WO2024081639A1 - Novel lipid nanoparticle compositions for the delivery of nucleic acids - Google Patents

Novel lipid nanoparticle compositions for the delivery of nucleic acids Download PDF

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Publication number
WO2024081639A1
WO2024081639A1 PCT/US2023/076457 US2023076457W WO2024081639A1 WO 2024081639 A1 WO2024081639 A1 WO 2024081639A1 US 2023076457 W US2023076457 W US 2023076457W WO 2024081639 A1 WO2024081639 A1 WO 2024081639A1
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lipid
lipid nanoparticle
nanoparticle
phospholipid
peg
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PCT/US2023/076457
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French (fr)
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Amit Sagi
Paige DAVIS
Rob BURKE
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Seawolf Therapeutics, Inc.
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Publication of WO2024081639A1 publication Critical patent/WO2024081639A1/en

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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—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links

Definitions

  • nucleic acid there are many instances in which nuclear delivery of a nucleic acid is desired, where such instances include research, diagnostic and therapeutic applications.
  • a therapeutic application is gene therapy.
  • viral vectors such as vectors based on the virus AAV
  • AAV AAV genome is limited in size, so any gene greater than 4.7kB will not be suitable for use AAV vectors, which limits the utility of such vectors for many indications.
  • viral vectors, such as AAV induce an antibody response, such that they can only be delivered once, which is not suitable for some indications, such as indications in the liver where the cells are slowly dividing and will lose the transduced genome, thereby requiring redosing.
  • viral vector such as AAV are toxic at the doses that are required for a therapeutic benefit in some indications. Accordingly, what is needed is a new delivery vehicle for delivering nucleic acids, such as DNA, to cells, particularly in patients in need of gene therapy but also in vitro during research.
  • That next generation delivery vehicle is a nanoparticle.
  • lipid nanoparticles LNPs
  • the problem is that the nucleic acid material packaged in a LNP will impact the physiochemical properties of the LNP, which in turn will impact the LNP pharmacokinetic profile, that is, the effect of the LNP on the body.
  • nucleic acids e.g., DNA and/or RNA
  • novel LNP formulations that are more efficacious and less immunostimulatory.
  • Novel lipid nanoparticle (LNP) compositions are provided for the delivery of nucleic acid such as DNA and/or RNA to cells in vitro and in vivo with different and improved pharmacokinetic profiles as compared to what is typically observed in the art. Also provided are methods for using compositions of the invention in research and as therapeutics.
  • nucleic acid such as DNA and/or RNA
  • FIGs. 1 A-1C describe studies performed to assess how the mol percentage of ionizable lipid and the type of phospholipid impact efficacy and toxicity of DNA-LNPs.
  • FIG. 1 A Formulation details for the test articles.
  • the ionizable lipid in all formulations is ALC-0315, varied to be 40%, 50%, or 60% of the lipid composition.
  • ALC-0315 is an exemplary cationic lipid [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2- hexyl decanoate).
  • the phospholipid is DSPC or DOPE.
  • Plasmid DNA comprising a CBH promoter driving the expression of an EPO transgene was formulated into LNPs. Good encapsulation efficiency and small size were observed of all test articles.
  • EPO (FIG. IB)
  • IL-6 (FIG. 1C) serum levels in wild type BALB/c mice were measured 4 hours post-i.v. administration of test articles at a dose of 1 mg/kg.
  • FIGs. 2A-2N describe the test articles and results of an experiment to assess the impact of different phospholipids, different PEG lipids, and the inclusion of the livertargeting moiety, GalNAc, on the efficacy and toxicity of DNA-LNPs.
  • FIG. 2A Formulation details for the test articles.
  • the ionizable lipid in all formulations is ALC-0315, although it will be appreciated that other ionizable lipids may be used.
  • LNPs in groups 1-3 and 7-9 comprise phospholipid DSPC while LNPs in groups 4-6 and 10-12 comprise phospholipid DOPE.
  • LNPs in groups 1-6 comprise PEG-DMG[2K], while LNPs in groups 7-12 comprise PEG-DSG[2K] and GalNAc-PEG-lipid.
  • Nanoplasmid DNA comprising an expression cassette with a human AAT promoter driving expression of an EPO transgene was formulated into LNPs.
  • FIG. 2B The structure of the GalNAc-PEG-lipid used in groups 7-12.
  • EPO levels in serum were recorded 3 days post-administration (FIG. 2C) and in a time course over 28 days post-administration (l.Omg/kg, FIG. 2E; 0.3 mg/kg, FIG. 2F; 0.1 mg/kg, FIG. 2G).
  • Cytokine levels in serum were recorded 4 hours postadministration (FIGs. 2D, 2H, 21, 2J, 2K, 2L, 2M).
  • Benchmark levels of EPO achieved by other therapeutic modalities are indicated as (i) RETACRIT dose for chronic kidney disease, (ii) AAV5.EPO 2E13 vg injected i.v. and assessed at 4 weeks.
  • FIGs. 3 A-3I describe the test articles and results of an experiment to assess how varying the length and saturation of the carbon chain of the phospholipid phosphatidylcholine (PC) used as a helper lipid in the DNA-LNP impact the efficacy and toxicity of the DNA- LNP.
  • PC variants comprising 16-, 18-, 20-, 22-, or 24-carbon tails and 0, 1, or 2 double bonds were assessed.
  • Nanoplasmid DNA comprising an expression cassette with a human AAT promoter and EPO transgene was formulated into LNPs.
  • FIGs. 3B, 3C, 3D, 3E, 3F, 3G, 3H, 31 EPO and cytokine levels in serum of wild type mice were measured post- i.v. administration of test articles. EPO levels were recorded 4 hours (FIG. 3B) and 2 days (FIG. 3C) post-administration, and cytokine levels were recorded 4 hours post-administration (IL-lbeta, FIG. 3D; IL-12, FIG.3E; IFNgamma, FIG. 3F; IL-6, FIG. 3G; KC, FIG. 3H; TNF alpha, FIG. 31).
  • FIGs. 4A-4I describe the test articles of an experiment to assess how varying the length and saturation of the carbon chain of the phospholipid phosphatidylethanolamine (PE) used as a helper lipid in the DNA-LNP impact the efficacy and toxicity of the DNA-LNP.
  • PE variants comprising 16-, 18-, 20-, 22-, or 24-carbon tails and 0, 1, or 2 double bonds were assessed.
  • Nanoplasmid DNA comprising an expression cassette with a human AAT promoter and EPO transgene was formulated into LNPs.
  • FIGs. 4B, 4C, 4D, 4E, 4F, 4G, 4H, 4J EPO and cytokine levels in serum of wild type mice were measured post-i.v. administration of test articles. EPO levels were recorded 4 hours (FIG. 4B) and 2 days (FIG. 4C) post-administration, and cytokine levels were recorded 4 hours post-administration (IL- lbeta, FIG. 4D; IL-12, FIG.4E; IFNgamma, FIG. 4F; IL-6, FIG. 4G; KC, FIG. 4H; TNFalpha, FIG. 41).
  • FIGs. 5A-5C describe the test articles of an experiment to assess the impact of introducing ring structures into the tails of the phospholipid phosphatidylcholine (PC) used as a helper lipid in the DNA-LNP on the efficacy and toxicity of the DNA-LNP.
  • FIGs. 5A-5C additionally describe the impact of varying the length and saturation of the carbon chain of the phospholipid phosphatidylcholine (PC) used as a helper lipid in the DNA-LNP on the efficacy and toxicity of the DNA-LNP.
  • PC variants PChcPC, PChemsPC, DChemsPC, and OChemsPC which contain ring structures in their tails, were assessed.
  • FIG. 5A Formulation details for the test articles.
  • the ionizable lipid in all formulations is ALC-0315, although it will be appreciated that other ionizable lipids may be used.
  • Nanoplasmid DNA (npDNA) comprising an expression cassette with a human AAT promoter and EPO transgene was formulated into LNPs.
  • FIGs. 5B-5C EPO and IL-6 cytokine levels in serum of wild type mice were measured post-i.v. administration of test articles. EPO levels were recorded 3 days post-administration (FIG 5B), and cytokine levels were recorded 4 hours post-administration (FIG. 5C).
  • FIGs. 6A-6B provide a summary of the efficacy and tolerability of LNPs made with some of the best-performing helper lipids.
  • FIG. 6A documents EPO expression 7 days postadministration.
  • FIG. 6B documents IL-6 levels at 4 hours post-administration.
  • FIGs. 7A-7B documents phospholipid transition temperature relative to EPO expression 7 days post-administration (FIG, 7A) and IL-6 levels at 4 hours postadministration (FIG. 7B).
  • FIGs 8A-8C illustrate that the observations made regarding the superiority of atypical helper lipids for formulating DNA payloads into LNPs also apply to co-formulating DNA+RNA payloads into LNPs.
  • PC and PE variants comprising 16- or 18-carbon tails with 0 or 1 double bond were assessed. (FIG.
  • the ionizable lipid used in these formulations is either ALC- 0315 or the novel ionizable lipid L-15.
  • Nanoplasmid DNA comprising an expression cassette with a human AAT promoter and EPO transgene, along with an mRNA payload were co-formulated into LNPs.
