere closed ended DNA, or dumbbell linear DNA), single stranded DNA (e.g., synthetically made AAV vectors), dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), mRNA, tRNA, rRNA, gRNA and DNA viral vectors, viral RNA vector, and any combination thereof. In any of the aspects and embodiments provided herein, the therapeutic nucleic acid can be a therapeutic DNA. Said therapeutic DNA can be ceDNA, CELiD, linear covalently closed DNA 103 ME146907648v.1 (“ministring” or otherwise), ssDNA, doggybone™, protelomere closed ended DNA, dumbbell linear DNA, minigenes, plasmids, single stranded DNA (e.g., synthetically made AAV vectors) or minicircles. According to some embodiments of any of the above aspects and embodiments, the TNA is selected from the group consisting of RNA, DNA, and derivatives and analogues thereof. According to some embodiments of any of the above aspects and embodiments, the TNA encodes a therapeutic gene and/or a therapeutic protein. According to some embodiments of any of the above aspects and embodiments, the e TNA is selected from the group consisting of mRNA, siRNA, synthetic ribozymes, antisense RNA, and gRNA. According to some embodiments of any of the above aspects and embodiments, the TNA is mRNA. According to some embodiments of any of the above aspects and embodiments, the TNA is selected from the group consisting of single-stranded-DNA (ssDNA) and double-stranded DNA (dsDNA). According to some embodiments of any of the above aspects and embodiments, the TNA is ssDNA. According to some embodiments of any of the above aspects and embodiments, the TNA is linear ssDNA. According to some embodiments of any of the above aspects and embodiments, the TNA is dsDNA. According to some embodiments of any of the above aspects and embodiments, the TNA is a non-viral capsid-free DNA vector with covalently-closed ends (ceDNA vector). siRNA or miRNA that can downregulate the intracellular levels of specific proteins through a process called RNA interference (RNAi) are also contemplated by the present disclosure to be nucleic acid therapeutics. After siRNA or miRNA is introduced into the cytoplasm of a host cell, these double-stranded RNA constructs can bind to a protein called RISC. The sense strand of the siRNA or miRNA is removed by the RISC complex. The RISC complex, when combined with the complementary mRNA, cleaves the mRNA and release the cut strands. RNAi is by inducing specific destruction of mRNA that results in downregulation of a corresponding protein. Antisense oligonucleotides (ASO) and ribozymes that inhibit mRNA translation into protein can be nucleic acid therapeutics. For antisense constructs, these single stranded deoxy nucleic acids have a complementary sequence to the sequence of the target protein mRNA, and Watson - capable of binding to the mRNA by Crick base pairing. This binding prevents translation of a target mRNA, and / or triggers RNaseH degradation of the mRNA transcript. As a result, the antisense oligonucleotide has increased specificity of action (i.e., down-regulation of a specific disease-related protein). In any of the aspects and embodiments provided herein, the therapeutic nucleic acid can be a therapeutic RNA. Said therapeutic RNA can be messenger RNA (mRNA) encoding a protein or peptide, an inhibitor of mRNA translation, agent of RNA interference (RNAi), catalytically active RNA molecule (ribozyme), transfer RNA (tRNA) or an RNA that binds an mRNA transcript (ASO), protein or other molecular ligand (aptamer). In any of the methods provided herein, the agent of RNAi can be a double-stranded RNA, single-stranded RNA, microRNA, short interfering RNA, short hairpin RNA, or a triplex-forming oligonucleotide. 104 ME146907648v.1 Single-Stranded DNA (ssDNA) According to one aspect, the disclosure provides an isolated, linear single stranded deoxyribonucleic acid (ssDNA) molecule comprising at least one nucleic acid sequence of interest flanked by at least one stem-loop structure at the 3’ end. According to one embodiment, the at least one stem-loop structure at the 3’ end is sufficient to prime replication and/or transcription. According to some embodiments, the stem structure at the 3’ end comprises a partial DNA duplex of between 4- 500 nucleotides. According to a further embodiment, the stem structure at the 3’ end comprises a partial DNA duplex of between 4-5 nucleotides. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 3’ end comprises between 3-500 unbound nucleotides. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 3’ end comprises a minimum of 3 unbound nucleotides. According to one embodiment of any of the aspects and embodiments herein, the ssDNA comprises at least two stem- loop structures at the 3’ end. According to one embodiment of any of the aspects and embodiments herein, the ssDNA comprises at least three stem-loop structures at the 3’ end. According to one embodiment of any of the aspects and embodiments herein, the ssDNA comprises at least four or more stem-loop structures at the 3’ end. According to one embodiment of any of the aspects and embodiments herein, the at least one stem-loop structure at the 3’ end comprises a hairpin DNA structure. According to one embodiment of any of the aspects and embodiments herein, the at least one stem-loop structure at the 3’ end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadraplex DNA structure, a bulged DNA structure, and a multibranched loop structure. According to one embodiment of any of the aspects and embodiments herein, the at least one stem-loop structure at the 3’ end does not comprise the A, A’, D, and D’ regions that would be present in a wild-type AAV ITR. According to one embodiment of any of the aspects and embodiments herein, the at least one stem-loop structure at the 3’ end does not comprise the A, A’, B, B’, C, C’, D, and D’ regions that would be present in a wild- type AAV ITR. According to one embodiment of any of the aspects and embodiments herein, the at least one stem-loop structure at the 3’ end does not comprise a rep binding element (RBE) that would be present in a wild-type ITR. According to one embodiment of any of the aspects and embodiments herein, the at least one stem-loop structure at the 3’ end does not comprise a terminal resolution site (trs) that would be present in a wild-type ITR. According to one embodiment of any of the aspects and embodiments herein, the stem structure at the 3’ end comprises four or more nucleotides that are modified to be exonuclease resistant. According to a further embodiment, the nucleotides are phosphorothioate-modified nucleotides. According to one embodiment of any of the aspects and embodiments herein, at least one stem-loop structure at the 3’ end further comprises a functional moiety. According to one embodiment of any of the aspects and embodiments herein, the ssDNA molecule further comprises a 5’ end, comprising at least one stem-loop structure. According to a further embodiment, the ssDNA comprises at least two stem-loop structures at the 5’ end. According 105 ME146907648v.1 to one embodiment of any of the aspects and embodiments herein, the ssDNA comprises at least three stem-loop structures at the 5’ end. According to one embodiment of any of the aspects and embodiments herein, the ssDNA comprises at least four or more stem-loop structures at the 5’ end. According to one embodiment of any of the aspects and embodiments herein, the at least one stem- loop structure at the 5’ end comprises a hairpin DNA structure. According to one embodiment of any of the aspects and embodiments herein, the at least one stem-loop structure at the 5’ end comprises a DNA structure selected from the group consisting of: a cruciform DNA structure, a hammerhead DNA structure, a quadraplex DNA structure, a bulged DNA structure, and a multibranched loop structure. According to one embodiment of any of the aspects and embodiments herein, the at least one stem-loop structure at the 5’ end does not comprise the A, A’, D, and D’ regions that would be present in a wild-type AAV ITR. According to one embodiment of any of the aspects and embodiments herein, the at least one stem-loop structure at the 5’ end does not comprise the A, A’, B, B’, C, C’, D, and D’ regions that would be present in a wild-type AAV ITR. According to one embodiment of any of the aspects and embodiments herein, the at least one stem-loop structure at the 5’ end does not comprise a rep binding element (RBE) that would be present in a wild-type ITR. According to one embodiment of any of the aspects and embodiments herein, the at least one stem- loop structure at the 5’ end does not comprise a terminal resolution site (trs) that would be present in a wild-type ITR. According to one embodiment of any of the aspects and embodiments herein, the stem structure at the 5’ end comprises four or more nucleotides that are modified to be exonuclease resistant. According to a further embodiment, the nucleotides are phosphorothioate-modified nucleotides. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more nucleic acids to stabilize the ends. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more nucleic acids that are chemically modified. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more aptamers. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more synthetic ribozymes. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more antisense oligonucleotides (ASOs). According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more short-interfering RNAs (siRNAs). According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more antiviral nucleoside analogues (ANAs). According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more triplex forming oligonucleotides. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more gRNAs or gDNAs. According to one embodiment of any of the aspects and embodiments herein, the loop structure at the 5’ end further comprises one or more 106 ME146907648v.1 molecular probes. According to one embodiment of any of the aspects and embodiments herein, the ssDNA molecule is devoid of any viral capsid protein coding sequences. According to one embodiment of any of the aspects and embodiments herein, the ssDNA molecule is synthetically produced in vitro. According to a further embodiment, the ssDNA molecule is synthetically produced in vitro in a cell-free environment. According to one embodiment of any of the aspects and embodiments herein, the ssDNA molecule does not activate or minimally activates an immune pathway. According to a further embodiment, the immune pathway is an innate immune pathway. According to another further embodiment, the innate immune pathway is selected from the group consisting of the cGAS/STING pathway, the TLR9 pathway, an inflammasome-mediated pathway, and a combination thereof. Closed-ended DNA (ceDNA) Vectors In some embodiments, LNPs provided by the present disclosure comprise closed-ended DNA (ceDNA). In some embodiments, the TNA comprises closed-ended linear duplexed (ceDNA) vectors that can express a transgene (e.g., a therapeutic nucleic acid (TNA)). The ceDNA vectors as described herein have no packaging constraints imposed by the limiting space within the viral capsid. ceDNA vectors represent a viable eukaryotically-produced alternative to prokaryote-produced plasmid DNA vectors, as opposed to encapsulated AAV genomes. This permits the insertion of control elements, e.g., regulatory switches as disclosed herein, large transgenes, multiple transgenes etc. ceDNA vectors preferably have a linear and continuous structure rather than a non- continuous structure. The linear and continuous structure is believed to be more stable from attack by cellular endonucleases, as well as less likely to be recombined and cause mutagenesis. Thus, a ceDNA vector in the linear and continuous structure is a preferred embodiment. The continuous, linear, single strand intramolecular duplex ceDNA vector can have covalently bound terminal ends, without sequences encoding AAV capsid proteins. These ceDNA vectors are structurally distinct from plasmids (including ceDNA plasmids described herein), which are circular duplex nucleic acid molecules of bacterial origin. The complimentary strands of plasmids may be separated following denaturation to produce two nucleic acid molecules, whereas in contrast, ceDNA vectors, while having complimentary strands, are a single DNA molecule and therefore even if denatured, it is likely to remain a single molecule. In some embodiments, ceDNA vectors can be produced without DNA base methylation of prokaryotic type, unlike plasmids. Therefore, the ceDNA vectors and ceDNA-plasmids are different both in term of structure (in particular, linear versus circular) and also in view of the methods used for producing and purifying these different objects, and also in view of their DNA methylation which is of prokaryotic type for ceDNA-plasmids and of eukaryotic type for the ceDNA vector. 107 ME146907648v.1 Provided herein are non-viral, capsid-free ceDNA molecules with covalently closed ends (ceDNA). These non-viral capsid free ceDNA molecules can be produced in permissive host cells from an expression construct (e.g., a ceDNA-plasmid, a ceDNA-bacmid, a ceDNA-baculovirus, or an integrated cell-line) containing a heterologous gene (e.g., a transgene, in particular a therapeutic transgene) positioned between two different inverted terminal repeat (ITR) sequences, where the ITRs are different with respect to each other. In some embodiments, one of the ITRs is modified by deletion, insertion, and/or substitution as compared to a wild-type ITR sequence (e.g., AAV ITR); and at least one of the ITRs comprises a functional terminal resolution site (trs) and a Rep binding site. The ceDNA vector is preferably duplex, e.g., self-complementary, over at least a portion of the molecule, such as the expression cassette (e.g., ceDNA is not a double stranded circular molecule). The ceDNA vector has covalently closed ends, and thus is resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III), e.g., for over an hour at 37°C. In one aspect, a ceDNA vector comprises, in the 5’ to 3’ direction: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (for example an expression cassette as described herein) and a second AAV ITR. In one embodiment, the first ITR (5’ ITR) and the second ITR (3’ ITR) are asymmetric with respect to each other - that is, they have a different 3D-spatial configuration from one another. As an exemplary embodiment, the first ITR can be a wild-type ITR and the second ITR can be a mutated or modified ITR, or vice versa, where the first ITR can be a mutated or modified ITR and the second ITR a wild- type ITR. In one embodiment, the first ITR and the second ITR are both modified but are different sequences, or have different modifications, or are not identical modified ITRs, and have different 3D spatial configurations. Stated differently, a ceDNA vector with asymmetric ITRs have ITRs where any changes in one ITR relative to the WT-ITR are not reflected in the other ITR; or alternatively, where the asymmetric ITRs have a the modified asymmetric ITR pair can have a different sequence and different three-dimensional shape with respect to each other. In one embodiment, a ceDNA vector comprises, in the 5’ to 3’ direction: a first adeno- associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (for example an expression cassette as described herein) and a second AAV ITR, where the first ITR (5’ ITR) and the second ITR (3’ ITR) are symmetric, or substantially symmetrical with respect to each other - that is, a ceDNA vector can comprise ITR sequences that have a symmetrical three-dimensional spatial organization such that their structure is the same shape in geometrical space, or have the same A, C- C’ and B-B’ loops in 3D space. In such an embodiment, a symmetrical ITR pair, or substantially symmetrical ITR pair can be modified ITRs (e.g., mod-ITRs) that are not wild-type ITRs. A mod- ITR pair can have the same sequence which has one or more modifications from wild-type ITR and are reverse complements (inverted) of each other. In one embodiment, a modified ITR pair are substantially symmetrical as defined herein, that is, the modified ITR pair can have a different sequence but have corresponding or the same symmetrical three-dimensional shape. In some 108 ME146907648v.1 embodiments, the symmetrical ITRs, or substantially symmetrical ITRs can be wild type ITRs (WT- ITRs) as described herein. That is, both ITRs have a wild-type sequence from the same AAV serotype. In some other embodiments, the two wild-type ITRs are from different AAV serotypes. For example, one WT-ITR can be derived from one AAV serotype, and the other WT-ITR can be derived from a different AAV serotype. In such an embodiment, a WT-ITR pair are substantially symmetrical as defined herein, that is, they can have one or more conservative nucleotide modification while still retaining the symmetrical three-dimensional spatial organization. The wild-type or mutated or otherwise modified ITR sequences provided herein represent DNA sequences included in the expression construct (e.g., ceDNA-plasmid, ceDNA Bacmid, ceDNA- baculovirus) for production of the ceDNA vector. Thus, ITR sequences actually contained in the ceDNA vector produced from the ceDNA-plasmid or other expression construct may or may not be identical to the ITR sequences provided herein as a result of naturally occurring changes taking place during the production process (e.g., replication error). In one embodiment, a ceDNA vector in the LNPs of the present disclosure comprising the expression cassette with a transgene which is a therapeutic nucleic acid sequence, can be operatively linked to one or more regulatory sequence(s) that allows or controls expression of the transgene. In one embodiment, the polynucleotide comprises a first ITR sequence and a second ITR sequence, wherein the nucleotide sequence of interest is flanked by the first and second ITR sequences, and the first and second ITR sequences are asymmetrical relative to each other, or symmetrical relative to each other. In one embodiment, an expression cassette is located between two ITRs in the following order with one or more of: a promoter operably linked to a transgene, a posttranscriptional regulatory element, and a polyadenylation and termination signal. In one embodiment, the promoter is regulatable - inducible or repressible. The promoter can be any sequence that facilitates the transcription of the transgene. In one embodiment the promoter is a CAG promoter, or variation thereof. The posttranscriptional regulatory element is a sequence that modulates expression of the transgene, as a non-limiting example, any sequence that creates a tertiary structure that enhances expression of the transgene which is a therapeutic nucleic acid sequence. In one embodiment, the posttranscriptional regulatory element comprises WPRE. In one embodiment, the polyadenylation and termination signal comprise BGHpolyA. Any cis regulatory element known in the art, or combination thereof, can be additionally used e.g., SV40 late polyA signal upstream enhancer sequence (USE), or other posttranscriptional processing elements including, but not limited to, the thymidine kinase gene of herpes simplex virus, or hepatitis B virus (HBV). In one embodiment, the expression cassette length in the 5’ to 3’ direction is greater than the maximum length known to be encapsidated in an AAV virion. In one embodiment, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb. Various expression cassettes are exemplified herein. 