  • FIG 8B-8C EPO and IL-6 cytokine levels in serum of wild type mice were measured post-i.v. administration of test articles. EPO levels were recorded 3 days post-administration (FIG. 8B), and IL-6 cytokine levels were recorded 4 hours post-administration (FIG. 8C). 5.
  • Novel lipid nanoparticle compositions are provided for the delivery of nucleic acid such as DNA to cells in vitro and in vivo with different and improved pharmacokinetic profiles as compared to what is typically observed in the art. Also provided are methods for using the lipid nanoparticle compositions of this disclosure in research and as therapeutics.
  • a “lipid nanoparticle” refers to a lipid composition that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., DNA and/or RNA), a protein, a small molecule, and the like to a target site of interest.
  • an active agent or therapeutic agent such as a nucleic acid (e.g., DNA and/or RNA), a protein, a small molecule, and the like.
  • a nucleic acid agent may be encapsulated in the lipid, thereby protecting the agent from enzymatic degradation.
  • the lipid nanoparticle includes several lipid components, including e.g., an ionizable lipid, one or more helper lipid(s) (e.g., non-cationic lipid(s)), and a lipid that prevents aggregation of the nanoparticle (also referred to as a coat lipid or conjugated lipid e.g., a PEG-lipid).
  • a lipid nanoparticle (LNP) composition as described herein comprising a nucleic acid, where the nucleic acid is substantially encapsulated by the lipid components of the LNP.
  • the lipid nanoparticles (LNPs) of this disclosure can include an ionizable lipid.
  • the ionizable lipid is typically employed in the LNP to condense the nucleic acid cargo, e.g., DNA, at low pH and to drive membrane association and fusogenicity.
  • the term “ionizable lipid” refers to a lipid comprising an ionizable group that carries a net charge at a selected pH (e.g., a pH of 6.5 or less), but which can remain neutral at, e.g., a higher pH, such as physiological pH.
  • the pH-sensitivity of such ionizable lipids can be desirable to provide for intracellular delivery of nucleic acid cargo.
  • Ionizable lipids can have less interactions with cell membranes when neutral, and then become charged when internalized into endosomes in a target cell, where the pH is lower than in the extracellular environment. Ionizable lipids which are protonated and, therefore, become positively charged, may promote membrane destabilization and facilitate endosomal escape of the nanoparticle.
  • the ionizable lipid is a cationic lipid.
  • the term “cationic lipid” refers to a lipid that carries a net positive charge at a selected pH (e.g., a pH of 6.5 or less).
  • the ionizable lipids are cationic lipids including at least one ionizable amino group that is positively charged or becomes protonated at a selected pH, for example at pH of 6.5 or lower.
  • the cationic lipid includes one or more tertiary amino groups, e.g., a trialkyl amino group.
  • the cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group, hydrocarbon chains (e.g., C8-C20 carbon chains, such as Cis alkyl chains), ether linkages between the head group and hydrocarbon chains, and 0 to 3 double bonds per hydrocarbon chain.
  • a protonatable tertiary amine e.g., pH titratable
  • hydrocarbon chains e.g., C8-C20 carbon chains, such as Cis alkyl chains
  • ether linkages between the head group and hydrocarbon chains e.g., 0 to 3 double bonds per hydrocarbon chain.
  • Non-limiting examples of cationic lipids are described in detail herein.
  • Cationic lipids and related analogs, which are useful in the lipid nanoparticles of the present disclosure, include but are not limited to, those lipids described in U.S. Patent Publication Nos.
  • Additional cationic lipids of interest include, but are not limited to, l,2-distearyloxy-N,N-dimethyl-3- ami nopropane (DSDMA), 1,2 -dilinol eyloxy-N,N -dimethyl -3 -aminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), l,2-dioleyloxy-N,N- dimethyl-3 -aminopropane (DODMA), and heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA).
  • DSDMA 1,2 -dilinol eyloxy-N,N -dimethyl -3-aminopropane
  • DLenDMA 1,2 -dilino
  • LNPs of this disclosure can also include one or more helper lipids in addition to the ionizable lipid component described herein.
  • the helper lipid is a neutral lipid.
  • the neutral lipid is zwitterionic, e.g., has an overall net zero charge.
  • a helper lipid comprises an internal anionic moiety and internal cationic moiety, e.g., a phosphate and an ammonium.
  • the LNPs of this disclosure include a helper lipid component that includes a neutral lipid (e.g., zwitterionic or uncharged) that is a phospholipid.
  • a neutral lipid e.g., zwitterionic or uncharged
  • the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG).
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PG phosphatidylglycerol
  • the phospholipid has a hydrocarbon chain, or “tail” having 12- 24 carbons, e.g. 16-20 carbons, 18-22 carbons, 12-18 carbons. In some embodiments, phospholipid has a carbon tail of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbons. In some embodiments, the phospholipid tail comprises no double bonds, i.e. the bonds are saturated bonds. In some embodiments, the phospholipid tail is unsaturated, that is, it comprises one or more double bonds, e.g. 1, 2, 3, 4 or 5 double bonds. In some embodiments, the phospholipid tail is unsaturated, that is, it comprises one or more triple bonds, e.g. 1, 2, 3, 4 or 5 triple bonds.
  • the phospholipid tail comprises one or more ring structures.
  • the one or more ring structures is selected from 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl; 5- to 6- membered aryl; 7- to 10-membered saturated or partially unsaturated bicyclic carbocyclyl; and 7- to 10-membered bicyclic aryl wherein each ring structure is independently substituted with 0-7 R A groups; each R A is independently selected from halogen, or an optionally substituted group selected from C1-12 aliphatic, phenyl, or 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl.
  • the ring structure is a cholesterol or cholesterol derivative.
  • the phospholipid is symmetric, i.e., all tails of the phospholipid are the same. In other embodiments, the phospholipid is asymmetric, i.e., the phospholipid comprises two or more different hydrocarbon chains.
  • a helper lipid is or comprises symmetric or asymmetric aliphatic phospholipid moieties that are each independently optionally substituted, branched or straight, partially unsaturated or saturated C9-C24 aliphatic.
  • the helper lipid comprises one or more optionally substituted and/or optionally bridged ring structures in the hydrophobic tail.
  • exemplary helper lipids of this class include:
  • the helper lipid includes a phosphatidylethanolamine (PE).
  • PE Phosphatidylethanolamines
  • the phosphatidylethanolamine selected from the group consisting of phosphatidylethanolamine, dioleoylphosphatidylethanolamine (1,2- dioleyl-sn-glycero-3-phosphoethanolamine) (18: 1(A9-Cis) PE, or DOPE), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine) (DPPE), dimyristo
  • 16-0-monom ethyl PE dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), dielaidoyl-phosphatidylethanolamine (DEPE), lysophosphatidylethanolamine, 1,2- dilauroyl-sn-glycero-3 -phosphoethanolamine (DLPE), and l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DiPPE).
  • SOPE l-stearoyl-2-oleoyl-phosphatidylethanolamine
  • DEPE dielaidoyl-phosphatidylethanolamine
  • DLPE 1,2- dilauroyl-sn-glycero-3 -phosphoethanolamine
  • DiPPE diphytanoyl-sn-glycero-3- phosphoethanolamine
  • the phosphatidylethanolamine is di oleoylphosphatidylethanolamine (also referred to as l ,2-dioleoyl-.s//-glycero-3- phosphoethanolamine, 18: 1(A9-Cis) PE, “18-1 PE”, “18: 1 PE”, or DOPE), having a tail of 18 carbons and one saturated bond as shown below:
  • the phosphatidylethanolamine is 1,2-dipalmitoleoyl-sn- glycero-3 -phosphoethanolamine (also referred to as “16-1 PE” or “16: 1 PE”), having a tail of 16 carbons and one saturated bond:
  • the phosphatidylethanolamine is l-stearoyl-2-oleoyl-sn- glycero-3 -phosphoethanolamine (also referred to as “18-0/18-1 PE”, “18:0/18: 1 PE”, or SOPE), an asymmetric lipid having one tail of 18 unsaturated hydrocarbons and a second tail of 18 carbons with one saturated bond:
  • the helper lipid includes a phosphatidylcholine (PC).
  • PC Phosphatidylcholines
  • phosphatidylcholine is selected from the group consisting of phosphatidylcholine, di stearoylphosphatidylcholine (l,2-distearoyl-sn-glycero-3- phosphocholine) (DSPC), dioleoylphosphatidylcholine (l,2-dioleoyl-sn-glycero-3- phosphocholine) (18: 1(A9-Cis) PC, or DOPC), dipalmitoylphosphatidylcholine (1,2- dipalmitoyl-sn-glycero-3 -phosphocholine) (DPPC), l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine
  • the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) (also referred to as l,2-distearoyl-sw-glycero-3-phosphocholine, “18-0 PC” or “18:0 PC”), having a tail of 18 carbons and no saturated bonds as shown below:
  • DSPC distearoylphosphatidylcholine
  • the phosphatidylcholine is dioleoylphosphatidycholine (also referred to as l,2-dioleoyl-sn-glycero-3 -phosphocholine, “18-1 PC”, “18: 1 PC”, or DOPC), in some instances 18: 1(A9-Cis) PC having a tail of 18 carbons and one saturated bond as shown below:
  • the phosphatidylcholine is l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine (also referred to as “16-1 PC” or “16: 1 PC”), in some embodiments 16: 1(A9- Cis) PC having a tail of 16 carbons and one saturated bond as shown below:
  • the phosphatidylcholine is an asymmetric lipid, having one tail of 16 carbons and a second tail of 18 carbons.
  • the tail of the phosphatidylcholine having 18 carbons has one saturated bond, e,g, it is l-palmitoyl-2- oleoyl-glycero-3 -phosphocholine (also referred to as “16-0/18-1 PC”, “16:0/18: 1 PC” or POPC) as shown below:
  • the phosphatidylcholine is 1,2-dicholesterylhemisuccinoyl- sn-glycero-3 -phosphocholine (DChemsPC), as shown below:
  • the phosphatidylcholine is l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), as shown below: [0037] In certain embodiments, the phosphatidylcholine is l-palmitoyl-2- cholesterylcarbonoyl-sn-glycero-3-phosphocholine (PChcPC), as shown below:
  • the phosphatidylcholine is l-palmitoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), as shown below:
  • the helper lipid includes a phosphatidylglycerol selected from the group consisting of phosphatidylglycerol, dioleoylphosphatidylglycerol (1,2-dioleoyl-sn- glycero-3- phospho-(l’-rac-glycerol) (DOPG), dipalmitoylphosphatidylglycerol, (DPPG), dimyristoyl phosphatidylglycerol (DMPG), di stearoylphosphatidylglycerol (DSPG), and palmitoyloleyolphosphatidylglycerol (POPG).
  • DOPG dipalmitoylphosphatidylglycerol
  • DPPG dimyristoyl phosphatidylglycerol
  • DSPG di stearoylphosphatidylglycerol
  • POPG palmitoyloleyolphosphatidylglycerol
  • the helper lipid includes a phosphatidylserine, e.g. phosphatidyl serine or dioleoylphosphatidylserine (DOPS).
  • a phosphatidylserine e.g. phosphatidyl serine or dioleoylphosphatidylserine (DOPS).
  • DOPS dioleoylphosphatidylserine
  • the helper lipid includes a lecithin, e.g. lecithin or lysolecithin.
  • the helper lipid includes a sphingomyelin (SM), e.g., egg sphingomyelin (ESM).
  • SM sphingomyelin
  • ESM egg sphingomyelin
  • the helper lipid is cephalin, cardiolipin, phosphatidic acid, cerebrosides, or dicetylphosphate. In some embodiments, the helper lipid is cephalin. In some embodiments, the helper lipid is cardiolipin. In some embodiments, the helper lipid is phosphatidicacid. In some embodiments, the helper lipid is cerebroside. In some embodiments, the helper lipid is ganglioside. In some embodiments, the helper lipid is dicetylphosphate. [0044] In some embodiments, the helper lipid has a transition temperature between about - 50°C and 50°C.
  • LNPs comprising phospholipids having phase transition temperatures within about -50°C and 50°C are better tolerated by the organism, promoting more efficacious delivery of the nucleic acid cargo and/or eliciting lower levels of cytokines such as IL-6 than LNPs produced with phospholipids having phase transition temperatures outside of this range.
  • LNPs comprising phospholipids having phase transition temperatures within -50°C and 50°C elicit only about 3, 4, or 5-fold more cytokines than baseline (baseline being defined as untreated or treated with buffer or excipient), and 5- to 10-fold, in some instances 15-fold, in certain cases 25-fold, less cytokines than comparable LNPs formulated with a phospholipid having a transition temperature outside of this range, e.g. DSPC (transition temperature of 55°C).
  • the helper lipid has a transition temperature between about -50°C and 50°C.
  • the LNP of the present disclosure comprises a phospholipid having a transition temperature between about -40°C and 30°C.
  • the LNP of the present disclosure comprises a phospholipid having a transition temperature between about -35°C and 25°C.
  • the LNP is not 18: 1(A9-Cis) PE (DOPE).
  • Phase transition temperatures of phospholipids are well known in the art and can be determined by referencing various publicly available databases such as the Avanti Polar website and the Encyclopedia of Biophysics, Gordon C.K. Roberts editor, Vol. 1, pages 1841-1854, or by calculation based on chain length and degree of saturation.
  • the phase transition temperatures of exemplary phospholipids are provided in Table 1.
  • the LNP of the present disclosure comprises a phospholipid that is selected from the group consisting of 16: 1 PC (e.g.
  • A9-Cis 16:0/22:6 PC, 18: 1 PC (e.g A9-Cis, A6- Cis, A9-trans), 12:0 PC, 16:0/18: 1 PC, 20: 1 PC, 18:0/18: 1 PC, 18: 1/18:0 PC, 22: 1 PC (e.g. A13-Cis), 13:0 PC, 14:0 PC, 16:0/14:0 PC, 18:0/14:0 PC, 16: 1 PE, 18: 1 PE (e.g. A9-Cis), 16:0/18: 1 PE, 18:0/18: 1 PE and 12:0 PE.
  • A9-Cis 16:0/18: 1 PE, 18:0/18: 1 PE and 12:0 PE.
  • the LNP of the present disclosure comprises a phospholipid that is selected from the group consisting of 16: 1(A9- Cis) PC, 18: 1(A9-Cis) PC, 16:0/18: 1 PC, 20:1 PC, 16: 1 PE, 18: 1(A9-Cis) PE (DOPE), and 18:0/18: 1 PE.
  • the LNP is not 18: 1(A9-Cis) PE (DOPE). [0045] Table 1. Phospholipid phase transition temperatures
  • the LNP comprising the atypical phospholipid achieves 5-fold more expression or more, e.g. 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold more expression or more, in some instances 12-fold, 15-fold, 20-fold more expression or more, of the delivered nucleic acid cargo one week after delivery of the same dose of a comparable LNP comprising DSPC instead of the atypical phospholipid.
  • the LNP comprising the atypical phospholipid achieves 5-fold more expression or more, e.g.
  • the LNP comprising the atypical phospholipid achieves 5-fold more expression or more, e.g.
  • RNA levels for example by RT-PCR, qRT-PCR, Northern blot and the like
  • protein levels by, for example, ELISA, Western blot, and the like
  • measuring a functional change as the result of a new activity e.g. increased hematocrit with more EPO expression, an increase in clotting with more clotting factor, improvement in symptoms in a disease, etc.
  • the LNP comprising the atypical phospholipid elicits 2-fold less cytokines in serum or less, e.g. 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15- fold, 20-fold, 40-fold, 80-fold, in some instances 100-fold less cytokines, 4 hours after delivery of the same dose of a comparable LNP comprising DSPC instead of the atypical phospholipid.
  • the LNP comprising the atypical phospholipid elicits 2- fold less cytokines in serum or less, e.g.
  • the LNP comprising the atypical phospholipid elicits 5-fold less cytokines in serum or less, e.g. e.g.
  • RNA levels for example by RT-PCR, qRT-PCR, Northern blot and the like; or measuring protein levels by, for example, ELISA, Western blot, and the like; or measuring a functional change as the result of a new activity (e.g.
  • Changes in cytokines elicited may likewise be measured by any of a number of methods know in the art, including, e.g. measuring levels in serum by, e.g. ELISA.
  • the LNP can further comprise a component, such as a sterol, to provide membrane integrity.
  • a component such as a sterol
  • a component such as a sterol
  • the component providing membrane integrity, such as a sterol can comprise 0-50% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20- 50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.
  • the neutral lipid component of the LNPs can further include cholesterol or a derivative or analog thereof.
  • the helper lipid component includes cholesterol.
  • the LNP includes a neutral lipid component that includes a mixture of phospholipid and cholesterol or a derivative or analog thereof.
  • the LNP includes a neutral lipid component that includes a phosphatidylethanolamine phospholipid and cholesterol or a derivative or analog thereof.
  • the LNP includes a neutral lipid component that includes DOPE phospholipid and cholesterol. In some embodiments, the LNP includes a neutral lipid component that includes DSPC phospholipid and cholesterol. In some embodiments, the LNP includes a neutral lipid component that includes DOPC phospholipid and cholesterol.
  • LNPs of this disclosure can also include one or more additional lipid components.
  • Such lipids can be selected to provide for a desirable profile of nanoparticle properties, such as particle stability, delivery efficacy, tolerability and biodistribution.
  • the LNP can further comprise a non-cationic lipid.
  • Non-ionic lipids include amphipathic lipids, neutral lipids and anionic lipids. Accordingly, the non-cationic lipid can be a neutral uncharged, zwitterionic, or anionic lipid.
  • Non-cationic lipids are typically employed to enhance fusogenicity. Exemplary non-cationic lipids envisioned for use in the methods and compositions are described in International Application PCT/US2018/050042 published as WO2019051289A1. Exemplary non-cationic lipids are described in International application Publication WO2017/099823 and US patent publication US2018/0028664.
  • the LNP includes one or more lipids capable of reducing aggregation.
  • a lipids capable of reducing aggregation includes at least a hydrocarbon tail or chain linked to a hydrophilic group which is capable of being configured at the surface of the LNP and provide for reduced LNP aggregation.