109 ME146907648v.1 In one embodiment, the expression cassette can comprise more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides or 50,000 nucleotides, or any range between about 4000-10,000 nucleotides or 10,000-50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 50,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 75,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 10,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 1000 to 10,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 5,000 nucleotides in length. The ceDNA vectors do not have the size limitations of encapsidated AAV vectors, and thus enable delivery of a large-size expression cassette to the host. In one embodiment, the ceDNA vector is devoid of prokaryote-specific methylation. In one embodiment, the rigid therapeutic nucleic acid can be a plasmid. In one embodiment, the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., for medical, diagnostic, or veterinary uses) or immunogenic polypeptides. The expression cassette can comprise any transgene which is a therapeutic nucleic acid sequence. In certain embodiments, the ceDNA vector comprises any gene of interest in the subject, which includes one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen binding fragments, or any combination thereof. In one embodiment, the ceDNA expression cassette can include, for example, an expressible exogenous sequence (e.g., open reading frame) that encodes a protein that is either absent, inactive, or insufficient activity in the recipient subject or a gene that encodes a protein having a desired biological or a therapeutic effect. In one embodiment, the exogenous sequence such as a donor sequence can encode a gene product that can function to correct the expression of a defective gene or transcript. In one embodiment, the expression cassette can also encode corrective DNA strands, encode polypeptides, sense or antisense oligonucleotides, or coding RNAs or non-coding RNAs (e.g., siRNAs, guide RNAs, shRNAs, micro-RNAs, and their antisense counterparts (e.g., antagoMiR)). In one embodiment, expression cassettes can include an exogenous sequence that encodes a reporter protein to be used for experimental or diagnostic purposes, such as b-lactamase, b-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art. Accordingly, the expression cassette can include any gene that encodes a protein, polypeptide or RNA that is either reduced or absent due to a mutation or which conveys a therapeutic benefit 110 ME146907648v.1 when overexpressed is considered to be within the scope of the disclosure. The ceDNA vector may comprise a template or donor nucleotide sequence used as a correcting DNA strand to be inserted after a double-strand break (or nick) provided by a nuclease. The ceDNA vector may include a template nucleotide sequence used as a correcting DNA strand to be inserted after a double-strand break (or nick) provided by a guided RNA nuclease, meganuclease, or zinc finger nuclease. III. Production of a ceDNA Vector Methods for the production of a ceDNA vector as described herein comprising an asymmetrical ITR pair or symmetrical ITR pair as defined herein is described in section IV of PCT/US 18/49996 filed September 7, 2018, which is incorporated herein in its entirety by reference. As described herein, the ceDNA vector can be obtained, for example, by the process comprising the steps of: a) incubating a population of host cells (e.g. insect cells) harboring the polynucleotide expression construct template (e.g., a ceDNA-plasmid, a ceDNA-Bacmid, and/or a ceDNA- baculovirus), which is devoid of viral capsid coding sequences, in the presence of a Rep protein under conditions effective and for a time sufficient to induce production of the ceDNA vector within the host cells, and wherein the host cells do not comprise viral capsid coding sequences; and b) harvesting and isolating the ceDNA vector from the host cells. The presence of Rep protein induces replication of the vector polynucleotide with a modified ITR to produce the ceDNA vector in a host cell. The following is provided as a non-limiting example. According to some embodiments, synthetic ceDNA is produced via excision from a double- stranded DNA molecule. Synthetic production of the ceDNA vectors is described in Examples 2-6 of International Application PCT/US19/14122, filed January 18, 2019, which is incorporated herein in its entirety by reference. One exemplary method of producing a ceDNA vector using a synthetic method that involves the excision of a double-stranded DNA molecule. In brief, a ceDNA vector can be generated using a double stranded DNA construct, e.g., see FIGS.7A-8E of PCT/US19/14122. In some embodiments, the double stranded DNA construct is a ceDNA plasmid, e.g., see, e.g., FIG.6 in International patent application PCT/US2018/064242, filed December 6, 2018). In some embodiments, a construct to make a ceDNA vector comprises additional components to regulate expression of the transgene, for example, regulatory switches, to regulate the expression of the transgene, or a kill switch, which can kill a cell comprising the vector. A molecular regulatory switch is one which generates a measurable change in state in response to a signal. Such regulatory switches can be usefully combined with the ceDNA vectors described herein to control the output of expression of the transgene. In some embodiments, the ceDNA vector comprises a regulatory switch that serves to fine tune expression of the transgene. For example, it can serve as a biocontainment function of the ceDNA vector. In some embodiments, the switch is an “ON/OFF” switch that is designed to start or stop (i.e., shut down) expression of the gene of interest in the ceDNA vector in a controllable and regulatable fashion. In some embodiments, the 111 ME146907648v.1 switch can include a “kill switch” that can instruct the cell comprising the synthetic ceDNA vector to undergo cell programmed death once the switch is activated. Exemplary regulatory switches encompassed for use in a ceDNA vector can be used to regulate the expression of a transgene, and are more fully discussed in International application PCT/US18/49996, which is incorporated herein in its entirety by reference and described herein. Another exemplary method of producing a ceDNA vector using a synthetic method that involves assembly of various oligonucleotides, is provided in Example 3 of PCT/US19/14122, where a ceDNA vector is produced by synthesizing a 5’ oligonucleotide and a 3’ ITR oligonucleotide and ligating the ITR oligonucleotides to a double-stranded polynucleotide comprising an expression cassette. FIG.11B of PCT/US19/14122, incorporated by reference in its entirety herein, shows an exemplary method of ligating a 5’ ITR oligonucleotide and a 3’ ITR oligonucleotide to a double stranded polynucleotide comprising an expression cassette. An exemplary method of producing a ceDNA vector using a synthetic method is provided in Example 4 of PCT/US19/14122, incorporated by reference in its entirety herein, and uses a single- stranded linear DNA comprising two sense ITRs which flank a sense expression cassette sequence and are attached covalently to two antisense ITRs which flank an antisense expression cassette, the ends of which single stranded linear DNA are then ligated to form a closed-ended single-stranded molecule. One non-limiting example comprises synthesizing and/or producing a single-stranded DNA molecule, annealing portions of the molecule to form a single linear DNA molecule which has one or more base-paired regions of secondary structure, and then ligating the free 5’ and 3’ ends to each other to form a closed single-stranded molecule. In yet another aspect, the disclosure provides for host cell lines that have stably integrated the DNA vector polynucleotide expression template (ceDNA template) described herein, into their own genome for use in production of the non-viral DNA vector. Methods for producing such cell lines are described in Lee, L. et al. (2013) Plos One 8(8): e69879, which is herein incorporated by reference in its entirety. For example, the Rep protein is added to host cells at an MOI of 3. In one embodiment, the host cell line is an invertebrate cell line, preferably insect Sf9 cells. When the host cell line is a mammalian cell line, preferably 293 cells the cell lines can have polynucleotide vector template stably integrated, and a second vector, such as herpes virus can be used to introduce Rep protein into cells, allowing for the excision and amplification of ceDNA in the presence of Rep. In one embodiment, the capsid free non-viral DNA vector comprises or is obtained from a plasmid comprising a polynucleotide template comprising in this order: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (for example an expression cassette of an exogenous DNA) and a modified AAV ITR, wherein said template nucleic acid molecule is devoid of AAV capsid protein coding. In a further embodiment, the nucleic acid template of the disclosure is devoid of viral capsid protein coding sequences (i.e., it is devoid of AAV capsid genes but also of capsid genes of other viruses). In addition, in a particular embodiment, the template 112 ME146907648v.1 nucleic acid molecule is also devoid of AAV Rep protein coding sequences. Accordingly, in a preferred embodiment, the nucleic acid molecule of the disclosure is devoid of both functional AAV cap and AAV rep genes. In one embodiment, ceDNA can include an ITR structure that is mutated with respect to the wild type AAV2 ITR disclosed herein, but still retains an operable RBE, TRS and RBE´ portion. ceDNA Plasmid A ceDNA-plasmid is a plasmid that contains ceDNA sequences and can be used for later production of a ceDNA vector. In one embodiment, a ceDNA-plasmid can be constructed using known techniques to provide at least the following as operatively linked components in the direction of transcription: (1) a modified 5’ ITR sequence; (2) an expression cassette containing a cis-regulatory element, for example, a promoter, inducible promoter, regulatory switch, enhancers and the like; and (3) a modified 3’ ITR sequence, where the 3’ ITR sequence is symmetric relative to the 5’ ITR sequence. In some embodiments, the expression cassette flanked by the ITRs comprises a cloning site for introducing an exogenous sequence. The expression cassette replaces the rep and cap coding regions of the AAV genomes and ceDNA can be used as an expression vector. In one embodiment, a ceDNA vector is obtained from a plasmid, referred to herein as a “ceDNA-plasmid” encoding in this order: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), an expression cassette comprising a transgene, and a mutated or modified AAV ITR, wherein said ceDNA-plasmid is devoid of AAV capsid protein coding sequences. In alternative embodiments, the ceDNA-plasmid encodes in this order: a first (or 5’) modified or mutated AAV ITR, an expression cassette comprising a transgene, and a second (or 3’) modified AAV ITR, wherein said ceDNA-plasmid is devoid of AAV capsid protein coding sequences, and wherein the 5’ and 3’ ITRs are symmetric relative to each other. In alternative embodiments, the ceDNA-plasmid encodes in this order: a first (or 5’) modified or mutated AAV ITR, an expression cassette comprising a transgene, and a second (or 3’) mutated or modified AAV ITR, wherein said ceDNA-plasmid is devoid of AAV capsid protein coding sequences, and wherein the 5’ and 3’ modified ITRs have the same modifications (i.e., they are inverse complement or symmetric relative to each other). In one embodiment, the ceDNA-plasmid system is devoid of viral capsid protein coding sequences (i.e., it is devoid of AAV capsid genes but also of capsid genes of other viruses). In one embodiment, the ceDNA-plasmid is also devoid of AAV Rep protein coding sequences. In one embodiment, ceDNA-plasmid is devoid of functional AAV cap and AAV rep genes GG-3’ for AAV2) plus a variable palindromic sequence allowing for hairpin formation. IV. Preparation of Lipid Nanoparticles (LNPs) Lipid nanoparticles (LNPs) can form spontaneously upon mixing of ceDNA and the lipid(s). Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded 113 ME146907648v.1 through a membrane (e.g., 100 nrn cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. In one embodiment, the lipid nanoparticles are formed from mixing a high concentration organic (EtOH) mixture of lipid components and TNA with an aqueous buffer as described in WO 2022/016089, the content of which is incorporated herein by reference in its entirety. Generally, LNPs can be formed by any method known in the art. For example, the LNPs can be prepared by the methods described, for example, in US2013/0037977, US2010/0015218, US2013/0156845, US2013/0164400, US2012/0225129, and US2010/0130588, content of each of which is incorporated herein by reference in its entirety. In some embodiments, LNPs can be prepared using a continuous mixing method, a direct dilution process, or an in-line dilution process. The processes and apparatuses for apparatuses for preparing lipid nanoparticles using direct dilution and in-line dilution processes are described in US2007/0042031, the content of which is incorporated herein by reference in its entirety. The processes and apparatuses for preparing lipid nanoparticles using step-wise dilution processes are described in US2004/0142025, the content of which is incorporated herein by reference in its entirety. According to some embodiments, the disclosure provides for an LNP comprising a DNA vector, including a ceDNA vector as described herein and an ionizable lipid. For example, a lipid nanoparticle formulation that is made and loaded with therapeutic nucleic acid like ceDNA obtained by the process as disclosed in International Patent Application No. PCT/US2018/050042, filed on September 7, 2018, which is incorporated by reference in its entirety herein. In one embodiment, the LNPs can be prepared by an impinging jet process. Generally, the particles are formed by mixing lipids dissolved in alcohol (e.g., ethanol) with ceDNA dissolved in a buffer, e.g., a citrate buffer, a sodium acetate buffer, a sodium acetate and magnesium chloride buffer, a malic acid buffer, a malic acid and sodium chloride buffer, or a sodium citrate and sodium chloride buffer. The mixing ratio of lipids to ceDNA can be about 45-55% lipid and about 65-45% ceDNA. The lipid solution can contain an ionizable lipid, a lipid-anchored polymer and a sterol (e.g., cholesterol) at a total lipid concentration of 5-30 mg/mL, more likely 5-15 mg/mL, most likely 9-12 mg/mL in an alcohol, e.g., in ethanol. In the lipid solution, mol ratio of the lipids can range from about 25-98% for the cationic lipid, preferably about 35-65%; about 0-15% for the non-ionic lipid, preferably about 0-12%; about 0-15% for the PEG or PEG conjugated lipid molecule, preferably about 1-6%; and about 0-75% for the sterol, preferably about 30-50%. The ceDNA solution can comprise the ceDNA at a concentration range from 0.3 to 1.0 mg/mL, preferably 0.3-0.9 mg/mL in buffered solution, with pH in the range of 3.5-5. For forming the LNPs, in one exemplary but nonlimiting embodiment, the two liquids are heated to a temperature in the range of about 15-40°C, preferably about 30-40°C, and then mixed, for example, in an impinging jet mixer, instantly forming the LNP. The mixing flow rate can range from 114 ME146907648v.1 10-600 mL/min. The tube ID can have a range from 0.25 to 1.0 mm and a total flow rate from 10-600 mL/min. The combination of flow rate and tubing ID can have the effect of controlling the particle size of the LNPs between 30 and 200 nm. The solution can then be mixed with a buffered solution at a higher pH with a mixing ratio in the range of 1:1 to 1:3 vol:vol, preferably about 1:2 vol:vol. If needed this buffered solution can be at a temperature in the range of 15-40°C or 30-40°C. The mixed LNPs can then undergo an anion exchange filtration step. Prior to the anion exchange, the mixed LNPs can be incubated for a period of time, for example 30mins to 2 hours. The temperature during incubating can be in the range of 15-40°C or 30-40°C. After incubating the solution is filtered through a filter, such as a 0.8µm filter, containing an anion exchange separation step. This process can use tubing IDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000 mL/min. After formation, the LNPs can be concentrated and diafiltered via an ultrafiltration process where the alcohol is removed and the buffer is exchanged for the final buffer solution, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. The ultrafiltration process can use a tangential flow filtration format (TFF) using a membrane nominal molecular weight cutoff range from 30-500 kD. The membrane format is hollow fiber or flat sheet cassette. The TFF processes with the proper molecular weight cutoff can retain the LNP in the retentate and the filtrate or permeate contains the alcohol; citrate buffer and final buffer wastes. The TFF process is a multiple step process with an initial concentration to a ceDNA concentration of 1-3 mg/mL. Following concentration, the LNPs solution is diafiltered against the final buffer for 10-20 volumes to remove the alcohol and perform buffer exchange. The material can then be concentrated an additional 1-3-fold. The concentrated LNP solution can be sterile filtered. V. Pharmaceutical Compositions and Formulations The present disclosure also provides a pharmaceutical composition comprising the LNPs of the present disclosure and at least one pharmaceutically acceptable excipient. According to some embodiments, the TNA (e.g., ceDNA) is encapsulated in the LNP. In one embodiment, the LNPs of the disclosure are provided with full encapsulation, partial encapsulation of the therapeutic nucleic acid. In one embodiment, the nucleic acid therapeutics is fully encapsulated in the LNPs to form a nucleic acid containing lipid particle. In one embodiment, the nucleic acid may be encapsulated within the lipid portion of the particle, thereby protecting it from enzymatic degradation. In one embodiment, encapsulation of TNA (e.g., ceDNA) in the LNPs of the present disclosure can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid, for example, an Oligreen® assay or