  • the lipid capable of reducing aggregation is sometimes referred to as a conjugated lipid or coat lipid.
  • a lipid capable of reducing aggregation of particles may comprise a conjugated lipid molecule, such as a polyethylene glycol (PEG). Generally, these are used to inhibit aggregation of lipid nanoparticles and/or provide steric stabilization.
  • exemplary conjugated lipids include, but are not limited to, polyethyleneglycol (PEG)-lipid conjugate, polyoxazoline (POZ)-lipid conjugates, a polyamide (ATTA)-lipid conjugate, a cationic- polymer-lipid conjugates (CPLs), or mixtures thereof.
  • the LNPs comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate.
  • the PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL.
  • the lipid capable of reducing aggregation is a PEG-lipid.
  • a PEG-lipid refers to a lipid having one or more hydrocarbon tail(s) linked to one or more polyethylene glycol (PEG) moiety(ies) via an optional linker.
  • the PEG moieties may include terminal modification(s) to provide for e.g., conjugation to the lipid tails via the optional linker.
  • the PEG moiety may be terminated in as a hydroxyl group, or an alkyl ether (e.g., a methoxy terminal group).
  • the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.
  • PEG-lipids of interest include, but are not limited to, a PEG-diacylglycerol (DAG), a PEG di alkyl oxy propyl (DAA), a PEG- phospholipid, a PEG-ceramide (Cer), or mixtures thereof.
  • the PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG- dipalmityloxypropyl (C 16), a PEG-distearyloxypropyl (C 18), or mixtures thereof.
  • Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol
  • DAG (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG- DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a PEGylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS- DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-l-O-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl- methoxypolyethylene glycol 2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof.
  • DAG PEG-dialkyloxypropy
  • PEG-lipid conjugates are described, for example, in U.S. Pat. Nos. 5,885,613, 6,287,591, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2016/0376224, and US2017/0119904.
  • a PEG-lipid is a compound disclosed in US2018/0028664.
  • a PEG-lipid is disclosed in US20150376115 or in US2016/0376224.
  • the PEG-DAA conjugate can be, 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- omegal-methyl-poly(ethylene glycol), PEG- DMB (3,4-Ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-
  • lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid.
  • PEG-lipid conjugates polyoxazoline (POZ)-lipid conjugates, polyamidelipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (CPL) conjugates can be used in place of or in addition to the PEG-lipid.
  • POZ polyoxazoline
  • CPL cationic-polymer lipid
  • conjugated lipids i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymerlipids are described in the International patent application publications WO 1996/010392, WO1998/051278, W02002/087541, W02005/026372, WO2008/147438, W02009/086558, WO20 12/000104, WO2017/117528, WO2017/099823, WO2015/199952, WO2017/004143, WO2015/095346, WO2012/000104, WO2012/000104, and WO2010/006282, US patent application publications US2003/0077829, US2005/0175682, US2008/0020058,
  • the liver can be a target organ of interest in part due to its central role in metabolism and production of proteins and accordingly diseases which are caused by defects in liver-specific gene products (e.g., the urea cycle disorders) and may benefit from specific targeting of cells (e.g., hepatocytes).
  • the LNP further includes a component including a targeting ligand.
  • the targeting ligand can be selected as desired based on a target sell or tissue to which it is desired to direct the LNPs of this disclosure.
  • the targeting ligand is a ligand of a cell surface receptor.
  • the cell surface receptor is asialoglycoprotein receptor (ASGPR).
  • the ASGPR is expressed on the surface of hepatocyte cells.
  • the targeting ligand is a ligand for ASGPR, such as a N- acetylgalactosamine (GalNAc) containing ligand.
  • GalNAc N- acetylgalactosamine
  • GalNAc containing ligands and ligands are available for use in the LNP of this disclosure, including, e.g. those disclosed in WO2021178725, the full disclosure of which is incorporated herein by reference
  • the PEG-lipid is linked to the targeting ligand.
  • the targeting ligand of interest e.g., as described herein
  • a trisGalNac ligand conjugated to a PEG-lipid can provide for binding of the LNP to the ASGPR receptor of a target cell and result in endocytosis of the LNP.
  • the LNPs include an ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group; a phospholipid that is DOPE, cholesterol, and a lipid capable of reducing aggregation that is PEG-DMG.
  • the LNPs include an ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group; a phospholipid that is a phosphatidylethanolamine, (e.g. DOPE), cholesterol, and a lipid capable of reducing aggregation that is PEG-DMG, and/or PEG-DSG-GalNAc.
  • the LNPs include PEG-DMG.
  • the LNPs include PEG-DSG-GalNAc.
  • the LNPs include an ionizable that is a cationic lipid comprising a tertiary amino ionizable group, a phospholipid that is a phosphatidylcholine (e.g. l,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol and a coat lipid (polyethylene glycol-dimyristolglycerol, PEG-DMG), for example as disclosed by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3): 498-507.
  • a cationic lipid comprising a tertiary amino ionizable group
  • a phospholipid that is a phosphatidylcholine e.g. l,2-distearoyl-sn-glycero-3-phosphocholine, DSPC
  • cholesterol e.g. l,2-distearoyl-sn-glycer
  • a total lipid-to-nucleic acid e.g., DNA and/or RNA
  • N nitrogen
  • P nucleic acid phosphate
  • the N/P ratio (mass/mass ratio; w/w ratio) can be in the range of from about 1 : 1 to about 50: 1, from about 7: 1 to about 25: 1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9:1.
  • the N/P ratio (mass/mass ratio; w/w ratio) can be in the range of 5 to 10, 11 to 20, or 21 to 30.
  • the N/P ratio (mass/mass ratio; w/w ratio) can be in the range of 5 to 10.
  • the N/P ratio (mass/mass ratio; w/w ratio) can be in the range of 11 to 20. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) can be in the range of 21 to 30. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) is about 7. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) is about 10. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) is about 14. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) is about 28.
  • the amounts of lipids and nucleic acid can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3: 1 (“3”), 4: 1 (“4”), 5: 1 (“5”), 6: 1 (“6”), 7: 1 (“7”), 8: 1 (“8”), 9: 1 (“9”), 10: 1 (“10”), 11 : 1 (“11”), 12:1 (“12”), 13: 1 (“13”), 14: 1 (“14”) or higher.
  • N/P ratio 3: 1 (“3”), 4: 1 (“4”), 5: 1 (“5”), 6: 1 (“6”), 7: 1 (“7”), 8: 1 (“8”), 9: 1 (“9”), 10: 1 (“10”), 11 : 1 (“11”), 12:1 (“12”), 13: 1 (“13”), 14: 1 (“14”) or higher.
  • the lipid particle formulation's overall lipid content can range from about 5 mg/mL to about 30 mg/mL.
  • a lipid nanoparticle has a mean diameter between about 10 and about 1000 nm. In some embodiments, a lipid nanoparticle has a diameter that is less than 300 nm. In some embodiments, a lipid nanoparticle has a diameter between about 10 and about 300 nm. In some embodiments, a lipid nanoparticle has a diameter that is less than 200 nm. In some embodiments, a lipid nanoparticle has a diameter between about 25 and about 200 nm.
  • a lipid nanoparticle preparation (e.g., composition comprising a plurality of lipid nanoparticles) has a size distribution in which the mean size (e.g., diameter) is about 70 nm to about 200 nm, and more typically the mean size is about 100 nm or less.
  • the mean size e.g., diameter
  • an LNP has a mean diameter of 25 to 250 nm, 25 to 240 nm, 25 to 230 nm, 25 to 220 nm, 25 to 210 nm, 25 to 200 nm, 25 to 190 nm, 25 to 180 nm, 25 to 170 nm, 25 to 160 nm, 25 to 150 nm, 25 to 140 nm, 25 to 130 nm, 25 to 120 nm, 25 to 110 nm, 25 to 100 nm, 25 to 90 nm, 25 to 80 nm, 25 to 70 nm, 25 to 60 nm, or 25 to 50 nm.
  • an LNP has a mean diameter of 60 to 250 nm, 70 to 250 nm, 80 to 250 nm, 90 to 250 nm, 100 to 250 nm, 110 to 250 nm, 120 to 250 nm, 130 to 250 nm, 140 to 250 nm, 150 to 250 nm, 160 to 250 nm, 170 to 250 nm, 180 to 250 nm, 190 to 250 nm, 200 to 250 nm, 210 to 250 nm, 220 to 250 nm, 230 to 250 nm, or 240 to 250 nm
  • an LNP has a mean diameter of 60 to 250 nm, 70 to 240 nm, 80 to 230 nm, 90 to 220 nm, 100 to 210 nm, 110 to 200 nm, 120 to 190 nm, 130 to 180 nm, 140 to 170 nm, or 150 to 160 nm.
  • the structural characteristics of the target tissue may be exploited to direct the distribution of the LNPs to such target tissues.
  • a LNP may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; accordingly the LNP can readily penetrate such endothelial fenestrations to reach the target hepatocytes.
  • a LNP may be sized such that the dimensions of the particles are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues.
  • a LNP may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the LNPs to hepatocytes.
  • large LNPs will not easily penetrate the endothelial fenestrations, and would instead be cleared by the macrophage Kupffer cells that line the liver sinusoids.
  • the size of the LNPs is within the range of about 25 to 250 nm or 25 nm to 100 nm, preferably less than 250 nm, less than 175 nm, less than 150 nm, less than 125 nm, or less than 100 nm.
  • ionizable lipid can comprise 20-90% (mol) of the total lipid present in the lipid nanoparticle.
  • ionizable lipid molar content can be 20-70% (mol), 30-60% (mol) 40-60% (mol) or 40-50% (mol) of the total lipid present in the lipid nanoparticle.
  • ionizable lipid comprises from about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle.
  • the neutral lipid components can comprise 0-30% (mol) of the total lipid present in the lipid nanoparticle.
  • the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle.
  • the molar ratio of ionizable lipid to the neutral lipid ranges from about 2: 1 to about 8: 1.
  • the lipid nanoparticles do not comprise any phospholipids.
  • a given lipid nanoparticle may include a cargo, or payload, to be delivered to cells.
  • cargos that comprise a polynucleotide.
  • the polynucleotide is a DNA.
  • DNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure.
  • the DNA may be circular, e.g. a plasmid, a nanoplasmid, a minicircle, a covalently closed circular DNA, a circular viral genome, and the like.
  • the DNA may be linear, e.g. a doggybone or other closed-end DNA, a linear viral genome, and the like.
  • the DNA may be multivalent, e.g. a 3DNA.
  • the DNA may be single stranded or double stranded or a hybrid of single and double stranded.
  • the DNA may be chemically modified.
  • the polynucleotide is an RNA.
  • RNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure.
  • the RNA may be linear or it may be circular. It may be an mRNA, an siRNA, an shRNA, a guide RNA (gRNA), a microRNA (miRNA), a circular RNA (circRNA). It may be chemically modified.
  • the RNA may be single stranded or double stranded or a hybrid of single and double stranded.
  • the RNA may be chemically modified.
  • the cargo comprises both DNA and RNA.
  • DNA and RNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure.
  • the one or more additional compounds can be a therapeutic agent.
  • the therapeutic agent can be selected from any class suitable for the therapeutic objective.
  • the therapeutic agent can be selected from any class suitable for the therapeutic objective.
  • the therapeutic agent can be selected according to the treatment objective and biological action desired.
  • the additional compound can be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate).
  • the additional compound can be an antimicrobial agent (e.g., an antibiotic or antiviral compound).
  • the additional compound can be a compound that modulates an immune response (e.g., an immunosuppressant, immunostimulatory compound, or compound modulating one or more specific immune pathways).
  • an immunosuppressant e.g., an immunosuppressant, immunostimulatory compound, or compound modulating one or more specific immune pathways.
  • different cocktails of different lipid nanoparticles containing different compounds, such as a DNA encoding a different protein or a different compound, such as a therapeutic may be used in the compositions and methods of the invention.
  • the additional compound is an immune modulating agent.
  • the additional compound is an immunosuppressant.
  • the additional compound is immune stimulatory agent.
  • lipid nanoparticle-encapsulated nucleic acid e.g., DNA, RNA, DNA and RNA
  • a pharmaceutically acceptable carrier or excipient e.g., a pharmaceutically acceptable carrier or excipient.
  • the disclosure provides for a lipid nanoparticle formulation further comprising one or more pharmaceutical excipients.
  • the lipid nanoparticle formulation further comprises sucrose, tris, trehalose and/or glycine.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
  • Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water
  • “Pharmaceutically acceptable salt” includes both acid and base addition salts.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4- acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-l,2-disulfonic acid, ethanesulfonic acid, 2-hydroxye
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methyl
  • a “dosing regimen” is a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
  • a “reference” compound or composition is one that is sufficiently similar to a particular compound of interest to permit a relevant comparison.
  • information about a reference compound or composition is obtained simultaneously with information about a particular compound.
  • comparison of a particular compound of interest with a reference compound or composition establishes identity with, similarity to, or difference of the particular compound or composition of interest relative to the compound.
  • the present invention provides pharmaceutical compositions comprising a compound or composition of the present disclosure, in combination with a pharmaceutically acceptable excipient (e.g., carrier).
  • a pharmaceutically acceptable excipient e.g., carrier
  • the pharmaceutical compositions include optical isomers, diastereomers, or pharmaceutically acceptable salts of the composition disclosed herein.
  • a compound or composition may be covalently attached a carrier moiety, as described above.
  • a compound or composition of the pharmaceutical composition is not covalently linked to a carrier moiety.
  • a “pharmaceutically acceptable carrier”, as used herein refers to pharmaceutical excipients, for example, pharmaceutically, physiologically, acceptable organic or inorganic carrier substances suitable for enteral or parenteral application that do not deleteriously react with the active agent.
  • suitable pharmaceutically acceptable carriers include water, salt solutions (such as Ringer’s solution), alcohols, oils, gelatins, and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone.
  • Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
  • compositions of the present disclosure can be administered alone or can be coadministered to the subject. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound).
  • the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation).
  • a compound or composition as described herein can be incorporated into a pharmaceutical composition for administration by methods known to those skilled in the art and described herein for provided compounds or compositions.
  • Compounds or compositions of the present invention can be prepared and administered in a wide variety of oral, parenteral, and topical dosage forms.
  • the compounds or compositions of the present invention can be administered by injection (e.g., intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally).
  • compounds or composition of the present disclosure are administered orally.
  • the compounds or compositions described herein can be administered by inhalation, for example, intranasally.
  • compositions of the present disclosure can be administered transdermally. It is also envisioned that multiple routes of administration (e.g., intramuscular, oral, transdermal) can be used to administer compounds or compositions of the present disclosure.
  • routes of administration e.g., intramuscular, oral, transdermal
  • the present disclosure also provides pharmaceutical compositions comprising pharmaceutically acceptable carrier or excipient and one or more compounds or compositions of the disclosure.
  • pharmaceutically acceptable carriers can be either solid or liquid.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier can be one or more substances that may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
  • the carrier is finely divided solid in a mixture with the finely divided active component.
  • the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • compositions provided by the present disclosure include compositions wherein the active ingredient, i.e. the nucleic acid, is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose.
  • the actual amount effective for a particular application will depend, inter alia, on the condition being treated.
  • such compositions when administered in methods to treat phenylketonuria (PKU), such compositions will contain an amount of active ingredient effective to achieve the desired result of increasing the amount of phenylalanine hydroxylase made by cells that have been contacted with a pharmaceutical composition of the present disclosure, which in turn will increase the amount of phenylalanine that gets processed and will decrease the amount of phenylalanine that accumulates in tissues.
  • PKU phenylketonuria
  • the dosage and frequency (single or multiple doses) of compound or composition administered can vary depending upon a variety of factors, including route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of the symptoms of the disease being treated (e.g., the disease responsive treatment; and complications from any disease or treatment regimen.
  • Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of the invention.
  • the therapeutically effective amount can be initially determined from cell culture assays.
  • Target concentrations will be those concentrations of active compound(s), i.e. nucleic acid, that are capable of increasing the amount of gene product, e.g. RNA or protein, that is encoded by the nucleic acid
  • Therapeutically effective amounts for use in humans may be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals to treat the disease. Additionally or alternatively, a dose for humans can be formulated to achieve a concentration of protein typically found in individuals that are unaffected by disease, or that has been found to be effective in treating individuals having the disease, e.g. during protein or enzyme replacement therapy.
  • the dosage in humans can be adjusted by methods well understood by the ordinarily skilled artisan as they pertain to the disease being treated, including but not limited to monitoring the amount of gene product, e.g. RNA or protein, that is expressed in the contacted tissue, monitoring the amount of metabolite of the disease, monitoring the progression of the disease, and so on following administration of the pharmaceutical composition and adjusting the dosage upwards or downwards, as described above.
  • Dosages may be varied depending upon the requirements of the patient and the compound being employed.
  • the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side effects.
  • compositions provided herein display one or more improved pharmacokinetic (PK) properties (e.g., Cmax, tmax, Cmin, tl/2, AUC, CL, bioavailability, etc.) or one or more improved pharmacodynamic (PD) properties (e.g. cytokines secreted, or changes in expression level of one or more RNAs or proteins in the body) when compared to a reference composition.
  • PK pharmacokinetic
  • PD pharmacodynamic
  • cytokines secreted e.g. cytokines secreted, or changes in expression level of one or more RNAs or proteins in the body
  • a reference composition is a viral gene therapy known in the art.
  • a reference composition is an enzyme replacement therapy.
  • a reference composition is an LNP comprising a specific lipid that differs from the composition provided herein.
  • the reference composition is an LNP comprising a phospholipid that differs from the composition provided herein.
  • the reference composition is an LNP comprising DSPC.
  • the reference composition is an LNP comprising DOPE.
  • a compound of the disclosure or a pharmaceutical composition comprising the same is provided as a unit dose.
  • the LNP compositions can be prepared by high energy mixing of ethanolic lipids with aqueous DNA at low pH which protonates the ionizable lipid and provides favorable energetics for DNA / lipid association and nucleation of particles.
  • the particles can be further stabilized through aqueous dilution and removal of the organic solvent. The particles can be concentrated to the desired level.