PicoGreen® assay. Generally, encapsulation is determined by adding the dye to the lipid particle formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent. Detergent- 115 ME146907648v.1 mediated disruption of the lipid bilayer releases the encapsulated TNA (e.g., ceDNA), allowing it to interact with the membrane-impermeable dye. Encapsulation of ceDNA can be calculated as E= (Io - I)/Io, where I and Io refer to the fluorescence intensities before and after the addition of detergent. Depending on the intended use of the LNPs, the proportions of the components can vary and the delivery efficiency of a particular formulation can be measured using, for example, an endosomal release parameter (ERP) assay. In one embodiment, the TNA (e.g., ceDNA) can be complexed with the lipid portion of the particle or encapsulated in the lipid position of the lipid particle (e.g., lipid nanoparticle). In one embodiment, the TNA can be fully encapsulated in the lipid position of the lipid particle (e.g., lipid nanoparticle), thereby protecting it from degradation by a nuclease, e.g., in an aqueous solution. In one embodiment, the TNA in the lipid particle (e.g., lipid nanoparticle) is not substantially degraded after exposure of the lipid particle (e.g., lipid nanoparticle) to a nuclease at 37°C. for at least about 20, 30, 45, or 60 minutes. In some embodiments, the TNA in the lipid particle (e.g., lipid nanoparticle) is not substantially degraded after incubation of the particle in serum at 37°C. for at least about 30, 45, or 60 minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In one embodiment, the LNPs are substantially non-toxic to a subject, e.g., to a mammal such as a human. In one embodiment, the pharmaceutical composition comprising LNPs of the disclosure is an aqueous solution. In one embodiment, the pharmaceutical compostion comprising LNPs of the disclosure is a lyophilized powder. According to some aspects, the at least one pharmaceutically acceptable excipient in the pharmaceutical compositions of the present disclosure is a sucrose, tris, trehalose and/or glycine. In one embodiment, the pharmaceutical compositions comprising LNPs of the disclosure are suitable for administration to a subject for in vivo delivery to cells, tissues, or organs of the subject. In some embodiments, the pharmaceutical composition is suitable for a desired route of therapeutic administration (e.g., parenteral administration). The pharmaceutical compositions for therapeutic purposes can be formulated as a solution, microemulsion, dispersion, liposomes, or other ordered structure suitable for high TNA (e.g., ceDNA) vector concentration. Sterile injectable solutions can be prepared by incorporating the TNA (e.g., ceDNA) vector compound in the required amount in an appropriate buffer with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Pharmaceutical compositions comprising LNPs of the disclosure are suitable for topical, systemic, intra-amniotic, intrathecal, intracranial, intraarterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intra-tissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (e.g., extra-orbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, sub-choroidal, intrastromal, intracameral and 116 ME146907648v.1 intravitreal), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via high pressure intravenous or intraarterial infusion, as well as intracellular injection, such as intranuclear microinjection or intracytoplasmic injection, are also contemplated. Pharmaceutical compositions for therapeutic purposes typically must be sterile and stable under the conditions of manufacture and storage. In one embodiment, LNPs are solid core particles that possess at least one lipid bilayer. In one embodiment, the LNPs have a non-bilayer structure, i.e., a non-lamellar (i.e., non-bilayer) morphology. Without limitations, the non-bilayer morphology can include, for example, three dimensional tubes, rods, cubic symmetries, etc. The non-lamellar morphology (i.e., non-bilayer structure) of the LNPs can be determined using analytical techniques known to and used by those of skill in the art. Such techniques include, but are not limited to, Cryo-Transmission Electron Microscopy (“Cryo-TEM”), Differential Scanning calorimetry (“DSC”), X-Ray Diffraction, and the like. For example, the morphology of the lipid particles (lamellar vs. non-lamellar) can readily be assessed and characterized using, e.g., Cryo-TEM analysis as described in US2010/0130588, the content of which is incorporated herein by reference in its entirety. In one embodiment, the LNPs having a non-lamellar morphology are electron dense. In one embodiment, the LNPs provided by the present disclosure is either unilamellar or multilamellar in structure. In some aspects, the pharmaceutical composition of the disclosure comprises multi-vesicular particles and/or foam-based particles. By controlling the composition and concentration of the lipid components, one can control the rate at which the lipid conjugate exchanges out of the lipid particle and, in turn, the rate at which the LNP becomes fusogenic. In addition, other variables including, for example, pH, temperature, or ionic strength, can be used to vary and/or control the rate at which the LNP becomes fusogenic. Other methods which can be used to control the rate at which the LNP becomes fusogenic will be apparent to those of ordinary skill in the art based on this disclosure. It will also be apparent that by controlling the composition and concentration of the lipid conjugate, one can control the lipid particle size. According to some embodiments, for ophthalmic delivery, interfering RNA-ligand conjugates and nanoparticle-ligand conjugates may be combined with ophthalmologically acceptable preservatives, co-solvents, surfactants, viscosity enhancers, penetration enhancers, buffers, sodium chloride, or water to form an aqueous, sterile ophthalmic suspension or solution. Unit Dosage In one embodiment, the pharmaceutical compositions can be presented in unit dosage form. A unit dosage form will typically be adapted to one or more specific routes of administration of the pharmaceutical composition. In some embodiments, the unit dosage form is adapted for administration by inhalation. In some embodiments, the unit dosage form is adapted for administration by a vaporizer. In some embodiments, the unit dosage form is adapted for administration by a nebulizer. In some embodiments, the unit dosage form is adapted for 117 ME146907648v.1 administration by an aerosolizer. In some embodiments, the unit dosage form is adapted for oral administration, for buccal administration, or for sublingual administration. In some embodiments, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, the unit dosage form is adapted for intrathecal or intracerebroventricular administration. In some embodiments, the pharmaceutical composition is formulated for topical administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. VI. Methods of Treatment In some aspects, the present disclosure provides methods of treating a disorder in a subject that comprise administering to the subject an effective amount of an LNP of the disclosure of the pharmaceutical composition comprising the LNP of the disclosure. In some embodiments, the disorder is a genetic disorder. As used herein, the term “genetic disease” or “genetic disorder” refers to a disease, partially or completely, directly or indirectly, caused by one or more abnormalities in the genome, including and especially a condition that is present from birth. The abnormality may be a mutation, an insertion or a deletion in a gene. The abnormality may affect the coding sequence of the gene or its regulatory sequence. Provided herein are methods for treating genetic disorders by administering the LNP of the disclosure or the pharmaceutical composition comprising LNPs of the disclosure. There are a number of inherited diseases in which defective genes are known, and typically fall into two classes: deficiency states, usually of enzymes, which are generally inherited in a recessive manner, and unbalanced states, which may involve regulatory or structural proteins, and which are typically but not always inherited in a dominant manner. For deficiency state diseases, the LNPs and LNP compositions of the disclosure can be used to deliver transgenes to bring a normal gene into affected tissues for replacement therapy, as well, in some embodiments of any of the aspects and embodiments herein, to create animal models for the disease using antisense mutations. For unbalanced disease states, the LNPs and LNP compositions of the disclosure can be used to create a disease state in a model system, which could then be used in efforts to counteract the disease state. Thus, the LNPs or LNP compositions of the disclosure and methods disclosed herein permit the treatment of genetic diseases. As used herein, a disease state is treated by partially or wholly remedying the deficiency or imbalance that causes the disease or makes it more severe. In general, the LNPs and LNP compositions of the disclosure can be used to deliver any transgene in accordance with the description above to treat, prevent, or ameliorate the symptoms associated with any disorder related to gene expression. Illustrative disease states include, but are not- limited to: cystic fibrosis (and other diseases of the lung), hemophilia A, hemophilia B, thalassemia, 118 ME146907648v.1 anemia and other blood disorders, AIDS, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, epilepsy, and other neurological disorders, cancer, diabetes mellitus, muscular dystrophies (e.g., Duchenne, Becker), Hurler’s disease, adenosine deaminase deficiency, metabolic defects, retinal degenerative diseases (and other diseases of the eye), mitochondriopathies (e.g., Leber’s hereditary optic neuropathy (LHON), Leigh syndrome, and subacute sclerosing encephalopathy), myopathies (e.g., facioscapulohumeral myopathy (FSHD) and cardiomyopathies), diseases of solid organs (e.g., brain, liver, kidney, heart), and the like. In some embodiments of any of the aspects and embodiments herein, the ceDNA vectors as disclosed herein can be advantageously used in the treatment of individuals with metabolic disorders (e.g., ornithine transcarbamylase deficiency). In one embodiment of any of the aspects or embodiments herein, the LNPs of the disclosure or the pharmaceutical compositions comrpsing the LNPs of the disclosure can be used to treat, ameliorate, and/or prevent a disease or disorder caused by mutation in a gene or gene product. Exemplary diseases or disorders that can be treated with the LNPs or the LNP compositions of the disclosure include, but are not limited to, metabolic diseases or disorders (e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage disease); urea cycle diseases or disorders (e.g., ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy (MLD), mucopolysaccharidosis Type II (MPSII; Hunter syndrome)); liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis (PFIC); blood diseases or disorders (e.g., hemophilia A and B, thalassemia, and anemia); cancers and tumors, and genetic diseases or disorders (e.g., cystic fibrosis). In one embodiment, the LNPs or LNP compositions of the disclosure may be employed to deliver a heterologous nucleotide sequence in situations in which it is desirable to regulate the level of transgene expression (e.g., transgenes encoding hormones or growth factors). In one embodiment of any of the aspects or embodiments herein, the LNPs or LNP compositions of the disclosure can be used to correct an abnormal level and/or function of a gene product (e.g., an absence of, or a defect in, a protein) that results in the disease or disorder. The LNPs or LNP compositions of the disclosure can produce a functional protein and/or modify levels of the protein to alleviate or reduce symptoms resulting from, or confer benefit to, a particular disease or disorder caused by the absence or a defect in the protein. For example, treatment of OTC deficiency can be achieved by producing functional OTC enzyme; treatment of hemophilia A and B can be achieved by modifying levels of Factor VIII, Factor IX, and Factor X; treatment of PKU can be achieved by modifying levels of phenylalanine hydroxylase enzyme; treatment of Fabry or Gaucher disease can be achieved by producing functional alpha galactosidase or beta glucocerebrosidase, respectively; treatment of MFD or MPSII can be achieved by producing functional arylsulfatase A or iduronate-2-sulfatase, respectively; treatment of cystic fibrosis can be achieved by producing functional cystic fibrosis transmembrane conductance regulator; treatment of glycogen storage disease 119 ME146907648v.1 can be achieved by restoring functional G6Pase enzyme function; and treatment of PFIC can be achieved by producing functional ATP8B1, ABCB11, ABCB4, or TJP2 genes. In some embodiments, the LNPs or LNP compositions of the disclosure can be used to provide a DNA-based therapeutic to a cell in vitro or in vivo. Examples of DNA-based therapeutics include, but are not limited to, minicircle DNA, minigene, viral DNA (e.g., Lentiviral or AAV genome) or non-viral synthetic DNA vectors, closed-ended linear duplex DNA (ceDNA/CELiD), plasmids, bacmids, doggybone™ DNA vectors, minimalistic immunological-defined gene expression (MIDGE)-vector, nonviral ministring DNA vector (linear-covalently closed DNA vector), or dumbbell-shaped DNA minimal vector (“dumbbell DNA”). In one embodiment of any of the aspects or embodiments herein, exemplary transgenes encoded by ceDNA in the LNPs or LNP compositions of the disclosure include, but are not limited to: X, lysosomal enzymes (e.g., hexosaminidase A, associated with Tay-Sachs disease, or iduronate sulfatase, associated, with Hunter Syndrome/MPS II), erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, as well as cytokines (e.g., a interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4, interleukin 12, granulocyte- macrophage colony stimulating factor, lymphotoxin, and the like), peptide growth factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors 1 and 2, platelet derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), neurotrophic factor-3 and 4, brain-derived neurotrophic factor (BDNF), glial derived growth factor (GDNF), transforming growth factor-a and -b, and the like), receptors (e.g., tumor necrosis factor receptor). In some exemplary embodiments, the transgene encodes a monoclonal antibody specific for one or more desired targets. In some exemplary embodiments, more than one transgene is encoded by the ceDNA vector. In some exemplary embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments of any of the aspects and embodiments herein, the transgene encodes an antibody, including a full-length antibody or antibody fragment, as defined herein. In some embodiments of any of the aspects and embodiments herein, the antibody is an antigen-binding domain or an immunoglobulin variable domain sequence, as that is defined herein. Other illustrative transgene sequences encode suicide gene products (thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, and tumor necrosis factor), proteins conferring resistance to a drug used in cancer therapy, and tumor suppressor gene products. In one embodiment of any of the aspects or embodiments herein, this disclosure provides a method of providing anti-tumor immunity in a subject, the method comprising administering to the subject an effective amount of any embodiment of an LNP contemplated herein or any embodiment of a pharmaceutical composition comprising an LNP contemplated herein. Furthermore, this disclosure provides a method of treating a subject having a disease, disorder or condition associated with an elevated expression of a tumor antigen, the method comprising administering to the subject an 120 ME146907648v.1 effective amount of any embodiment of an LNP contemplated herein or any embodiment of a pharmaceutical composition comprising an LNP contemplated herein. In some embodiments, the TNA is retained in the spleen for at least about 6 hours, or at least about 9 hours, or at least about 12 hours, or at least about 15 hours, or at least about 18 hours, or at least about 21 hours, or at least about 24 hours, or at least about 27 hours, or at least about 30 hours, or at least about 33 hours, or at least about 36 hours after dosing of an LNP of this disclosure, for example, via intravenous or intratumoral administration. In some embodiments, the amount (i.e., number of copies) of the TNA at the start of a 12, 18, or 24-hour time window post-dosing and the amount of the TNA at the end of the time window are within the same order of magnitude (e.g., 10-7 copies, 10-6 copies, 10-5 copies, 10-4 copies, 10-3 copies, 10-2 copies, 10-1 copies, 100 copies, 101 copies, 102 copies, 103 copies, etc. or any other suitable therapeutic levels). In other words, there is minimal reduction in concentrations of the TNA in the spleen within a 12, 18, or 24-hour time window post-dosing. In some embodiments, the TNA is a messenger RNA (mRNA). Examples of solid tumors treatable with an LNP disclosed herein or a pharmaceutical composition comprising the same include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g., colon), genitourinary tract (e.g., renal, urothelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. According to some embodiments, the tumor or cancer is a melanoma, e.g., an advanced stage melanoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the disclosure. Examples of other solid tumors or cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers. In further embodiments, the present disclosure provides a method of treating a blood disease, disorder or condition in a subject, the method comprising administering to the subject an effective amount of any embodiment of an LNP contemplated herein or any embodiment of a pharmaceutical composition comprising an LNP contemplated herein. Non-limiting examples of the blood disease, 121 ME146907648v.1 disorder or condition include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Hodgkin lymphoma (HL), multiple myeloma, a myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), adrenoleukodystrophy (ALD), Hurler syndrome, Krabbe disease (Globoid-cell leukodystrophy or GLD), metachromatic leukodystrophy (MLD), severe aplastic anemia (SAA), severe combined immunodeficiency (SCID), sickle cell disease (SCD), thalassemia, Wiskott-Aldrich syndrome, Diamond-Blackfan anemia, essential thrombocytosis, Fanconi anemia, hemophagocytic lymphohistiscytosis (HLH), juvenile myelomonocytic leukemia (JMML), myelofibrosis, polycythemia vera, and a combination thereof. In some embodiments, the TNA is a messenger RNA (mRNA). In some embodiments, the TNA is retained in the bone marrow for at least about 6 hours, or at least about 9 hours, or at least about 12 hours, or at least about 15 hours, or at least about 18 hours, or at least about 21 hours, or at least about 24 hours, or at least about 27 hours, or at least about 30 hours, or at least about 33 hours, or at least about 36 hours after dosing of an LNP of this disclosure, for example, via intravenous or intratumoral administration. In some embodiments, the amount (i.e. number of copies) of the TNA at the start of a 12, 18, or 24-hour time window post-dosing and the number of the TNA at the end of the time window are within the same order of magnitude (e.g., 10-7 copies, 10-6 copies, 10-5 copies, 10-4 copies, 10-3 copies, 10-2 copies, 10-1 copies, 100 copies, 101 copies, 102 copies, 103 copies, etc. or any other suitable therapeutic levels) or are reduced for less than one order of magnitude. In other words, there is minimal or insignificant reduction in concentrations of the TNA in the bone marrow within a 12, 18, or 24-hour time window post-dosing. Administration In some embodiments, an LNP or an LNP composition of the disclosure can be administered to an organism for transduction of cells in vivo. In some embodiments, an LNP or an LNP composition of the disclosure can be administered to an organism for transduction of cells ex vivo. Generally, administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. Exemplary modes of administration of an LNP or an LNP composition of the disclosure include oral, rectal, transmucosal, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intraendothelial, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular [including administration to skeletal, diaphragm and/or cardiac muscle], intrapleural, intracerebral, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, and the like, as well as direct tissue or organ injection (e.g., to liver, eye, skeletal muscle, cardiac muscle, diaphragm muscle or brain). 122 ME146907648v.1 Administration of the LNP or LNP compositions of the disclosure can be to any site in a subject, including, without limitation, a site selected from the group consisting of the brain, a skeletal muscle, a smooth muscle, the heart, the diaphragm, the airway epithelium, the liver, the kidney, the spleen, the pancreas, the skin, and the eye. The most suitable route in any given case will depend on the nature and severity of the condition being treated, ameliorated, and/or prevented and on the nature of the particular ceDNA LNP that is being used. Additionally, ceDNA permits one to administer more than one transgene in a single vector, or multiple ceDNA vectors (e.g., a ceDNA cocktail). In one embodiment of any of the aspects or embodiments herein, the LNPs or LNP compositions of the disclosure can be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intra-ocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra-aural, intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon’s region) delivery as well as intramuscular delivery with retrograde delivery to motor neurons. In some embodiments, the LNPs of the disclosure or the pharmaceutical compositions comprising the LNPs of the disclosure, when administered to a subject, is characterized by a lower immunogenicity than a reference LNP or a pharmaceutical composition comprising a reference LNP. In some embodiments, the immunogenicity of the LNP of the disclosure or the pharmaceutical composition comprising the LNP of the disclosure may be measured by measuring levels of one or more proinflammatory cytokines. Accordingly, in some embodiments, the LNPs of the disclosure or the pharmaceutical compositions comprising the LNPs of the disclosure, when administered to a subject, elicits a lower pro-inflammatory cytokine respose than a reference LNP or a pharmaceutical composition comprising a reference LNP. The term “elicits a lower pro-inflammatory cytokine response than a reference LNP or a pharmaceutical composition comprising a reference LNP”, as used herein, means that the LNP of the disclosure or the pharmaceutical composition comprising the LNP of the disclosure, when administered to a subject, causes a smaller increase in the levels of one or more pro-inflammatory cytokines as compared to a reference LNP or a pharmaceutical composition comprising a reference LNP. Exemplary pro-inflammatory cytokines include, but are not limited to, granulocyte colony stimulating factor (G-CSF), interleukin 1 alpha (IL-1α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8 or CXCL8), interleukin 11 (IL-11), interleukin 17 (IL-17), interleukin 18 (IL-18), interferon α (IFN-α), interferon β (IFN-β), interferon γ (IFN-γ), C-X-C motif chemokine ligand 10 (CXCL10 or IP-10), monocyte chemoattractant protein 1 (MCP-1), CD40L, CCL2, CCL3, CCL4, CCL5, CCL11, tumor necrosis factor α (TNF-α), and combinations thereof. In some embodiments, the reference LNP is an LNP that comprises a helper lipid; and /or does not comprise the first lipid-anchored polymer and comprises a reference lipid-anchored polymer comprising one or more hydrophobic tails that each independently comprise less than 16 carbon atoms in a single aliphatic chain backbone. In one embodiment, the reference LNP comprises an ionizable lipid, DSPC, cholesterol, a lipid-anchored polymer comprising PEG attached to a lipid 123 ME146907648v.1 moiety which has two hydrophobic tails, each comprising of 14 carbon atoms (e.g., DMG-PEG2000, see below for the structure)
Figure imgf000125_0001
EXAMPLES The following examples are provided by way of illustration not limitation. Example 1. LNP formulations containing no helper lipid and containing C18 lipid-anchored polymer have physicochemical properties that are suitable for therapeutic use Exemplary LNP formulations of the present disclosure, e.g., containing no helper lipid and containing one or two C18 lipid-anchored polymers, were prepared using the methods as described herein, using a ceDNA vector having a luciferase transgene as the nucleic acid cargo. Control LNP A that contains DSPC as a helper lipid were also prepared and the physicochemical properties of all control LNP and test LNP formulations (i.e., LNP1, LNP2, LNP3, LNP4, LNP5) with varying amounts of cholesterol were compared (see Table 8). Table 8. Physicochemical properties of Control LNP A and test LNP formulations with varying amounts of cholesterol
Figure imgf000125_0002
124 ME146907648v.1
Figure imgf000126_0001
As can be seen in the formulation analytics in Table 8, all of the test LNP formulations (i.e., LNP1, LNP2, LNP3, LNP4, LNP5), which lack a helper lipid such as DSPC, were prepared successfully with an encapsulation efficiency of greater than 90%. This is surprising because DSPC is known to function to enhance encapsulation efficiency in liposomes and lipid nanoparticles. Moreover, it was surprisingly found that by increasing the amounts of cholesterol included in these LNP formulations, smaller average particle sizes could be achieved. Smaller particles with high encapsulation efficiencies are generally desirable and considered to be suitable for therapeutic use, due to the fenestration size of the target organ such as the liver and specifically, the hepatocytes. It was surprisingly found that LNP formulations containing no helper lipid (e.g., DSPC) and containing greater than 30 mol% of cholesterol each had an average diameter of < 100 nm. Of note, at 39.5 mol% and 44.5 mol% of cholesterol respectively, LNP4 and LNP5 had average diameters that were equivalent or smaller than the average diameter of the Control LNP A, and both LNP4 and LNP5 exhibited the highest encapsulation efficiencies among the test formulations that were comparable to the encapsulation efficiency of Control LNP A. When formulated as a different batch, LNP4 had an average diameter of < 80 nm (see Table 10 in Example 2). Example 2. LNP formulations containing no helper lipid and containing C18 lipid- anchored polymer reduced immunogenicity in mice The goal of this experiment was to demonstrate tolerability of the exemplary LNPs of the present disclosure, which are devoid of helper lipid. LNP formulations (with ceDNA vector having a luciferase transgene as the nucleic acid cargo) were prepared using ionizable lipid (e.g., Ionizable Lipid 87 and another Ionizable Lipid Z, cholesterol, and first lipid-anchored polymer (DSG-PEG- OMe or DODA-PG or bis-DSG-PEG which are all C18, i.e., having two hydrophobic tails that each contain 18 carbon atoms) to examine the effect of helper lipids on tolerability. The structure of Ionizable Lipid 87 is shown in Table 6. Ionizable Lipid Z (structure not shown) belongs to a different class of ionizable lipids compared to Ionizable Lipid 87, where both the headgroup and lipid tail moieties are structurally different from those of Ionizable Lipid 87. For control, LNPs having DSPC as a helper lipid were employed. Table 9 provides formulation analytics of the resultant LNPs. Table 9. Physicochemical properties of Control LNP B and test LNP formulations having different ionizable lipids 125 ME146907648v.1
Figure imgf000127_0001
* C14 PEG2000 Lipid is a lipid-anchored polymer having two hydrophobic tails that each contain 14 carbon atoms, conjugated to PEG2000. The resultant LNPs listed in Table 9 which encapsulate ceDNA nucleic acid comprising a luciferase reporter construct were administered to CD-1 mice intravenously (IV) at a dose of 0.5 mg/kg and 2.0 mg/kg (0 day). As shown in FIG.1, all LNPs formulated without helper lipid and with C18 lipid-anchored polymers exhibited equivalent or slightly improved body weight loss profiles to Control LNP A at Day 1 post-dosing and elicited no significant adverse reaction. The immunogenicity profiles of LNP4, LNP6, LNP7, and Control LNP B were compared by analyzing, at 6 hours post-dosing, the blood serum levels of multiple types of cytokines implicated in the regulation of innate immune response, i.e., IFN-α, IL-6, IFN-γ, TNF-α, IL-18, and IP-10. As shown in FIGs.2A-2F, the LNP formulations that were devoid of helper lipid and that contained C18 lipid-anchored polymers exhibited an overall reduction in cytokine levels as compared to the DSPC- and C14 PEG2000 Lipid-containing Control LNP B. This reduction was more pronounced in LNP formulations containing Ionizable Lipid 87 as the ionizable lipid and in particular, LNP4 that contains DSG-PEG2000-OMe as a lipid-anchored polymer. For example, LNP4 exhibited reduction in all six tested cytokines, as compared to Control LNP B. Overall, the results in FIGs.2A-2F indicate that LNP formulations of this disclosure that contain no helper lipid and contain a lipid-anchored polymer having hydrophobic tails that are longer than 14 carbon atoms may mitigate pro-inflammatory immune responses as compared to a conventional LNP formulation that contains a lipid-anchored polymer having hydrophobic tails that are 14 carbon atoms in length. The mitigated pro-inflammatory 126 ME146907648v.1 immune response is often characteristic of a stealth LNP formulation, in which the nucleic acid cargo is protected from degradation prior to delivery to a target organ. Example 3. LNP formulations containing no helper lipid and containing C18 lipid-anchored polymer have higher retention in blood The pharmacokinetics properties of DSPC- and C14 PEG2000 Lipid-containing Control LNP B and LNP4 (i.e., no DSPC or no helper lipid and with DSG-PEG2000-OMe as lipid-anchored polymer) were measured to assess clearance of the ceDNA nucleic acid cargo from the whole blood and plasma. The LNPs were formulated as disclosed above. The Control LNP B and LNP4 formulations were injected as an IV bolus injection via the tail vein of CD-1 mice. Whole blood samples were collected with K2EDTA as anticoagulant 150 μL/aliquot for qPCR quantitative analysis at 2-min, 1-hour, 3-hour and 6-hour timepoints. The plasma portion of the blood was separated and tested for the ceDNA amount using qPCR. Surprisingly, whole blood and plasma concentrations of ceDNA collected from the mice group treated with LNP4 showed a much higher level of plasma retention than the control group treated with Control LNP B in the first hour post-dosing (FIG.3). This suggests that eliminating DSPC or a helper lipid from an LNP can increase the plasma retention rate of the LNP formulation and the nucleic acid cargo, indicative of a stealth LNP as discussed above in Example 2. For example, the high retention of ceDNA in the bloodstream or slower clearance of the ceDNA cargo from the bloodstream as shown by LNP4 with stealth properties could be beneficial in that off-target delivery to non-target cells, including but not limited to a blood cell or a macrophage) may be reduced. Example 4. LNP formulations with mRNA cargo exhibit in vitro expression and hepatocyte uptake All of the LNP formulations described in the foregoing Examples 1-3 were prepared using ceDNA vector as the nucleic acid cargo. The goal of this study was to explore the characteristics of LNP formulations that contain no helper lipid (e.g., no DSPC), and that contain lipid-anchored polymers having hydrophobic tails that are longer than 14 carbon atoms and mRNA as the nucleic acid cargo. mRNA structurally differs from a ceDNA vector at least in that mRNA is single-stranded and is likely less negatively charged than the covalently closed-ended and double-stranded ceDNA vector. mRNA is also known to be less stable than DNA and is less rigid than DNA. To this end, the LNP formulations listed in Table 10 were prepared using luciferase mRNA as the nucleic cargo. All of these LNP formulations included the DiD (DiIC18(5); 1,1′-dioctadecyl- 3,3,3′,3′- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt) for the purpose of measuring and analyzing the uptake of the particles by primary mouse hepatocytes. Like the ceDNA/LNP formulations, mRNA/LNP formulations of this disclosure (i.e., containing no helper lipid, and containing lipid-anchored polymers having hydrophobic tails that are longer than 14 carbon atoms) 127 ME146907648v.1 exhibited average diameters and encapsulation efficiencies that are considered to be suitable for therapeutic use, including delivery to the hepatocytes. Table 10. Physicochemical properties of test LNP formulations having mRNA cargo and with or without GalNAc3 targeting moiety
Figure imgf000129_0001
The in vitro expression of luciferase encoding mRNA as delivered in the exemplary LNP formulations of the disclosure was also investigated. Briefly, primary mouse hepatocytes from C57BL/6 mice were plated at 50,000 cells per well in William’s E attachment media (Thermo fisher# A1217601). Four hours after plating, the cells were treated with 200 ng mRNA/LNP in Hepatocyte Culture Medium.1 hour following the mRNA/LNP treatments, the cells were washed twice with DPBS and the hepatocyte culture media was added to the wells. Approximately 18 hours later, relative cell viability was measured by CellTiter-Fluor Cell viability assay (Promega #G6080). Subsequently, luciferase acitivity was measured using One Step Luciferease assay system (BPS Bioscience #60690-1). To analyze the hepatocyte uptake of the mRNA/LNP, the cells were imaged using Opera High Content imaging for DiD (Ex/Em: 650/665) and Hoechst (Ex/Em: 350/460 nm) signals. FIG.4A indicates that the luciferase activity was significantly higher in LNP9, LNP10, and LNP11 that were each formulated with DSPE-PEG2000-GalNAc3, as compared to LNP8 that was formulated without GalNAc3. FIG.4B indicates that the presence of GalNAc3 resulted in significantly higher uptake by the cells. 128 ME146907648v.1 Example 5: LNP formulations with mRNA cargo exhibit in vivo expression, improved half-life in whole blood, and superior cargo concentration and retention in certain organs The following LNP formulations as listed in Table 11 were prepared using luciferase mRNA as the nucleic acid cargo. Table 11. Physicochemical properties of LNP formulations having mRNA-luciferase cargo
Figure imgf000130_0001
CD-1 mice were injected IV bolus via the tail vein at a dose of 0.3 mg/kg of any one of the LNP formulations listed in Table 11. Whole blood samples were collected with K2EDTA as anticoagulant 150 uL/aliquot for qPCR at 2 min, 1-hour and 6-hour timepoints and also at the 24-hour terminal timepoint. The total fluorescence (IVIS) in the liver was also measured at the 24-hour terminal timepoint. As can be seen in FIG.5, LNP101, which is an exemplary LNP formulation of the invention, i.e., LNP formulation that does not include a helper lipid (e.g., DSPC) and includes a lipid- anchored polymer having two hydrophobic tails that each comprises 16 to 22 carbon atoms in a single aliphatic chain backbone (e.g., DSG-PEG2000-OMe), showed robust in vivo luciferase expression in mice. Furthermore, pharmacokinetic (PK) studies investigating luciferase mRNA levels over the 24- hour period in whole blood (FIG.6A), liver (FIG.6B), spleen (FIG.6C), and bone marrow (FIG. 6D) were also conducted. Table 13 below summarizes the half-life (t1/2), area under the curve at the terminal timepoint which is 24 hours (AUClast), and clearance rate (Cl) parameters of the tested luciferase mRNA LNP formulations. 129 ME146907648v.1 Table 12. PK parameters of luciferase mRNA LNP formulations
Figure imgf000131_0001