  • the LNPs and LNP pharmaceutical composition of the present disclosure when formulated with nucleic acids, are less toxic in vivo as compared to a reference LNP, for example an industry standard LNP comprising DSPC (e.g. comprising 50% ionizable lipid ALC-0315, 10% DSPC, 38.5% cholesterol, and 1.5% PEG lipid) administered at the same dose, e.g. at least 2-fold less toxic, e.g. 3-fold, 4-fold or 5-fold less toxic, in some instances 10-fold, 20-fold, or 50-fold less toxic, in certain instances 100-fold less toxic.
  • DSPC e.g. comprising 50% ionizable lipid ALC-0315, 10% DSPC, 38.5% cholesterol, and 1.5% PEG lipid
  • DSPC e.g. comprising 50% ionizable lipid ALC-0315, 10% DSPC, 38.5% cholesterol, and 1.5% PEG lipid
  • DSPC e.g. comprising 50% ionizable
  • the LNPs and LNP pharmaceutical composition of the present disclosure have been observed to be equally or more efficacious at delivering their nucleic acid cargo to the target cell of interest as that same reference LNP administered at the same dose, e.g. having 2-fold the efficacy or more, e.g. 3-fold, 4-fold or 5-fold the efficacy or more, in some instances 10-fold, 20-fold or 50-fold the efficacy, in certain instances 100-fold more efficacious or more.
  • more efficacious it is meant able to deliver more nucleic acid cargo to the cell, resulting in an increase in the amount of mRNA transcribed from that nucleic acid cargo or an increase in the amount of protein translated, for example a 2-fold increase or more, e.g. a 3-fold, 4-fold, 5-fold increase, e.g. 10-fold, 20-fold, 50-fold increase, in some instances a 100-fold increase or more.
  • the LNPs and the LNP pharmaceutical compositions of the present disclosure achieve 5-fold more expression or more, e.g.
  • Changes in expression may be assessed by any method known by the ordinarily skilled artisan, including, for example, measuring RNA levels, for example by RT-PCR, qRT-PCR, Northern blot and the like; or measuring protein levels by, for example, ELISA, Western blot, and the like; or measuring a functional change as the result of a new activity (e.g. increased hematocrit with more EPO expression, an increase in clotting with more clotting factor, improvement in symptoms in a disease, etc.).
  • the LNPs and the LNP pharmaceutical compositions of the present disclosure may elicit 4-fold less cytokines in serum or less, e.g.
  • cytokines 5-fold, 6-fold, 7- fold less cytokines, 4 hours after delivery of a 0.3 mg/kg dose i.v. or 5-fold less cytokines in serum or less, e.g. 5-fold, 7-fold, 10-fold, and in some instance 15-fold less cytokines or less, 4 hours after delivery of a 1.0 mg/kg dose i.v. than the reference LNP, e.g. the comparable LNP comprising DSPC instead of the phospholipid of the present disclosure.
  • Changes in cytokines may be measured by any of a number of methods know in the art, including for example measuring levels in serum by, e.g. ELISA.
  • the LNPs of the present disclosure demonstrate an improved pharmacokinetics (PK) profile that broadens the therapeutic index of the composition.
  • PK pharmacokinetics
  • a therapeutic index or therapeutic ratio, it is meant the range of doses at which a medication is effective without unacceptable adverse events, calculated as the ratio that compares the blood concentration at which a drug becomes toxic and the concentration at which the drug is effective.
  • This improvement over the art makes them more amenable to delivering nucleic acids to cells in vitro and in vivo, and accordingly they find many uses in many applications, including in the delivery of nucleic acids to cells for research and for therapeutic applications.
  • the cells are typically contacted with the composition, e.g. LNP or pharmaceutical composition thereof, in amount effective to deliver the agent into the cytoplasm of the cell.
  • the contacting is in vitro. In other embodiments, the contacting is in vivo.
  • the method further comprises measuring the amount of protein produced.
  • the present disclosure further provides methods of treating or preventing diseases in a subject in need thereof wherein an effective amount of the therapeutic compositions described herein is administered to the subject.
  • the route of administration will vary, naturally, with the location and nature of the disease being treated, and may include, for example intradermal, transdermal, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration.
  • the encapsulated polynucleotide compositions described herein are useful in the treatment of any of any indication in which it is beneficial to deliver a therapeutic cargo into the target cell.
  • the present disclosure further provides methods of immunizing a subject against a disease wherein an effective amount of a therapeutic composition described herein is administered to the subject.
  • the route of administration will vary, naturally, with the location and nature of immunization agent, and may include, for example intradermal, transdermal, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration.
  • the present disclosure further provides a particle of the disclosure, a vector of the disclosure, a recombinant DNA of the disclosure, or compositions thereof, for use as a medicament.
  • the medicament is for expressing a protein in a cell.
  • the expressing of a protein is for the treatment of a disease in which the cell is deficient for the protein.
  • the expressing of a protein is for the treatment of a disease in which another cell is deficient for the protein.
  • the medicament is for the treatment of a cancer.
  • the medicament is for immunization against a disease.
  • the subject methods and compositions can be used in any application where delivery of a cargo nucleic acid is desired.
  • Applications of interest include both research and therapeutic applications. Applications of interest include, but are not limited to: research applications, diagnostic applications and therapeutic applications.
  • cargo nucleic acids that may be introduced into a nucleus via methods of the invention include those encoding research proteins, diagnostic proteins and therapeutic proteins.
  • Research proteins are proteins whose activity finds use in a research protocol. As such, research proteins are proteins that are employed in an experimental procedure. The research protein may be any protein that has such utility, where in some instances the research protein is a protein domain that is also provided in research protocols by expressing it in a cell from an encoding vector.
  • transcription modulators of inducible expression systems include, but are not limited to: transcription modulators of inducible expression systems, members of signal production systems, e.g., enzymes and substrates thereof, hormones, prohormones, proteases, enzyme activity modulators, perturbimers and peptide aptamers, antibodies, modulators of protein-protein interactions, genomic modification proteins, such as CRE recombinase, meganucleases, Zinc-finger nucleases, CRISPR/Cas-9 nuclease, TAL effector nucleases, etc., cellular reprogramming proteins, such as Oct 3/4, Sox2, Klf4, c-Myc, Nanog, Lin-28, etc., and the like.
  • transcription modulators of inducible expression systems include, but are not limited to: transcription modulators of inducible expression systems, members of signal production systems, e.g., enzymes and substrates thereof, hormones, prohormones, proteases, enzyme activity modulators, perturbimers and
  • Diagnostic proteins are proteins whose activity finds use in a diagnostic protocol. As such, diagnostic proteins are proteins that are employed in a diagnostic procedure.
  • the diagnostic protein may be any protein that has such utility. Examples of specific types of diagnostic proteins include, but are not limited to: members of signal production systems, e.g., enzymes and substrates thereof, labeled binding members, e.g., labeled antibodies and binding fragments thereof, peptide aptamers and the like.
  • Proteins of interest further include therapeutic proteins.
  • Therapeutic proteins of interest include without limitation, hormones and growth and differentiation factors, fibrinolytic proteins, transcription factors, and enzymes.
  • Target cells to which nucleic acids may be delivered in accordance with embodiments of this disclosure may vary widely.
  • Target cells of interest include, but are not limited to: cell lines, HeLa, HEK, CHO, 293 and the like, Mouse embryonic stem cells, human stem cells, mesenchymal stem cells, primary cells, tissue samples and the like.
  • Some non-limiting examples of a mammalian cell include, without limitation, a mouse cell, a rat cell, hamster cell, a rodent cell, and a nonhuman primate cell.
  • the target cell is a human cell. It should also be appreciated that the target cell may be of any cell type.
  • the target cell may be a stem cell, which may include embryonic stem cells, induced pluripotent stem cells (iPS cells), fetal stem cells, cord blood stem cells, or adult stem cells (i.e., tissue specific stem cells).
  • the target cell may be any differentiated cell type found in a subject.
  • Cells of interest include both dividing cells and non-dividing cells. Examples of specific target cells of interest include, but are not limited to: hepatocytes, stellate cells, T lymphocytes, B lymphocytes, NK cells, skeletal muscle cells, cardiomyocytes, neurons, astrocytes, oligodendrocytes, dendritic cells, skin cells, etc.
  • the application of interest is a therapeutic application, for example, in the treatment of a disease.
  • the compositions and methods of the present application may be used to deliver a nucleic acid sequence to the nucleus of a cell to complement a genetic deficiency.
  • compositions of the present application may be used in the treatment of a genetic deficiency that impacts the function of hepatocytes, or in the treatment of a genetic deficiency elsewhere in the body that can be remedied by leveraging hepatocytes as a biofactory to secrete the deficient protein.
  • the terms "individual,” “subject” and “host” are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired.
  • the subject is a mammal.
  • the subject is a human being.
  • the subject is a patient.
  • the subject is a human patient.
  • the subject can have or is suspected of having a disorder or health condition associated with a gene-of-interest (GOI).
  • GOI gene-of-interest
  • the subject is a human who is diagnosed with a risk of disorder or health condition associated with a GOI at the time of diagnosis or later.
  • the diagnosis with a risk of disorder or health condition associated with a GOI can be determined based on the presence of one or more mutations in the endogenous GOI or genomic sequence near the GOI in the genome that may affect the expression of GOI.