Overall, the various PK parameters and values shown in Table 12 indicate that LNP101 (having no helper lipid, and having the C18 DSG-PEG2000-OMe as a lipid-anchored polymer) and Control LNP C having the combination of DSPC as a helper lipid and C14 DMG-PEG2000 as a lipid- anchored polymer possessed opposite PK profiles: longer half-life (t1/2), higher blood exposure at the terminal timepoint which is 24 hours (AUClast) and slower clearance from the systemic circulation in the inventive LNP formulations, as opposed to shorter half-life (t1/2), lower blood exposure at the terminal timepoint (AUClast) and faster clearance from the systemic circulation in Control LNP C.For example, in whole blood (FIG.6A), LNP101 exhibited excellent stealth properties in that the half-life (t1/2) of LNP101 is greater than 5 hours. In contrast, Control LNP C that incorporates C14 tailed DMG-PEG2000 as a lipid-anchored polymer exhibited a half-life (t1/2) of about 2.5 hours in whole blood. From a clearance rate (Cl) standpoint, LNP101 exhibited Cl rates of about 10-40 mL/min/kg, whereas Control LNP C had a significantly higher Cl rate of 307 ml/min/kg. A higher clearance rate (Cl) is indicative of a quicker rate of the drug substance (i.e., luciferase mRNA) being cleared from the systemic circulation. Hence, the Cl rates indicated that the luciferase mRNA delivered by Control LNP C was rapidly cleared from the bloodstream. High Cl rates correlate with low blood exposures (AUClast) of the drug substance at the terminal timepoint, which in this case is 24 hours. Accordingly, the AUClast values in Table 12 indicate that a blood exposure (AUClast) of only 27.2 hour*ng/mL, as compared to significantly higher AUClast values of greater than 500 hour*ng/mL of all of the inventive LNP formulations of LNP101. 130 ME146907648v.1 The calculated Cl rates and AUClast values of the tested LNP formulations are further corroborated by the PK curves of FIG.6A. Of note, FIG.6A indicates that in LNP101, the luciferase mRNA concentrations as detected by qPCR steadily dropped from 10-1 µg/mL to 10-3 µg/mL over the 24-hour period; whereas in Control LNP C the luciferase mRNA concentrations dropped from 10-1 µg/mL to almost 10-4 µg/mL within the first hour, which continued to significantly drop to 10-6 µg/mL at 6 hours and further, to almost 10-7 µg/mL at 24 hours. As discussed above in Example 3, the higher retention of ceDNA in the bloodstream or less rapid clearance of the ceDNA cargo from the bloodstream as delivered by the LNP4 could be beneficial in that off-target delivery to non-target cells. In the liver (FIG.6B), LNP101 exhibited slightly higher amounts of mRNA copies of luciferase and at least equivalent or higher retention rates of luciferase mRNA from 6 hours to 24 hours post-dosing, as compared to Control LNP C. The observation of higher luciferase mRNA amounts in the inventive LNP formulation is surprising and unexpected, considering that the IVIS fluorescence data as shown in FIG.5 suggested that the luciferase mRNA expression levels of these inventive LNP formulations were lower than that of Control LNP C. Similarly, in the spleen (FIG.6C), LNP101 exhibited excellent retention rates of luciferase mRNA from 6 hours to 24 hours post-dosing with negligible reductions in copy amounts whereby the amount at 6 hours and the amount at 24 hours post-dosing were within the same order of magnitude. In the spleen, T-cells including CD8+ T-cells are primed to generate precursors with an enhanced ability to differentiate into long-lived, stem-like memory T cells. Stem-like T-cells (TSC) are a subpopulation of mature T-cells that display stem cell-like properties, maintaining long-lasting immune effect even among exhausting clones. In the bone marrow where hematopoietic stems cells (HSC) are present (FIG.6D), the amounts of luciferase mRNA throughout the 6-24 hour post-dosing period, as delivered by LNP101 were consistently found to be about 2 orders of magnitude higher than the amounts of luciferase mRNA as delivered by Control LNP C. Moreover, as indicated in FIG.6D, the retention rates of luciferase mRNA in the mice bone marrow during the 6-24 hour post-dosing period, as delivered by LNP101, were also superior to the retention rate of the mRNA as delivered by Control LNP C. Specifically, within the 6-24 hour post-dosing period, the amount of copies of luciferase mRNA in the mice bone marrow merely dropped for less than one order of magnitude in mice dosed with the inventive LNP formulation, as compared to a drop of greater than one order of magnitude within the same time window for mice dosed with Control LNP C. Example 6: LNPs formulated with no helper lipid and a variety of ionzable lipids can result in increased expression in vivo and in vitro Stealth LNPs with no helper lipid were formulated using ionizable lipids with head or tail group variations of Ionizable Lipid 87. This study compares expression in vivo and in vitro to identify 131 ME146907648v.1 ionizable lipids with activity comparable to Ionizable Lipid 87. In vivo, LNPs were injected at 0.5 mpk as an IV bolus injection via the tail vein of CD-1 mice. Expression was measured by IVIS on day 7 post-dose administration. Body weights were measured on day 1 post-dose administration. Referring to FIG.7B, body weight loss was comparable among all groups. Referring to FIG.7A, LNP Formulation 497 containing Ionizable Lipid 114 showed about 0.5 log increase in expression compared to Formulation 495 containing Ionizable Lipid 87 and the other variants. Similarly, referring to FIG.9A, LNPs containing mRNA cargo were administered in vitro to primary mouse hepatocytes. LNPs were uptaken uniformly, with no significative differences between the Ionizable Lipid 87 variants (e.g., Ionizable Lipids 113, 114 and 115). However, referring to FIG.9B LNP containing, Ionizable Lipid 114 and mRNA cargo showed about 2-fold increase in expression compared to Ionizable Lipid 87 containing LNP, while other ionizable lipids variants showed slightly lower expression compared to Ionizable Lipid 87. Altogether, these results demonstrate that stealth LNP with no helper lipid of the present disclosure can be formulated with a variety of ionizable lipids, which can result in increased expression in vivo and in vitro. Table 13. Physicochemical properties of LNP formulations having ceDNA-luciferase cargo
Figure imgf000133_0001
Example 7: Stealth LNPs formulated with no helper lipid and different variants of Ionizable Lipid 87, and with or without a GalNAc-based targeting ligand Stealth LNPs with no helper lipid with either Ionizable Lipid 87 or Ionizable Lipid 114 were formulated. LNPs with Ionizable Lipid Z and C2 ceramide as helper lipid were formulated as control. This study aims to further investigate expression of stealth LNP formulations of the present disclosure 132 ME146907648v.1 containing Ionizable Lipid 114, and with or without a GalNAc-based targeting ligand (i.e. +G or -G), in vivo. Both +G and -G versions were dosed to verify that stealth properties were maintained. LNPs were injected at 1.0 mpk as an IV bolus injection via the tail vein of CD-1 mice. Expression was measured by IVIS on day 7 post-dose administration. Body weights were measured on day 1 post- dose administration. Referring to FIG.8B, body weight loss was comparable among all groups. Referring to FIG.8A, LNPs with Ionizable Lipid 87 of the present disclosure without or with GalNAc (LNP Formulation 624 vs.619) clearly showed stealth properties. LNPs without GalNAc had low expression as they are long circulating and do not have a targeting agent to reach the target organ. When GalNAc was added as active targeting ligand, expression levels were increased notably, which indicates that the LNP was then capable of reaching the target organ due to the presence of the targeting ligand (i.e., GalNAc). LNPs with Ionizable Lipid 114 of the present disclosure behaved similarly to LNPs with Ionizable Lipid 87 of the present disclosure. This behavior was less prominent when using LNPs with Ionizable Lipid Z and C2 ceramide as helper lipid as control, where the difference between +G and -G was less pronounced. This indicates that due to the presence of helper lipid, LNPs without GalNAc, but with helper lipid were able to reach the liver in some amount through opsonization based delivery. Table 14. Physicochemical properties of LNP formulations having ceDNA-luciferase cargo
Figure imgf000134_0001
133 ME146907648v.1 Example 8: Smaller LNPs were formulated by adding alternative lipid-conjugated polymer to the base LNPs of the present disclosure Referring to FIG.10A, smaller ceDNA LNPs containing anchored polymer (e.g., DSG-PEG) were formulated through addition of dissociable polymer (e.g., 5% DMG-PEG or mono-C18-PEG having the structure
Figure imgf000135_0001
. This study compares these smaller LNPs to control LNPs to observe if smaller particles lead to higher expression and improved tolerability. The LNPs were injected as an IV bolus injection via the tail vein of CD-1 mice. Expression was measured by IVIS on day 7 post-dose administration. Referring to FIG.10C, body weights were measured on days 1, 2, 3 and 7 post-dose administration. Referring to FIG.10B, while two of the smaller formulations (LNP Formulations 620 and 623) had 0.5 to 1 log decrease in IVIS expression with additional PEG (5% DMG-PEG or 5% mono-C18-PEG, respectively) compared to control (LNP F 433), one of the formulations (LNP Formulation 621) showed comparable IVIS expression with the addition of 5% DMG-PEG. Small single-stranded DNA LNPs were also formulated with Ionizable Lipid Z, as well as Ionizable Lipid 87, and the addition of 5% dissociable DMG-PEG. These formulations were evaluated for uptake in primary human hepatocytes. The study demonstrated that single-stranded DNA LNPs of the present disclosure with 0% or 3% DMG-PEG are capable of GalNAc-mediated uptake. Taken together, these studies demonstrate that LNPs with anchored PEG and dissociable PEG, but no helper lipid, can be formulated, while maintaining their ability to be internalized by hepatocytes and express in vio in mice. Table 15. Physicochemical properties of LNP formulations having ceDNA-luciferase cargo
Figure imgf000135_0002
134 ME146907648v.1
Figure imgf000136_0001
Example 9: Consistent formulation of the LNPs of the present disclosure, in vivo luciferase expression from ceDNA cargo, and tolerability when employing different processes Previous experiments by the inventors have identified the LNPs of the present disclosure as stealthy formulations that lack a helper lipid, yet possess extended blood residence time in the absence of a targeting ligand, but rapid distribution to the liver in the presence of a targeting ligand. The purpose of this experiment was to confirm consistent formulations of the LNPs of the present disclosure, in vivo mouse expression and tolerability when employing “Old Process” (OP) or “New Process” (NP), as these terms are defined below, targeting dose levels of 0.5, 2.0, and 4.0 mg/kg. Test articles are compared against a control LNP Formulation 241 containing dissociable PEG, e.g., Ac- PEG, instead of anchored PEG, e.g., DSG-PEG, as its expression and tolerability profiles are well- documented and will confirm successful test article administration in mice. The LNPs of the present disclosure were confirmed to possess comparable physicochemical characteristics across processes based on particle size and polydispersity, while encapsulation efficiency was improved with New Process (NP). In CD-1 mice, there was no obvious difference observed in Luciferase expression, cytokine induction, or body weight loss across dose levels when comparing the LNPs of the present disclosure prepared by either OP or NP, or when prepared with or without a targeting ligand. Expression of ceDNA luciferase cargo of the LNPs of the present disclosure was uniformly less than that of the control LNP Formulation 241, attributable to the control formulation’s incorporation of dissociable PEG-lipid and an ionizable lipid known to promote endosomal escape. For Old Process (OP), individual lipid components were dissolved in tert-butanol at stock concentrations. Lipid mixtures were prepared according to described molar ratios of individual components, targeting a final molar concentration of 3.94 mM in tert-butanol.1.0 mg/mL ceDNA solution was prepared in water. Prior to formulation, the final lipid mixture was mixed at ratio of 10:1 (v/v) with ceDNA in water, resulting in a final solvent of 90.9% tert-butanol and 9.1% water. Nanoassemblr was used to prepare the lipid nanoparticle formulation. The aqueous stream identity was 25 mM sodium acetate, pH 4, and the organic stream identity was the lipid/ceDNA mixture in 90.9% tert-butanol, a final lipid concentration of 3.59 mM, and a final ceDNA concentration of 0.091 mg/mL. Mixing through the Nanoassemblr targeted a flow rate ratio of 3:1 (aqueous:organic). Formulations were dialyzed against 1X DPBS for at least two hours at 4°C. Dialyzed formulations were concentrated via Amicon spin filters (100 kDa MWCO) spun at 2000 g via centrifuge. Concentrated formulations were sterile filtered through a 0.2 µm nylon filter. New Process (NP) follows a similar procedure that incorporates ethanol/methanol (EtOH/MeOH) for tert-butanol (t-BuOH) as the organic solvent. Individual lipid components were 135 ME146907648v.1 dissolved in ethanol at stock concentrations. Lipid mixtures were prepared according to described molar ratios of individual components, targeting a final molar concentration of 3.94 mM in EtOH/MeOH.1.0 mg/mL ceDNA solution was prepared in water. Prior to formulation, the final lipid mixture was mixed at ratio of 10:1 (v/v) with ceDNA in water, resulting in a final solvent of 90.9% EtOH/MeOH and 9.1% water. Nanoassemblr was used to prepare the lipid nanoparticle formulation. The aqueous stream identity was 25 mM sodium acetate, pH 4, and the organic stream identity was the lipid/ceDNA mixture in 90.9% EtOH/MeOH, a final lipid concentration of 3.59 mM, and a final ceDNA concentration of 0.091 mg/mL. Mixing through the Nanoassemblr targeted a flow rate ratio of 3:1 (aqueous:organic). The LNP formulations were dialyzed against 1X DPBS for at least two hours at 4°C, performed a total of three times. Dialyzed formulations were concentrated via Amicon spin filters (100 kDa MWCO) spun at 2000 g via centrifuge. Concentrated LNP formulations were sterile filtered through a 0.2 µm nylon filter. Table 16. Physicochemical properties of LNP formulations having ceDNA-luciferase cargo
Figure imgf000137_0001
FIG.11A shows CD-1 mouse body weight loss recorded on day 1 post-dose administration. Loss associated with 0.5 mpk dose groups was negligible when compared to PBS mice, while 2.0 mpk and 4.0 mpk dose groups had more variable impact on body weight loss. As shown in FIG.11B, mouse body weights trended toward full recovery by day 3 post-dose administration, with exception to LNP Formulation 117 that employed NP. FIGS.12A and 12B show IVIS readouts performed at day 4 and day 7 post-dose administrating measuring the level of Luciferase protein expressed in CD-1 mouse liver tissue. The LNPs of the present disclosure prepared without a targeting ligand resulted in near-baseline Luciferase expression in a dose-independent manner while the LNPs of the present disclosure containing a targeting ligand resulted in a modest boost in expression in a dose-independent manner. FIGs.13A-13F show cytokine profile of test articles comparing 0.5 mpk and 2.0 mpk doses. Decreased induction of IL-18, IFN-gamma, and TNF-alpha was observed for the LNPs of the present 136 ME146907648v.1 disclosure prepared without a targeting ligand, and similarly for the liver-targeted LNP of the present disclousred prepared by Old Process (OP). The liver-targeted LNP of the present disclosure prepared New Process (NP) induced control-like cytokines with exception to TNF-alpha at 2.0 mpk. Example 10: PK profiles of the ceDNA cargo of the stealth LNP formulations of the present disclosure prepared by different processes This study aims to compare whole blood and plasma PK profiles of the stealth LNP formulations of the present disclosure prepared by Old Process (OP) or New Process (NP), with or without a GalNAc-based targeting ligand, as listed in Table 16 above, to better understand if formulation process impacts these properties of stealth LNPs. Test articles were compared against a control LNP prepared with dissociable DMG-PEG, serving as a non-stealthy LNP control formulation that rapidly distributes from the blood compartment toward tissue compartments. PK of the test articles was determined by quantifying by qPCR the amount of genetic cargo present in blood and plasma samples drawn at 2 minutes, 60 minutes, 3 hours, and 6 hours post-dosing. Similarly prolonged blood and plasma PK profiles were observed for untargeted stealth LNP formulations of the present disclosure regardless of the LNP formulation process. When paired with a GalNAc targeting ligand, both stealth LNP formulations underwent faster clearance from the blood compartment compared to untargeted stealth LNP formulations. In contrast, the control LNP formulation containing dissociable PEG (e.g., Ac-PEG) underwent the most rapid clearance. The GalNAc-targeted stealth LNP formulation of the present disclosure prepared by OP appeared to distribute away from blood and into tissues more rapidly than the targeted LNP prepared by NP, though this difference may be a result of in vivo biological variability. In this study, formulation process (e.g., OP or NP) did not have an observable effect on blood PK of stealth LNP formulations. FIGs.14A and 14B show pharmacokinetics of LNP-encapsulated ceDNA in mouse whole blood and plasma. ceDNA encapsulated in the control LNP formulation containing dissociable Ac- PEG was cleared from both compartments rapidly within the first hour post-administration, while ceDNA encapsulated within stealth LNP formulations lacking a targeting ligand underwent prolonged residence time in both blood and plasma over a period of six hours. The same stealth formulations prepared with a GalNAc targeting ligand were cleared from both compartments at a faster rate. Example 11: The stealth LNPs of the present disclosure formulated with higher amount of cholesterol, and without a targeting ligand, produced smaller particles that retained prolonged PK profiles In earlier work by the inventors, LNPs prepared with 2.9% DSG-PEG2000, 67.6% ionizable lipid, and 29.5% cholesterol, and lacking a helper lipid, demonstrated prolonged blood residence time in the absence of a targeting ligand, and quick clearance from blood when containing a GalNAc targeting ligand. These are desired stealth-like properties of the LNP; however, these formulations 137 ME146907648v.1 were not optimized for particle size, which is believed to play a role in efficient delivery of the LNP to certain target cell types, in this case hepatocytes. Compositional exploration by the inventors has led to the discovery that LNPs incorporating 2.9% DSG-PEG2000, 57.6% ionizable lipid, and 39.5% cholesterol produce 10-15 nm smaller particles, prepared by New Process (NP). The purpose of this study is to confirm that untargeted LNPs prepared with this composition retain prolonged whole blood and plasma PK profiles, as well as the expected GalNAc-mediated blood clearance. In-situ hybridization (ISH) analysis and payload concentration was carried out on extracted livers at 1 hour and 6 hours post-dosing to confirm that differences in blood PK are reflected in payload distribution to liver tissue. Test articles were compared against a control LNP formulation prepared with dissociable DMG-PEG2000, serving as a non-stealthy LNP control formulation that rapidly distributes from the blood compartment toward tissue compartments. PK of the test articles was determined by quantifying by qPCR the amount of genetic cargo present in blood and plasma samples drawn at 2 minutes, 60 minutes, 3 hours, and 6 hours post-dosing. The untargeted LNP of the present disclosure was observed to have prolonged blood residence and cleared from blood quickly when containing a GalNAc targeting ligand, suggesting that 57.6% ionizable lipid and 39.5% cholesterol does not impact these stealth-like properties. The dissociable PEG control LNP formulation containing dissociable Ac-PEG expectedly underwent rapid distribution out of the blood compartment. Differences in blood clearance at 1 and 6 hours did not translate to payload concentration in liver tissue, but were qualitatively observed by liver ISH. qPCR analysis of liver tissue suggested similar payload concentrations across all test groups with no statistical outliers. However, clear rank-ordering of LNP distribution to the liver could be established by ISH analysis of liver tissue. At both time points, the highest payload density in liver was observed with the dissociable Ac-PEG control LNP, followed by the liver-targeted stealth LNP of the present disclosure, while the lowest payload density was observed with the untargeted stealth LNP. This relationship is the inverse of blood PK observations, implying that more rapid clearance of payload from blood must lead to higher distribution to liver tissue, though this data set cannot address distribution to other tissue compartments. It is suspected that technical error occurred during qPCR quantification of payload in liver, resulting in stark disagreement between liver ISH and liver qPCR data. Table 17. Physicochemical properties of LNP formulations having ceDNA-luciferase cargo