  • treatment used referring to a disease or condition means that at least an amelioration of the symptoms associated with the condition afflicting an individual is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom, associated with the condition (e.g., hemophilia A) being treated.
  • a parameter e.g., a symptom
  • treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or eliminated entirely such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition.
  • treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease.
  • an effective amount means a sufficient amount of the composition to provide the desired utility when administered to a subject having a particular condition.
  • the term “therapeutically effective amount” therefore refers to an amount of therapeutic cells or a composition having therapeutic cells that is sufficient to promote a particular effect when administered to a subject in need of treatment.
  • An effective amount would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate "effective amount” can be determined by one of ordinary skill in the art using routine experimentation.
  • pharmaceutically acceptable excipient refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive or diluent for administration of a compound(s) of interest to a subject.
  • pharmaceutically acceptable excipient can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.
  • aliphatic or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocyclyl”, “cycloaliphatic”, or “cycloalkyl”), that has a single point of attachment to the rest of the molecule.
  • aliphatic groups contain 1-6 aliphatic carbon atoms.
  • aliphatic groups contain 1-5 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms.
  • “cycloaliphatic” refers to a monocyclic C3-C7 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule.
  • Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
  • alkylene refers to a bivalent alkyl group.
  • An “alkylene chain” is a polymethylene group, i.e., -(CH2)n-, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3.
  • a substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
  • cyclopropylenyl refers to a bivalent cyclopropyl group of the following structure: .
  • halogen means F, Cl, Br, or I.
  • bridged bicyclic refers to any bicyclic ring system, i.e., carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge.
  • a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen).
  • a bridged bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • bridged bicyclic groups are well known in the art and include those groups set forth below where each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group is optionally substituted with one or more substituents as set forth for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include:
  • aryl refers to monocyclic and bicyclic ring systems having a total of five to ten ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members.
  • the term “aryl” may be used interchangeably with the term “aryl ring”.
  • 8- to 10-membered bicyclic aryl group is an optionally substituted naphthyl ring.
  • “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents.
  • aryl is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.
  • partially unsaturated refers to a ring moiety that includes at least one double or triple bond.
  • partially unsaturated is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.
  • a bivalent carbocycle is “ carb ocyclyl ene”, a bivalent aryl ring is “arylene”, a bivalent benzene ring is “phenylene”, a bivalent heterocycle is “heterocyclylene”, a bivalent heteroaryl ring is “heteroarylene”, a bivalent alkyl chain is “alkylene”, a bivalent alkenyl chain is “alkylene”, a bivalent alkynyl chain is “alkynylene”, and so forth.
  • compounds of the invention may, when specified, contain “optionally substituted” moieties.
  • substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either explicit or implicit from the structure (e.g., refers to at least ).
  • substituents may, unless otherwise indicated, replace a hydrogen on any individual ring (e.g., refers to at least Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their purification, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
  • Suitable monovalent substituents on R° are independently halogen; -(CH 2 )o- 2 R*; -(haloR*), -(CH 2 )o- 2 OH; -(CH 2 )o- 2 OR e ; -(CH 2 )o- 2 CH(OR’) 2 ; -O(haloR’); -CN; -N 3 ; - (CH 2 )O- 2 C(0)R*; -(CH 2 )O- 2 C(0)OH; -(CH 2 )O- 2 C(0)OR’; -(CH 2 )O- 2 SR’; -(CH 2 )O- 2 SH; -(CH 2 )O- 2 NH 2 ; -(CH 2 )O- 2 NHR*; -(CH 2 )O- 2 NR’ 2 ; -NO 2 , -S
  • R # is selected from hydrogen, Ci-6 aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR # 2)2-3O-, wherein each independent occurrence of R # is selected from hydrogen, Ci-6 aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • Suitable substituents on the aliphatic group of R # include halogen, -R*, -(haloR*), - OH, -OR*, -O(haloR’), -CN, -C(O)OH, -C(O)OR*, -NH 2 , -NHR*, -NR*2, or -NO 2 , wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci-4 aliphatic, -CH2PI1, -0(CH2)o-iPh, or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -R', -NR’2, -C(O)R f , -C(O)OR f , -C(O)C(O)R f , -C(O)CH 2 C(O)R f , -S(O) 2 R f , - S(O) 2 NR f 2, -C(S)NR f 2, -C(NH)NR f 2, or -N(R f )S(O)2R f 2; wherein each RUs independently hydrogen, Cl -6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the definition above, two independent occurrences or R 1 ', taken together
  • Suitable substituents on the aliphatic group of R' are independently halogen, -R*, - (haloR*), -OH, -OR*, -O(haloR’), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci-4 aliphatic, -CH2PI1, -0(CH2)o-iPh, or a 5- to 6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.
  • the neutral forms of the compounds are regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.
  • structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.
  • structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are with the scope of this invention.
  • Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.
  • compounds of the present disclosure are provided as a single enantiomer or single diastereomer.
  • Single enantiomer refers to an enantiomeric excess 80% or more, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.
  • oxo means an oxygen that is double bonded to a carbon atom thereby forming a carbonyl.
  • Embodiment 1 A lipid nanoparticle comprising: a. a nucleic acid, b. an ionizable lipid, c. a phospholipid that is not DSPC, d. cholesterol, and e. a lipid capable of reducing aggregation.
  • Embodiment 2 The lipid nanoparticle of embodiment 1, wherein the nucleic acid comprises DNA.
  • Embodiment 3 The lipid nanoparticle of embodiment 2, wherein the nucleic acid consists essentially of DNA.
  • Embodiment 4 The lipid nanoparticle of embodiment 2, wherein the nucleic acid further comprises RNA.
  • Embodiment 5 The lipid nanoparticle of embodiment 4, wherein the RNA is selected from mRNA, gRNA, and siRNA.
  • Embodiment 6 The lipid nanoparticle of embodiment 1, wherein the nucleic acid consists essentially of RNA.
  • Embodiment 7 The lipid nanoparticle of embodiment 6, wherein the RNA is selected from mRNA, gRNA, and siRNA.
  • Embodiment 8 The lipid nanoparticle of any one of embodiments 1-7, wherein the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG), and derivatives thereof.
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PG phosphatidylglycerol
  • Embodiment 9 The lipid nanoparticle of any one of embodiments 1-8, wherein the phospholipid comprises hydrocarbon chains each independently having 12-24 carbons.
  • Embodiment 10 The lipid nanoparticle of embodiment 9, wherein the phospholipid comprises hydrocarbon chains each independently having 16-20 carbons.
  • Embodiment 11 The lipid nanoparticle of embodiment 9 or 10, wherein the hydrocarbon chains are saturated.
  • Embodiment 12 The lipid nanoparticle of embodiment 9 or 10, wherein one or more of the hydrocarbon chains are unsaturated.
  • Embodiment 13 The lipid nanoparticle of embodiment 12, wherein the hydrocarbon chains each independently comprise 1-4 double bonds.
  • Embodiment 14 The lipid nanoparticle of any one of embodiments 6-13, wherein the phospholipid comprises two different hydrocarbon chains.
  • Embodiment 15 The lipid nanoparticle of embodiment 8, wherein the phospholipid has a phase transition temperature of -40 °C and 30 °C.
  • Embodiment 16 The lipid nanoparticle of any one of embodiments 6-15, wherein the phospholipid is a phosphatidylethanolamine.
  • Embodiment 17 The lipid nanoparticle of embodiment 16, wherein the phosphatidylethanolamine is selected from the group consisting of 1 -stearoyl -2 -oleoyl-sn- glycero-3 -phosphoethanolamine (18:0/18: 1 PE), l,2-dipalmitoleoyl-sn-glycero-3- phosphoethanolamine (16: 1 PE), and dioleoylphosphatidylethanolamine (18:1(A9-Cis) PE, or DOPE).
  • the phosphatidylethanolamine is selected from the group consisting of 1 -stearoyl -2 -oleoyl-sn- glycero-3 -phosphoethanolamine (18:0/18: 1 PE), l,2-dipalmitoleoyl-sn-glycero-3- phosphoethanolamine (16: 1 PE), and dioleoylphosphatidylethanolamine (18:1(A9-Cis) PE, or DOPE).
  • Embodiment 18 The lipid nanoparticle of embodiment 17, wherein the phosphatidylethanolamine is l-stearoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (18:0/18: 1 PE).
  • Embodiment 19 The lipid nanoparticle of embodiment 17, wherein the phosphatidylethanolamine is l,2-dipalmitoleoyl-sn-glycero-3 -phosphoethanolamine (16: 1 PE).
  • Embodiment 20 The lipid nanoparticle of any one of embodiments 6-15, wherein the phospholipid is a phosphatidylcholine.
  • Embodiment 21 The lipid nanoparticle of embodiment 20, wherein the phosphatidylcholine is selected from the group consisting of l,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (16: 1(A9-Cis) PC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (18: l(A9-cis) PC), and 1- palmitoyl-2-oleoyl-glycero-3-phosphocholine (16:0/18: 1 PC).