Figure imgf000139_0001
138 ME146907648v.1
Figure imgf000140_0001
FIGs.15A and 15B show pharmacokinetics of LNP-encapsulated payload in mouse whole blood and plasma. Approximately 99% of payload encapsulated in the control LNP formulation containing dissociable Ac-PEG is cleared from the whole blood compartment within the first hour post-administration. When encapsulated within the untargeted LNP containing 57.6% ionizable lipid and 39.5% cholesterol, approximately 5% of ceDNA payload was cleared within the first hour. Clearance of ceDNA payload in liver-targeted, dissociable PEG LNPs was significantly faster than that of liver-targeted stealth LNPs, implying stealth LNPs of the present disclosure possess fundamentally different GalNAc-mediated biodistribution away from the blood compartment. PK of ceDNA payload in plasma compartment generally followed that of the whole blood compartment in all test groups, though increased mouse-to-mouse variability was observed. FIGs.16A-16C show ISH staining of mouse livers collected at 1 hour post-administration. The highest ceDNA payload density was observed in the liver tissue dosed with the control LNP formulation containing dissociable Ac-PEG, while the lowest payload density was observed in the liver tissue dosed with untargeted stealth LNP formulations of the present disclosure. FIGs.16D-16F show SH staining of the mouse livers collected at 6 hours post-administration. ceDNA payload staining was less abundant in all test groups compared to the livers collected at 1 hour. The highest payload density was observed in the liver tissue dosed with control LNP formulation containining dissociable Ac-PEG, while the lowest payload density was observed in the liver tissue dosed with untargeted stealth LNPs. Example 12: Smaller LNPs were formulated by increasing the cholesterol content Stealth-like properties, such as prolonged blood PK and low cytokine induction, have been demonstrated for LNP formulations containing ionizable lipid, cholesterol and DSG-PEG2000 at a molar ratio of 67.6/29.5/2.9. However, resulting LNP physicochemical properties were sub-optimal as particle sizes were often larger than 90 nm (e.g., ~100 nm) while encapsulation efficiency was often below 90%. The purpose of this experiment was to observe murine-based in vivo Luciferase expression, body weight loss, and cytokine induction comparing this LNP formulation against LNP formulations containing higher cholesterol content, e.g., LNP formulations containing ionizable lipid, cholesterol and DSG-PEG2000 at a molar ratio of 57.6 : 39.5 : 2.9, which was demonstrated to produce LNPs less than 80 nm in size and greater than 90% encapsulated cargo. Comparable expression, body weight loss, and cytokine induction was observed between these two LNP formulations at both 0.5 and 2.0 mpk, implying that LNP potency and tolerability properties were not lost when modifying the lipid composition to improve physiochemical properties. Both stealth LNP 139 ME146907648v.1 formulations generally induced fewer cytokines compared to the control LNP containing a dissociable PEG lipid. Table 18. Physicochemical properties of LNP formulations having ceDNA-luciferase cargo
Figure imgf000141_0001
FIGs.17A and 17B show IVIS readouts performed at day 4 and day 7 post-dose administration measuring the level of Luciferase protein expressed in CD-1 mouse liver tissue. At 0.5 and 2.0 mpk, the LNPs of the present disclosure had comparable Luciferase expression at both time points. Expression data suggests that shifts from 29.5% to 39.5% cholesterol and from 67.6% to 57.6% ionizable lipid in the LNPs of the present disclosure did not significantly impact payload expression. FIGs.18A and 18B show CD-1 mouse body weight loss at day 1 post-dose administration. Mouse body weight loss at day 1 post-administration was generally consistent between PBS and 0.5 mpk groups. At the same dose level, the LNP Formulation 118 containing 29.5% cholesterol resulted in a small decrease in body weight compared to other groups, but values below 5% body weight loss may not be significant. At 2.0 mpk, mice across all dose groups averaged 5-10% body weight loss. Mouse body weight recovered by day 3 post-administration. FIGs.19A-19E show cytokine profiles of test articles comparing 0.5 and 2.0 mpk doses. Decreased induction of IFN-alpha, IL-18, IFN-gamma, and TNF-alpha were observed for both the LNP formulations of the present disclosure containing DSG-PEG compared to the control LNP formulation containing methoxypolyethyleneglycoloxy(2000)-N,N-ditetradecylacetamide (Ac-PEG). Cytokine readouts of stealth LNPs are consistent across dose levels, suggesting that compositional changes to cholesterol and ionizable lipid percent did not affect downstream cytokine response. There is no discernable trend in IL-6 induction across all test articles. Example 13: Antibody conjugation of the LNPs of the present disclosure leads to significant increase in uptake and expression as compared to the parental LNPs, regardless of ionizable lipid This example compares DiD uptake and mRNA luciferase expression in primary human hepatocytes of the LNPs of the present disclosure, with or without antibody conjugation (e.g., scFv, 140 ME146907648v.1 VHH, Fab, etc.), using the protocols as shown in FIG.20 (without an endocytosis inhibitor) and FIG. 23 (with an endocytosis inhibitor). The aim of the study using the protocol as shown in FIG.23 is to ensure that uptake and expression of conjugated LNPs with varying ionizable lipids is clathrin- mediated by using an endocytosis inhibitor. The media containing an endocytosis inhibitor is incomplete (no BSA or transferrin) media with 100 µM of DynGo 4a. Table 19. Physicochemical properties of LNP formulations having mRNA-luciferase cargo
Figure imgf000142_0001
Referring to FIGs.21A and 21B and Table 19, antibody (VHH) conjugation for targeting hepatic ASGPR1 protein led to significant increase in LNP uptake compared to the parental LNPs of the present disclosure without antibody conjugation, and regardless of the identity of the ionizable lipid. 141 ME146907648v.1 Referring to FIGs.22A and 22B and Table 19, antibody (VHH) conjugation for targeting hepatic ASGPR1 protein led to minimal increase in luciferase expression as compared to the parental LNPs of the present disclosure without antibody conjugation. Parental (unconjugated) LNP formulations of the present disclosure and Control LNP 183 showed minimal luciferase expression. LNPs formulated with ionizable lipids MC3 and LP01 showed equivalent uptake but very low expression suggesting that these ionizable lipids adversely affect endosomal escape. Referring to FIGs.24A and 24B and Table 19, for both VHH and scFv conjugates (for targeting hepatic ASGPR1 protein) of the LNP formulations of the present disclosure, co-incubation with an endocytosis inhibitor resulted in complete inhibition of uptake in all ionizable lipids. Referring to FIGs.25A and 25B and Table 19, for both VHH and scFv conjugates (for targeting hepatic ASGPR1 protein) of the LNP formulations of the present disclosure, co-incubation with an endocytosis inhibitor resulted in complete inhibition of mRNA cargo expression in all ionizable lipids. Table 20. Physicochemical properties of LNP formulations having mRNA-luciferase cargo
Figure imgf000143_0001
142 ME146907648v.1
Figure imgf000144_0001
Referring to FIGs.26A and 26B and Table 20, antibody (VHH) conjugation for targeting hepatic ASGPR1 protein led to higher levels of uptake compared to the parental LNPs of the present disclosure without antibody conjugation, and regardless of the identity of the ionizable lipid, as well as when compared to Control LNPs 183 and 261. Referring to FIGs.27A and 27B and Table 20, antibody (VHH) conjugation for targeting hepatic ASGPR1 protein led to higher levels of expression of mLuc and rLuc luciferase cargo compared to the parental LNPs of the present disclosure without antibody conjugation, except for DOTAP. 143 ME146907648v.1

Claims

CLAIMS What is Claimed is: 1. A lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; an ionizable lipid; a sterol; a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises: a polymer; a lipid moiety comprising at least one hydrophobic tail; and optionally a linker connecting the polymer to the lipid moiety; wherein the at least one hydrophobic tail comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; and wherein the LNP does not comprise a helper lipid.
2. The lipid nanoparticle (LNP) of claim 1, wherein the LNP has an average particle size of about 50-100 nm in diameter.
3. The lipid nanoparticle (LNP) of claim 2, wherein the LNP has an average particle size of about 50-90 nm in diameter.
4. The lipid nanoparticle (LNP) of claim 3, wherein the LNP has an average particle size of about 50-80 nm in diameter.
5. The lipid nanoparticle (LNP) of claim 4, wherein the LNP has an average particle size of about 50-70 nm in diameter.
6. The lipid nanoparticle (LNP) of claim 5, wherein the LNP has an average particle size of about 50-60 nm in diameter.
7. The lipid nanoparticle (LNP) of any one of claims 1-6, wherein the lipid moiety comprises at least two hydrophobic tails.
8. The lipid nanoparticle (LNP) of claim 7, wherein the two hydrophobic tails each independently comprise 16, 17, 18, 19, 20, 21, or 22 carbon atoms. 144 ME146907648v.1
9. The lipid nanoparticle (LNP) of claim 8, wherein the two hydrophobic tails each independently comprise 16, 17, 18, 19, 20, or 21 carbon atoms.
10. The lipid nanoparticle (LNP) of claim 9, wherein the two hydrophobic tails each independently comprise 16, 17, 18, 19, or 20 carbon atoms.
11. The lipid nanoparticle (LNP) of claim 9, wherein the two hydrophobic tails each independently comprise 16, 17, 18, or 19 carbon atoms.
12. The lipid nanoparticle (LNP) of claim 9, wherein the two hydrophobic tails each independently comprise 16, 17, or 18 carbon atoms.
13. The lipid nanoparticle (LNP) of claim 9, wherein the two hydrophobic tails each independently comprise 18, 19, or 20 carbon atoms.
14. The lipid nanoparticle (LNP) of claim 9, wherein the two hydrophobic tails each comprise 16 carbon atoms.
15. The lipid nanoparticle (LNP) of claim 9, wherein the two hydrophobic tails each comprise 18 carbon atoms.
16. The lipid nanoparticle (LNP) of claim 9, wherein the two hydrophobic tails each comprise 20 carbon atoms.
17. The lipid nanoparticle (LNP) of any one of claims 1-16, wherein the at least one or two hydrophobic tails are each a fatty acid.
18. The lipid nanoparticle (LNP) of any one of claims 1-17, wherein the at least one or two hydrophobic tails are each independently selected from the group consisting of octadecane, octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and a derivative thereof.
19. The lipid nanoparticle (LNP) of any one of claims 1-18, wherein the first lipid-anchored polymer comprises a glycerolipid. 145 ME146907648v.1
20. The lipid nanoparticle (LNP) of any one of claims 1-19, wherein the first lipid-anchored polymer comprises a phospholipid.
21. The lipid nanoparticle (LNP) of any one of claims 1-20, wherein the first lipid-anchored polymer does not comprise distearoylphosphatidylcholine (DSPC).
22. The lipid nanoparticle (LNP) of any one of claims 1-21, wherein the first lipid-anchored polymer comprises a linker-lipid moiety selected from the group consisting of 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1- palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3- phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), dioctadecylamine (DODA), distearoyl- rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), a derivative thereof, and a combination of any of the foregoing.
23. The lipid nanoparticle (LNP) of claim 22, wherein the first lipid-anchored polymer comprises a linker-lipid moiety selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, a derivative thereof, and a combination of any of the foregoing.
24. The lipid nanoparticle (LNP) of any one of claims 1-23, wherein the polymer is selected from the group consisting of polyethylene (PE), polypropylene (PP), polyethylene glycol (PEG), polyglycerol (PG), polyvinyl alcohol (PVOH), polysarcosine (pSar), and a combination thereof.
25. The lipid nanoparticle (LNP) of claim 24, wherein the polymer is selected from the group consisting of polyethylene glycol (PEG), polyglycerol (PG), polysarcosine (pSar), and a combination thereof.
26. The lipid nanoparticle (LNP) of any one of claims 1-25, wherein the polymer has a molecular weight of between about 1000 Da and about 5000 Da.
27. The lipid nanoparticle (LNP) of claim 26, wherein the polymer has a molecular weight of between about 3200 Da and about 3500 Da. 146 ME146907648v.1
28. The lipid nanoparticle (LNP) of claim 27, wherein the polymer has a molecular weight of about 2000 Da.
29. The lipid nanoparticle (LNP) of any one of claims 24-28, wherein the polymer is polyethylene glycol (PEG).
30. The lipid nanoparticle (LNP) of any one of claims 1-29, wherein the sterol is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and a derivative of thereof, and a combination of any of the foregoing.
31. The lipid nanoparticle (LNP) of claim 30, wherein the sterol is cholesterol.
32. The lipid nanoparticle (LNP) of claim 30, wherein the sterol is beta-sitosterol.
33. The LNP of any of claims to 1-32, wherein the ionizable lipid is a lipid represented by: a) Formula (A):
Figure imgf000148_0001
Formula (A), or a pharmaceutically acceptable salt thereof, wherein: R1 and R1’ are each independently optionally substituted linear or branched C1-3 alkylene; R2 and R2’ are each independently optionally substituted linear or branched C1-6 alkylene; R3 and R3’ are each independently optionally substituted linear or branched C1-6 alkyl; or alternatively, when R2 is optionally substituted branched C1-6 alkylene, R2 and R3, taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; or alternatively, when R2’ is optionally substituted branched C1-6 alkylene, R2’ and R3', taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; R4 and R4’ are each independently –CRa, –C(Ra)2CRa, or –[C(Ra)2]2CRa; Ra, for each occurrence, is independently H or C1-3 alkyl; 147 ME146907648v.1 or alternatively, when R4 is –C(Ra)2CRa, or –[C(Ra)2]2CRa and when Ra is C1-3 alkyl, R3 and R4, taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; or alternatively, when R4’ is –C(Ra)2CRa, or –[C(Ra)2]2CRa and when Ra is C1-3 alkyl, R3’ and R4’, taken together with their intervening N atom, form a 4- to 8-membered heterocyclyl; R5 and R5’ are each independently hydrogen, C1-20 alkylene or C2-20 alkenylene; R6 and R6’, for each occurrence, are independently C1-20 alkylene, C3-20 cycloalkylene, or C2-20 alkenylene; and m and n are each independently an integer selected from 1, 2, 3, 4, and 5; or b) Formula (B):
Figure imgf000149_0001
Formula (B); or a pharmaceutically acceptable salt thereof, wherein: a is an integer ranging from 1 to 20; b is an integer ranging from 2 to 10; R1 is absent or is selected from (C2-C20)alkenyl, -C(O)O(C2-C20)alkyl, and cyclopropyl substituted with (C2-C20)alkyl; and R2 is (C2-C20)alkyl; or c) Formula (C):
Figure imgf000149_0002
Formula (C); or a pharmaceutically acceptable salt thereof, wherein: R1 and R1’ are each independently (C1-C6)alkylene optionally substituted with one or more groups selected from Ra; R2 and R2’ are each independently (C1-C2)alkylene; R3 and R3’ are each independently (C1-C6)alkyl optionally substituted with one or more groups selected from Rb; or alternatively, R2 and R3 and/or R2’ and R3’ are taken together with their intervening N atom to form a 4- to 7-membered heterocyclyl; R4 and R4’ are each a (C2-C6)alkylene interrupted by –C(O)O-; 148 ME146907648v.1 R5 and R5’ are each independently a (C2-C30)alkyl or (C2-C30)alkenyl, each of which are optionally interrupted with –C(O)O- or (C3-C6)cycloalkyl; and Ra and Rb are each halo or cyano; or d) Formula (D):
Figure imgf000150_0001
Formula (D), or a pharmaceutically acceptable salt thereof, wherein: R’ is absent, hydrogen, or C1-C6 alkyl; provided that when R’ is hydrogen or C1-C6 alkyl, the nitrogen atom to which R’, R1, and R2 are allpositively charged; R1 and R2 are each independently hydrogen, C1-C6 alkyl, or C2-C6 alkenyl; R3 is C1-C12 alkylene or C2-C12 alkenylene; R4 is C1-C18 unbranched alkyl, C2-C18 unbranched alkenyl, or
Figure imgf000150_0002
wherein: R4a and R4b are each independently C1-C16 unbranched alkyl or C2-C16 unbranched alkenyl; R5 is absent, C1-C8 alkylene, or C2-C8 alkenylene; R6a and R6b are each independently C7-C16 alkyl or C7-C16 alkenyl; provided that the total number of carbon atoms in R6a and R6b as combined is greater than 15; X1 and X2 are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(Ra)=N-, -N=C(Ra)-, -C(Ra)=NO-, -O-N=C(Ra)-, -C(=O)NRa-, -NRaC(=O)-, -NRaC(=O)NRa-, -OC(=O)O-, -OSi(Ra)2O-, -C(=O)(CRa2)C(=O)O-, or OC(=O)(CRa2)C(=O)-; wherein: Ra, for each occurrence, is independently hydrogen or C1-C6 alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6; or e) Formula (E):
Figure imgf000150_0003
149 ME146907648v.1 Formula (E), or a pharmaceutically acceptable salt thereof, wherein: R’ is absent, hydrogen, or C1-C3 alkyl; provided that when R’ is hydrogen or C1-C3 alkyl, the nitrogen atom to which R’, R1, and R2 are all attached ispositively charged; R1 and R2 are each independently hydrogen or C1-C3 alkyl; R3 is C3-C10 alkylene or C3-C10 alkenylene; R4 is C1-C16 unbranched alkyl, C2-C16 unbranched alkenyl, or
Figure imgf000151_0001
wherein: R4a and R4b are each independently C1-C16 unbranched alkyl or C2-C16 unbranched alkenyl; R5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; R6a and R6b are each independently C7-C14 alkyl or C7-C14 alkenyl; X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(Ra)=N-, -N=C(Ra)-, -C(Ra)=NO-, -O-N=C(Ra)-, -C(=O)NRa-, -NRaC(=O)-, -NRaC(=O)NRa-, -OC(=O)O-, -OSi(Ra)2O-, -C(=O)(CRa2)C(=O)O-, or OC(=O)(CRa2)C(=O)-; wherein: Ra, for each occurrence, is independently hydrogen or C1-C6 alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6; or f) an ionizable lipid selected from any of the ionizable lipids in Table 1, 4, 5, 6 or 7.
34. The lipid nanoparticle (LNP) of any one of claims 1-33, wherein the LNP further comprises a targeting moiety.
35. The lipid nanoparticle (LNP) of claim 34, wherein the LNP comprises a second lipid- anchored polymer and the targeting moiety is conjugated to the second lipid-anchored polymer.
36. The lipid nanoparticle (LNP) of claim 35, wherein the second lipid-anchored polymer comprises a linker-lipid moiety selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2- oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn- glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1- trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DPHyPE), dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2- dipalmitoyl-rac-glycerol (DPG), a derivative thereof, and a combination of any of the foregoing. 150 ME146907648v.1
37. The lipid nanoparticle (LNP) of claim 36, wherein the second lipid-anchored polymer comprises a linker-lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, a derivative thereof, and a combination of any of the foregoing.
38. The lipid nanoparticle of (LNP) of claim 37, wherein the first and the second lipid-anchored polymers are different lipid-anchored polymers; and wherein the first and the second lipid-anchored polymers comprise one of the following combinations: DSG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DSPE (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); and DMG (the first lipid-anchored polymer) and DPG (the second lipid-anchored polymer).
39. The lipid nanoparticle (LNP) of claim 37, wherein the first and the second lipid-anchored polymers are the same lipid-anchored polymers; and wherein the first and the second lipid-anchored polymers comprise one of the following combinations: DSG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DSPE (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); and DPG (the first lipid-anchored polymer) and DPG (the second lipid-anchored polymer).
40. The lipid nanoparticle (LNP) of any one of claims 34-39, wherein the targeting moiety is conjugated to a DSPE-anchored polymer.
41. The lipid nanoparticle (LNP) of claim 40, wherein the DSPE-anchored polymer is DSPE-PEG or a derivative thereof.
42. The lipid nanoparticle (LNP) of any one of claims 34-39, wherein the targeting moiety is conjugated to a DSG-anchored polymer. 151 ME146907648v.1
43. The lipid nanoparticle (LNP) of claim 42, wherein the DSG-anchored polymer is DSG-PEG or a derivative thereof.
44. The lipid nanoparticle (LNP) of any one of claims 34-43, wherein the targeting moiety is capable of binding to a liver cell.
45. The lipid nanoparticle (LNP) of claim 44, wherein the liver cell is a hepatocyte.
46. The lipid nanoparticle (LNP) of claim 45, wherein the targeting moiety is N-acetyl galactosamine (GalNAc) or a GalNAc derivative.
47. The lipid nanoparticle (LNP) of claim 46, wherein the targeting moiety is a tri-antennary GalNAc conjugate or a tetra-antennary GalNAc conjugate.
48. The lipid nanoparticle (LNP) of claim 45, where the targeting moiety is selected from the group consisting of an ApoE protein, an ApoE polypeptide, an ApoB protein, an ApoB polypeptide, a fragment thereof, and a derivative of any of the foregoing.
49. The lipid nanoparticle (LNP) of claim 45, wherein the targeting moiety is capable of binding to ASGPR1 protein.
50. The lipid nanoparticle (LNP) of claim 34-45, wherein the targeting moiety is an antibody, antibody fragment, or an antibody derivative.
51. The lipid nanoparticle (LNP) of claim 50, wherein the antibody, antibody fragment, or antibody derivative is selected from the group consisting of a full-length antibody, an Fab, an Fab’, a single-domain antibody, a single-chain antibody, a nanobody, and a VHH.
52. The lipid nanoparticle (LNP) of any one of claims 1-51, wherein the LNP further comprises a dissociable lipid-conjugated polymer; wherein the lipid-conjugated polymer comprises: a polymer; a lipid moiety comprising at least one hydrophobic tail; and a linker connecting the polymer to the lipid moiety; wherein the at least one hydrophobic tail comprises 10-15 carbon atoms in a single aliphatic chain backbone. 152 ME146907648v.1
53. The lipid nanoparticle (LNP) of claim 52, wherein the at least one hydrophobic tail comprises 14 carbon atoms in a single aliphatic chain backbone.
54. The lipid nanoparticle (LNP) of claim 53, wherein the dissociable lipid-conjugated polymer is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) or mono-C18-PEG.
55. The lipid nanoparticle (LNP) of claim 54, wherein the mono-C18-PEG is mono-C18- PEG2000.
56. The lipid nanoparticle (LNP) of any of claims 52-55, wherein the dissociable lipid-conjugated polymer is present in the LNP in an amount of about 5%.
57. The lipid nanoparticle (LNP) of any one of claims 1-56, wherein the ionizable lipid is Ionizable Lipid 87:
Figure imgf000154_0001
or a pharmaceutically acceptable salt thereof.
58. The lipid nanoparticle (LNP) of any one of claims 1-57, wherein the ionizable lipid is present in the LNP in an amount of about 35 mol% to about 60 mol% of the total lipid present in the LNP.
59. The lipid nanoparticle (LNP) of any one of claims 1-58, wherein the sterol is present in the LNP in an amount of no less than about 30 mol% (i.e., ≥ 30 mol%) of the total lipid present in the LNP.
60. The lipid nanoparticle (LNP) of any one of claims 1-58, wherein the sterol is present in the LNP in an amount of about 20 mol% to about 45 mol% of the total lipid present in the LNP.
61. The lipid nanoparticle (LNP) of claim 59 or claim 60, wherein the sterol is present in the LNP in an amount of about 30 mol% to about 45 mol% of the total lipid present in the LNP.
62. The lipid nanoparticle (LNP) of claim 61, wherein the sterol is present in the LNP in an amount of about 35 mol% to about 45 mol% of the total lipid present in the LNP. 153 ME146907648v.1
63. The lipid nanoparticle (LNP) of claim 62, wherein the sterol is present in the LNP in an amount of about 35 mol% to about 40 mol% of the total lipid present in the LNP.
64. The lipid nanoparticle (LNP) of claim 62, wherein the sterol is present in the LNP in an amount of about 40 mol% to about 45 mol% of the total lipid present in the LNP.
65. The lipid nanoparticle (LNP) of claim 62, wherein the sterol is present in the LNP in an amount of about 35 mol% of the total lipid present in the LNP.
66. The lipid nanoparticle (LNP) of claim 62, wherein the sterol is present in the LNP in an amount of about 40 mol% of the total lipid present in the LNP.
67. The lipid nanoparticle (LNP) of claim 62, wherein the sterol is present in the LNP in an amount of about 45 mol% of the total lipid present in the LNP.
68. The lipid nanoparticle (LNP) of any one of claims 1-67, wherein the first lipid-anchored polymer is present in the LNP in an amount of about 0.005 mol% to about 5 mol% of the total lipid present in the LNP.
69. The lipid nanoparticle (LNP) of claim 68, wherein the second lipid-anchored polymer is present in the LNP in an amount of about 0.05 mol% to about 2 mol% of the total lipid present in the LNP.
70. The lipid nanoparticle (LNP) of claim 69, wherein the second lipid-anchored polymer is present in the LNP in an amount of about 0.1 mol% to about 1 mol% of the total lipid present in the LNP.
71. The lipid nanoparticle (LNP) of claim 70, wherein the second lipid-anchored polymer is present in the LNP in an amount of about 0.5 mol% of the total lipid present in the LNP.
72. The lipid nanoparticle (LNP) of any one of claims 68-71, wherein the first lipid-anchored polymer and the second lipid anchored polymer are present in the LNP in an amount of about 2.5 mol% and 0.5 mol%, respectively, of the total lipid present in the LNP.
73. The lipid nanoparticle (LNP) of any one of claims 1-72, wherein the LNP is suitable for intravenous administration. 154 ME146907648v.1
74. The lipid nanoparticle (LNP) of claim 73, wherein the LNP is less immunogenic than a reference LNP that comprises a helper lipid; and/or does not comprise the first lipid-anchored polymer and comprises a reference lipid-anchored polymer comprising one or more hydrophobic tails that each independently comprise less than 16 carbon atoms in a single aliphatic chain backbone.
75. The lipid nanoparticle (LNP) of claim 74, wherein the reference lipid-anchored polymer is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) or methoxypolyethyleneglycoloxy-N,N-ditetradecylacetamide (Ac-PEG).
76. The lipid nanoparticle (LNP) of claim 75, wherein the LNP elicits lower pro-inflammatory cytokine response than the reference LNP.
77. The lipid nanoparticle (LNP) of claim 75 or claim 76, wherein the LNP results in a lower uptake level of the TNA by a blood cell than that of the reference LNP.
78. The lipid nanoparticle (LNP) of claim 77, wherein said blood cell is selected from the group consisting of a red blood cell, a macrophage, and a peripheral blood mononuclear cell.
79. The lipid nanoparticle (LNP) of any one of claims 1-78, wherein the therapeutic nucleic acid (TNA) is selected from the group consisting of a minigene, a plasmid, a minicircle, a small interfering RNA (siRNA), a microRNA (miRNA), a guide RNA (gRNA) an antisense oligonucleotide (ASO), a ribozyme, a closed-ended DNA (ceDNA), a ministring, a doggybone™, a protelomere closed ended DNA, a dumbbell linear DNA, synthetic single stranded AAV vector, a dicer-substrate dsRNA, a small hairpin RNA (shRNA), an asymmetrical interfering RNA (aiRNA), mRNA, tRNA, rRNA, a DNA viral vector, a viral RNA vector, a non-viral vector and any combination thereof.
80. The lipid nanoparticle (LNP) of any one of claims 1-79, wherein the TNA is greater than about 200 bp or greater than about 200 nt in length.
81. The lipid nanoparticle (LNP) of claim 80, wherein the TNA is greater than about 500 bp or greater than about 500 nt in length.
82. The lipid nanoparticle (LNP) of claim 81, wherein the TNA is greater than about 1000 bp or greater than about 1000 nt in length.
83. The lipid nanoparticle (LNP) of claim 82, wherein the TNA is greater than about 4000 bp or greater than about 4000 nt in length. 155 ME146907648v.1
84. The lipid nanoparticle (LNP) of any one of claims 1-83, wherein the TNA is a closed-ended DNA (ceDNA).
85. The lipid nanoparticle (LNP) of any one of claims 1-83, wherein the TNA is a messenger RNA (mRNA).
86. The lipid nanoparticle (LNP) of any one of claims 1-85, wherein the TNA is a single-stranded nucleic acid.
87. The lipid nanoparticle (LNP) of any one of claims 1-85, wherein the TNA is a double- stranded nucleic acid.
88. A pharmaceutical composition comprising the LNP of any one of claims 1-87 and a pharmaceutically acceptable carrier.
89. A method of treating a genetic disorder in a subject, said method comprising administering to said subject an effective amount of the LNP of any one of claims 1-87 or the pharmaceutical composition of claim 88.
90. The method of claim 89, wherein said subject is a human.
91. The method claim 89 or claim 90, wherein the genetic disorder is selected from the group consisting of sickle cell anemia; melanoma; hemophilia A (clotting factor VIII (FVIII) deficiency); hemophilia B (clotting factor IX (FIX) deficiency); cystic fibrosis (CFTR); familial hypercholesterolemia (LDL receptor defect); hepatoblastoma; Wilson disease; phenylketonuria (PKU); congenital hepatic porphyria; an inherited disorder of hepatic metabolism; Lesch Nyhan syndrome; a thalassaemia; xeroderma pigmentosum; Fanconi’s anemia; retinitis pigmentosa; ataxia telangiectasia; Bloom’s syndrome; retinoblastoma; a mucopolysaccharide storage disease; a Niemann- Pick Disease; Fabry disease; Schindler disease; GM2-gangliosidosis Type II (Sandhoff Disease); Tay- Sachs disease; Metachromatic Leukodystrophy; Krabbe disease; a mucolipidosis (ML); Sialidosis Type II, a glycogen storage disease (GSD); Gaucher disease; cystinosis; Batten disease; Aspartylglucosaminuria; Salla disease; Danon disease (LAMP-2 deficiency); Lysosomal Acid Lipase (LAL) deficiency; a neuronal ceroid lipofuscinoses (NCL); a sphingolipidoses, galactosialidosis; amyotrophic lateral sclerosis (ALS); Parkinson’s disease; Alzheimer’s disease; Huntington’s disease; spinocerebellar ataxia; spinal muscular atrophy (SMA); Friedreich’s ataxia; Duchenne muscular dystrophy (DMD); a Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB); 156 ME146907648v.1 ectonucleotide pyrophosphatase 1 deficiency; generalized arterial calcification of infancy (GACI); Leber Congenital Amaurosis; Stargardt disease; wet macular degeneration (wet AMD); ornithine transcarbamylase (OTC) deficiency; Usher syndrome; alpha-1 antitrypsin deficiency; a progressive familial intrahepatic cholestasis (PFIC); and Cathepsin A deficiency.
92. The method of claim 91, wherein said genetic disorder is phenylketonuria (PKU).
93. The method of claim 91, wherein the genetic disorder is hemophilia A (Factor VIII deficiency).
94. The method of claim 91, wherein said genetic disorder is Wilson disease.
95. The method of claim 91, wherein said genetic disorder is Gaucher disease.
96. The method of claim 91, wherein said genetic disorder is Gaucher disease Type I, Gaucher disease Type II or Gaucher disease type III.
97. The method of claim 91, wherein said genetic disorder is Leber congenital amaurosis (LCA).
98. The method of claim 91, wherein said genetic disorder LCA is LCA10.
99. The method of claim 91, wherein said genetic disorder is Stargardt disease.
100. The method of claim 91, wherein said genetic disorder is wet macular degeneration (wet AMD).
101. A method of treating providing anti-tumor immunity in a subject, the method comprising administering to the subject an effective amount of the LNP of any one of claims 1-87 or the pharmaceutical composition of claim 88.
102. A method of treating a subject having a disease, disorder or condition associated with an elevated expression of a tumor antigen, the method comprising administering to the subject an effective amount of the LNP of any one of claims 1-87 or the pharmaceutical composition of claim 88.
103. The method of claim 101 or claim 102, wherein the TNA is retained in the spleen for at least about 6 hours, or at least about 9 hours, or at least about 12 hours, or at least about 15 hours, or at 157 ME146907648v.1 least about 18 hours, or at least about 21 hours, or at least about 24 hours, or at least about 27 hours, or at least about 30 hours, or at least about 33 hours, or at least about 36 hours after dosing.
104. The method of claim 103, wherein the concentration of the TNA at the start of a 12, 18, or 24- hour time window post-dosing and the concentration of the TNA at the end of the time window are within the same order of magnitude.
105. A method of treating a blood disease, disorder or condition in a subject, the method comprising administering to the subject an effective amount of the LNP of any one of claims 1-87 or the pharmaceutical composition of claim 88.
106. The method of claim 105, wherein the blood disease, disorder or condition is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Hodgkin lymphoma (HL), multiple myeloma, a myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), adrenoleukodystrophy (ALD), Hurler syndrome, Krabbe disease (Globoid-cell leukodystrophy or GLD), metachromatic leukodystrophy (MLD), severe aplastic anemia (SAA), severe combined immunodeficiency (SCID), sickle cell disease (SCD), thalassemia, Wiskott-Aldrich syndrome, Diamond-Blackfan anemia, essential thrombocytosis, Fanconi anemia, hemophagocytic lymphohistiscytosis (HLH), juvenile myelomonocytic leukemia (JMML), myelofibrosis, polycythemia vera, and a combination thereof.
107. The method of any one of claims 101-106, wherein the TNA is a messenger RNA (mRNA).
108. A lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; wherein the TNA is a deoxyribonucleic acid (DNA) or a messenger ribonucleic acid (mRNA); an ionizable lipid; a sterol; a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises: a polymer; a lipid moiety comprising at least one or two hydrophobic tails; and a linker connecting the polymer to the lipid moiety; wherein each of the at least two hydrophobic tails independently comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; and wherein the LNP does not comprise a helper lipid. 158 ME146907648v.1
109. A lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; an ionizable lipid; a sterol; a first lipid-anchored polymer and a second lipid-anchored polymer; wherein the first lipid- anchored polymer and the second lipid-anchored polymer each comprise: a polymer; a lipid moiety comprising at least one or two hydrophobic tails; and a linker connecting the polymer to the lipid moiety; wherein each of the at least two hydrophobic tails independently comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; wherein a targeting moiety conjugated to the second lipid-anchored polymer; and wherein the LNP does not comprise a helper lipid.
110. A lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; an ionizable lipid; a sterol; a first lipid-anchored polymer and a second lipid-anchored polymer; wherein the first lipid- anchored polymer and the second lipid-anchored polymer each comprise: a polymer; a lipid moiety comprising at least one or two hydrophobic tails; and a linker connecting the polymer to the lipid moiety; wherein each of the at least two hydrophobic tails independently comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; wherein a targeting moiety conjugated to the second lipid-anchored polymer; and wherein the LNP does not comprise a helper lipid and wherein the LNP is prepared by a method comprising the following steps: a) adding the TNA to a first solution comprising one or more low molecular weight alcohols to result in a TNA solution having an alcohol content of >80%; b) adding the TNA solution to a second solution comprising the ionizable lipid, the sterol, the first lipid-anchored polymer and the second lipid-anchored polymer in one or more low molecular weight alcohols to result in a TNA/lipid solution having an alcohol content of between about 80% and about 95%; 159 ME146907648v.1 c) mixing the TNA/lipid solution with an acidic aqueous buffer to form a treated TNA/lipid solution; and d) subjecting the treated TNA/lipid solution to buffer exchange with a neutral-pH aqueous buffer to produce the LNP.
111. A lipid nanoparticle (LNP) comprising: a therapeutic nucleic acid (TNA) greater than about 100 base pairs or greater than about 100 nucleotides in length; an ionizable lipid; a sterol; a first lipid-anchored polymer and a second lipid-anchored polymer; wherein the first lipid- anchored polymer and the second lipid-anchored polymer each comprise: a polymer; a lipid moiety comprising at least one or two hydrophobic tails; and a linker connecting the polymer to the lipid moiety; wherein each of the at least two hydrophobic tails independently comprises 16 to 22 carbon atoms in a single aliphatic chain backbone; wherein a targeting moiety conjugated to the second lipid-anchored polymer; and wherein the LNP does not comprise a helper lipid and wherein the LNP is prepared by a method comprising the following steps: a) adding the TNA to a solution comprising the ionizable lipid, the sterol, the first lipid- anchored polymer and the second lipid-anchored polymer in one or more low molecular weight alcohols to result in a TNA/lipid solution having an alcohol content of between about 80% and about 95%; b) mixing the TNA/lipid solution with an acidic aqueous buffer to form a treated TNA/lipid solution; and c) subjecting the treated TNA/lipid solution to buffer exchange with a neutral-pH aqueous buffer to thereby produce the LNP. 160 ME146907648v.1
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Publication number Priority date Publication date Assignee Title
WO2024249954A1 (en) 2023-05-31 2024-12-05 Capstan Therapeutics, Inc. Lipid nanoparticle formulations and compositions
CN118634207B (en) * 2024-08-15 2024-11-26 北京悦康科创医药科技股份有限公司 Nucleic acid-lipid nanoparticles for atomized inhalation and preparation method and application thereof

Family Cites Families (83)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5885613A (en) 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
EP1027033B1 (en) 1997-05-14 2009-07-22 The University Of British Columbia High efficiency encapsulation of nucleic acids in lipid vesicles
US6320017B1 (en) 1997-12-23 2001-11-20 Inex Pharmaceuticals Corp. Polyamide oligomers
US20030077829A1 (en) 2001-04-30 2003-04-24 Protiva Biotherapeutics Inc.. Lipid-based formulations
FR2824431A1 (en) 2001-05-03 2002-11-08 Mitsubishi Electric Inf Tech METHOD AND DEVICE FOR RECEIVING SIGNAL
EP1519714B1 (en) 2002-06-28 2010-10-20 Protiva Biotherapeutics Inc. Method and apparatus for producing liposomes
NZ581166A (en) 2003-09-15 2011-06-30 Protiva Biotherapeutics Inc Polyethyleneglycol-modified lipid compounds and uses thereof
WO2005121348A1 (en) 2004-06-07 2005-12-22 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering rna
US7745651B2 (en) 2004-06-07 2010-06-29 Protiva Biotherapeutics, Inc. Cationic lipids and methods of use
WO2006007712A1 (en) 2004-07-19 2006-01-26 Protiva Biotherapeutics, Inc. Methods comprising polyethylene glycol-lipid conjugates for delivery of therapeutic agents
JP5292572B2 (en) 2004-12-27 2013-09-18 サイレンス・セラピューティクス・アーゲー Coated lipid complexes and their use
US7404969B2 (en) 2005-02-14 2008-07-29 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
AU2006274413B2 (en) 2005-07-27 2013-01-10 Arbutus Biopharma Corporation Systems and methods for manufacturing liposomes
ES2430206T3 (en) 2006-10-03 2013-11-19 Tekmira Pharmaceuticals Corporation Formulation containing lipids
JP2010519203A (en) 2007-02-16 2010-06-03 メルク・シャープ・エンド・ドーム・コーポレイション Compositions and methods for enhancing the activity of bioactive molecules
US20110117125A1 (en) 2008-01-02 2011-05-19 Tekmira Pharmaceuticals Corporation Compositions and methods for the delivery of nucleic acids
PL2279254T3 (en) 2008-04-15 2017-11-30 Protiva Biotherapeutics Inc. Novel lipid formulations for nucleic acid delivery
WO2009132131A1 (en) 2008-04-22 2009-10-29 Alnylam Pharmaceuticals, Inc. Amino lipid based improved lipid formulation
WO2010006282A2 (en) 2008-07-10 2010-01-14 Serina Therapeutics, Inc. Polyoxazolines with inert terminating groups, polyoxazolines prepared from protected initiating groups and related compounds
WO2010042877A1 (en) 2008-10-09 2010-04-15 Tekmira Pharmaceuticals Corporation Improved amino lipids and methods for the delivery of nucleic acids
WO2010048536A2 (en) 2008-10-23 2010-04-29 Alnylam Pharmaceuticals, Inc. Processes for preparing lipids
WO2010054384A1 (en) 2008-11-10 2010-05-14 Alnylam Pharmaceuticals, Inc. Lipids and compositions for the delivery of therapeutics
EP3757090B1 (en) 2008-11-10 2024-06-12 Arbutus Biopharma Corporation Novel lipids and compositions for the delivery of therapeutics
CA2751342C (en) 2009-01-29 2019-05-07 Alnylam Pharmaceuticals, Inc. Lipid formulations comprising cationic lipid and a targeting lipid comprising n-acetyl galactosamine for delivery of nucleic acid
KR102229618B1 (en) 2009-05-05 2021-03-18 알닐람 파마슈티칼스 인코포레이티드 Lipid compositions
SG10201912450XA (en) 2009-06-10 2020-03-30 Arbutus Biopharma Corp Improved lipid formulation
WO2011000106A1 (en) 2009-07-01 2011-01-06 Protiva Biotherapeutics, Inc. Improved cationic lipids and methods for the delivery of therapeutic agents
JP5766188B2 (en) 2009-07-01 2015-08-19 プロチバ バイオセラピューティクス インコーポレイティッド Lipid formulations for delivering therapeutic agents to solid tumors
US9181295B2 (en) 2009-08-20 2015-11-10 Sirna Therapeutics, Inc. Cationic lipids with various head groups for oligonucleotide delivery
CA2775092A1 (en) 2009-09-23 2011-03-31 Protiva Biotherapeutics, Inc. Compositions and methods for silencing genes expressed in cancer
US20130022649A1 (en) 2009-12-01 2013-01-24 Protiva Biotherapeutics, Inc. Snalp formulations containing antioxidants
AU2010328336B2 (en) 2009-12-07 2017-03-02 Arbutus Biopharma Corporation Compositions for nucleic acid delivery
EP2526113B1 (en) 2010-01-22 2016-08-10 Sirna Therapeutics, Inc. Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry
EP2558074B1 (en) 2010-04-08 2018-06-06 The Trustees of Princeton University Preparation of lipid nanoparticles
US20130156845A1 (en) 2010-04-29 2013-06-20 Alnylam Pharmaceuticals, Inc. Lipid formulated single stranded rna
WO2011141705A1 (en) 2010-05-12 2011-11-17 Protiva Biotherapeutics, Inc. Novel cationic lipids and methods of use thereof
WO2011141704A1 (en) 2010-05-12 2011-11-17 Protiva Biotherapeutics, Inc Novel cyclic cationic lipids and methods of use
DK2575767T3 (en) 2010-06-04 2017-03-13 Sirna Therapeutics Inc HOWEVER UNKNOWN LOW MOLECULAR CATIONIC LIPIDS TO PROCESS OIGONUCLEOTIDES
US9006417B2 (en) 2010-06-30 2015-04-14 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
US20130323269A1 (en) 2010-07-30 2013-12-05 Muthiah Manoharan Methods and compositions for delivery of active agents
AU2011291582A1 (en) 2010-08-20 2013-03-07 Cerulean Pharma Inc. Conjugates, particles, compositions, and related methods
PT3981427T (en) 2010-08-31 2022-06-27 Glaxosmithkline Biologicals Sa Pegylated liposomes for delivery of immunogen-encoding rna
JP5961170B2 (en) 2010-09-20 2016-08-02 サーナ・セラピューティクス・インコーポレイテッドＳｉｒｎａ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． Novel low molecular weight cationic lipids for oligonucleotide delivery
JP2013545723A (en) 2010-09-30 2013-12-26 メルク・シャープ・エンド・ドーム・コーポレイション Low molecular weight cationic lipids for oligonucleotide delivery
US9029590B2 (en) 2010-10-21 2015-05-12 Sirna Therapeutics, Inc. Low molecular weight cationic lipids for oligonucleotide delivery
US9999673B2 (en) 2011-01-11 2018-06-19 Alnylam Pharmaceuticals, Inc. PEGylated lipids and their use for drug delivery
WO2012162210A1 (en) 2011-05-26 2012-11-29 Merck Sharp & Dohme Corp. Ring constrained cationic lipids for oligonucleotide delivery
CN103764121A (en) 2011-07-06 2014-04-30 诺华股份有限公司 Liposomes having useful N:P ratio for delivery of RNA molecules
WO2013016058A1 (en) 2011-07-22 2013-01-31 Merck Sharp & Dohme Corp. Novel bis-nitrogen containing cationic lipids for oligonucleotide delivery
BR112014004607A2 (en) 2011-08-31 2017-03-21 Novartis Ag pegylated liposomes for immunogenic encoded RNA delivery
WO2013049328A1 (en) 2011-09-27 2013-04-04 Alnylam Pharmaceuticals, Inc. Di-aliphatic substituted pegylated lipids
KR102046968B1 (en) 2011-11-04 2019-12-02 닛토덴코 가부시키가이샤 Single use system for sterilely producing lipid-nucleic acid particles
CN103930398B (en) 2011-11-18 2016-08-24 日油株式会社 Cationic lipids with improved intracellular kinetics
EP2788316B1 (en) 2011-12-07 2019-04-24 Alnylam Pharmaceuticals, Inc. Branched alkyl and cycloalkyl terminated biodegradable lipids for the delivery of active agents
CA2856742A1 (en) 2011-12-07 2013-06-13 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
WO2013086373A1 (en) 2011-12-07 2013-06-13 Alnylam Pharmaceuticals, Inc. Lipids for the delivery of active agents
WO2013089151A1 (en) 2011-12-12 2013-06-20 協和発酵キリン株式会社 Lipid nanoparticles for drug delivery system containing cationic lipids
WO2013116126A1 (en) 2012-02-01 2013-08-08 Merck Sharp & Dohme Corp. Novel low molecular weight, biodegradable cationic lipids for oligonucleotide delivery
EP3473611B1 (en) 2012-02-24 2021-10-20 Arbutus Biopharma Corporation Trialkyl cationic lipids and methods of use thereof
EP2830594B1 (en) 2012-03-27 2018-05-09 Sirna Therapeutics, Inc. DIETHER BASED BIODEGRADABLE CATIONIC LIPIDS FOR siRNA DELIVERY
US20160151284A1 (en) 2013-07-23 2016-06-02 Protiva Biotherapeutics, Inc. Compositions and methods for delivering messenger rna
EP3871696B1 (en) 2013-10-22 2025-03-19 Translate Bio, Inc. Lipid formulations for delivery of messenger rna
CN105873902B (en) 2013-11-18 2019-03-08 阿克丘勒斯治疗公司 Ionizable cationic lipids for RNA delivery
US9365610B2 (en) 2013-11-18 2016-06-14 Arcturus Therapeutics, Inc. Asymmetric ionizable cationic lipid for RNA delivery
ES2774968T3 (en) 2013-12-19 2020-07-23 Novartis Ag Lipids and lipid compositions for the administration of active agents
EP4019506A1 (en) 2013-12-19 2022-06-29 Novartis AG Lipids and lipid compositions for the delivery of active agents
PT3766916T (en) 2014-06-25 2022-11-28 Acuitas Therapeutics Inc Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids
SI3221293T1 (en) 2014-11-18 2023-05-31 Arcturus Therapeutics, Inc. Ionizable cationic lipid for rna delivery
US11669953B2 (en) 2015-01-30 2023-06-06 Hitachi High-Tech Corporation Pattern matching device and computer program for pattern matching
WO2016121942A1 (en) 2015-01-30 2016-08-04 日油株式会社 Cationic lipid
AU2016285852B2 (en) 2015-06-29 2020-12-17 Acuitas Therapeutics Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2017049245A2 (en) 2015-09-17 2017-03-23 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
LT3368507T (en) 2015-10-28 2023-03-10 Acuitas Therapeutics Inc. NOVEL LIPIDS AND LIPID NANOPARTICLE COMPOSITIONS FOR NUCLEIC ACID DELIVERY
JP7080172B2 (en) 2015-12-10 2022-06-03 モデルナティエックス インコーポレイテッド Compositions and Methods for Delivery of Therapeutic Agents
WO2017117528A1 (en) 2015-12-30 2017-07-06 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US20180020547A1 (en) 2016-07-13 2018-01-18 Alcatel-Lucent Canada Inc. Underlying recessed component placement
US12064513B2 (en) 2018-03-27 2024-08-20 Nof Corporation Cationic lipid exhibiting improved intracellular dynamics
CN116437964A (en) 2020-07-17 2023-07-14 世代生物公司 Methods for encapsulation of polynucleotides into lipid nanoparticles of reduced size and novel formulations thereof
WO2022216787A2 (en) * 2021-04-08 2022-10-13 City Of Hope Lipid nanoparticles and methods of use
BR112023021504A2 (en) * 2021-04-20 2023-12-19 Generation Bio Co CATIONIC LIPIDS AND COMPOSITIONS THEREOF
AU2022288618A1 (en) 2021-06-07 2023-12-14 Generation Bio Co. Apoe and apob modified lipid nanoparticle compositions and uses thereof
CN113908292B (en) * 2021-10-13 2022-08-16 南京吉迈生物技术有限公司 Target-mediated nucleic acid nano preparation and preparation method thereof
US20250222129A1 (en) * 2022-03-21 2025-07-10 National Research Council Of Canada Lipid Based Nanoparticles for Targeted Gene Delivery to the Brain

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