  • the phosphatidylcholine is selected from the group consisting of l,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (16: 1(A9-Cis) PC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (18: l(A9-cis) PC), and 1- palmitoyl-2-oleoyl-glycero-3
  • Embodiment 22 The lipid nanoparticle of embodiment 21, wherein the phosphatidylcholine is 1 -palmitoyl -2 -oleoyl -glycero-3-phosphocholine (16:0/18: 1 PC).
  • Embodiment 23 The lipid nanoparticle of embodiment 21, wherein the phosphatidylcholine is
  • Embodiment 24 The lipid nanoparticle of embodiment 21, wherein the phosphatidylcholine is
  • Embodiment 25 The lipid nanoparticle of any one of embodiments 1-24 wherein the lipid capable of reducing aggregation is a PEG-lipid.
  • Embodiment 26 The lipid nanoparticle of embodiment 25, wherein the PEG-lipid is 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1, 2- distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).
  • PEG-DMG[2K] 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000
  • PEG-1 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000
  • PEG-DSG[2K] 2- distearoyl-rac-glycero-3-methylpolyoxyethylene 2000
  • Embodiment 27 The lipid nanoparticle of any one of embodiments 1-26, further comprising a targeting ligand.
  • Embodiment 28 The lipid nanoparticle of embodiment 27, wherein the targeting ligand comprises GalNAc.
  • Embodiment 30 The lipid nanoparticle of embodiment 29, wherein the lipid capable of reducing aggregation is PEG- l,2-distearoyl-rac-glycero-3 -methylpoly oxyethylene 2000 (PEG-DSG[2K]).
  • Embodiment 31 The lipid nanoparticle of any one of embodiments 1-30, wherein the N/P ratio
  • ratio of moles of the amine groups of cationic lipids to those of the phosphate ones of DNA is from 5 to 30.
  • Embodiment 32 The lipid nanoparticle of embodiment 31, wherein the N/P ratio is 5-10
  • Embodiment 33 The lipid nanoparticle of embodiment 32, wherein the N/P ratio is 7.
  • Embodiment 34 The lipid nanoparticle of embodiment 31, wherein the N/P ratio is 10.
  • Embodiment 35 The lipid nanoparticle of embodiment 31, wherein the N/P ratio is 11-20.
  • Embodiment 36 The lipid nanoparticle of embodiment 35, wherein the N/P ratio is 14.
  • Embodiment 37 The lipid nanoparticle of embodiment 31, wherein the N/P ratio is 21-30.
  • Embodiment 38 The lipid nanoparticle of embodiment 37, wherein the N/P ratio is 28.
  • Embodiment 39 The lipid nanoparticle of any one of embodiments 1-38, comprising: a. an ionizable lipid at 40 to 60 mol % of the total lipid present; b. a phospholipid at 5 to 20 mol % of the total lipid present; c. cholesterol at 25 to 50 mol % of the total lipid present; and d. a lipid capable of reducing aggregation at 1.5 to 3.0 mol % of the total lipid present.
  • Embodiment 40 The lipid nanoparticle of any one of embodiments 1-38, comprising: a. an ionizable lipid at 40 to 60 mol % of the total lipid present; b.
  • Embodiment 41 The lipid nanoparticle of any one of embodiments 1-38, comprising: a. an ionizable lipid at 40 to 60 mol % of the total lipid present; b. a phospholipid at 10 to 20 mol % of the total lipid present; c. cholesterol at 35 to 45 mol % of the total lipid present; and d.
  • Embodiment 42 The lipid nanoparticle of any one of embodiments 1-38, comprising: a. an ionizable lipid at 40 to 49 mol % of the total lipid present; b. a phospholipid at 10 to 20 mol % of the total lipid present; c. cholesterol at 35 to 45 mol % of the total lipid present; and d. a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
  • Embodiment 43 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b.
  • the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 44 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 45 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 46 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 47 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 48 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 49 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 50 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 51 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 52 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 53 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 54 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 55 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 56 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 57 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 58 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 59 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 60 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 61 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 62 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 63 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 64 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 65 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 66 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 67 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 68 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 69 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 70 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 71 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b.
  • the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 72 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 73 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 74 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 75 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 76 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 77 The lipid nanoparticle of any one of embodiments 29-38, wherein: a.
  • the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 78 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
  • Embodiment 79 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 80 The lipid nanoparticle of any one of embodiments 29-38, wherein: a.
  • the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 81 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 82 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
  • Embodiment 83 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 84 The lipid nanoparticle of any one of embodiments 29-38, wherein: a.
  • the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 85 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 86 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 87 The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
  • Embodiment 88 The lipid nanoparticle of any one of embodiments 1-87, wherein the nucleic acid encodes for a therapeutic agent.
  • Embodiment 89 A pharmaceutical composition comprising a lipid nanoparticle of any one of embodiments 1-88 and a pharmaceutically acceptable excipient, carrier, or diluent.
  • Embodiment 90 A method for delivering a nucleic acid into a cell, the method comprising contacting the cell with a lipid nanoparticle of any one of embodiments 1 to 88 or the pharmaceutical according to embodiment 89.
  • Embodiment 91 The method according to embodiment 90, wherein the cell is in vitro.
  • Embodiment 92 The method according to embodiment 90, wherein the cell is in vivo.
  • Embodiment 93 The method according to embodiment 90, wherein at least 8-fold more nucleic acid is delivered to the cell than a lipid nanoparticle comprising a DSPC phospholipid.
  • Embodiment 94 The method according to embodiment 90, wherein at least 5 -fold less cytokines are elicited than a lipid nanoparticle comprising the same nucleic acid, the same ionizable lipid, a DSPC phospholipid, the same cholesterol, and the same lipid capable of reducing aggregation.
  • Embodiment 95 A method for delivering a nucleic acid for in vivo production of target protein, the method comprising: administering systemically to a subject in need thereof a pharmaceutical composition of embodiment 89, wherein the nucleic acid encodes a target protein and is encapsulated within the lipid nanoparticles, and the administering of the pharmaceutical composition results in the prolonged stable expression of the target protein. 7.
  • LNP formulation LNPs encapsulating nucleic acid payloads were prepared by mixing an organic solution of lipids with an aqueous solution of nucleic acid (e.g., DNA only, mRNA only, or DNA/mRNA mixtures) as described in Prud’Neill et al. (J Pharm Sci. 2018). Briefly, the lipidic excipients mixture (ionizable lipid, helper lipid, cholesterol, PEG-lipid and potentially other targeting moieties) is dissolved in an organic solvent. An aqueous solution of the nucleic acid is prepared in a low pH buffer of range pH 3.0 - 4.0.
  • nucleic acid e.g., DNA only, mRNA only, or DNA/mRNA mixtures
  • the lipid mixture is then mixed with the aqueous nucleic acid solution at a flow ratio of 1 :3 (VW) using a commercially available mixer device.
  • the resulting solution is immediately diluted with a buffer pH range of 5.0-6.5.
  • the diluted LNP is subjected to dialysis purification against a secondary buffer with the pH range of 7.0-8.0.
  • the LNP solution is concentrated by using 100,000 MWCO Amicon Ultra centrifuge tubes (Millipore Sigma) followed by filtration through 0.2 pm PES sterilizing-grade filter. Particle size is determined by dynamic light scattering (Horiba nanoPartica SZ-100). Encapsulation efficiency is calculated by using Quant-it RiboGreen assay kit.
  • EPO and cytokine detection in serum Blood is collected via a retro-orbital bleed into serum separator tubes and processed to serum. The serum samples may be stored at -80C from collection until analysis.
  • the serum levels of human EPO protein driven by expression from the DNA payload are quantified using the U-PLEX Human EPO Assay from MSD according to the manufacturer’s instructions.
  • the serum levels of mouse cytokines resulting from exposure to DNA-LNPs were quantified using the Mouse Prolnflammatory 7-Plex Tissue Culture Kit from MSD according to the manufacturer’s instructions.
  • EXAMPLE 1 Preparation and analysis of lipid nanoparticle formulations with varying mol% of ionizable lipid and different helper lipids
  • DNA payloads were formulated into lipid nanoparticles (LNPs) comprising the ALC- 0315 ionizable lipid at several mol ratios and one of two helper lipids (DSPC or DOPE), as described above and detailed in FIG. 1 A.
  • LNPs lipid nanoparticles
  • Wild type female BALB/c mice (approximately 7- 8 weeks old) were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight.
  • the DNA-LNPs were administered at 1 mg/kg based on the weight of the DNA payload.
  • blood was collected via a retro-orbital bleed and EPO levels in serum determined as presented in in FIG. IB.
  • the serum levels of mouse cytokines resulting from exposure to DNA-LNPs were quantified, with the results of IL-6 as a representative cytokine shown in FIG. 1C.
  • Varying the ionizable lipid mol% and type of helper lipid only had a minor effect on the EPO expression levels, with most groups within a 2- to 3-fold range.
  • the ionizable lipid mol% and type of helper lipid had a very strong effect on cytokine release, with lower mol% of ionizable lipid resulting in lower cytokines and DOPE resulting in significantly less cytokines (approx. 10-fold to 15-fold less) compared to DSPC.
  • EXAMPLE 2 Preparation and analysis of lipid nanoparticle formulations with different helper phospholipids, different PEG-lipids, and with or without GalNAc-PEG-lipid [0149]
  • the sugar moiety N-Acetylgalactosamine (GalNAc) has been shown to increase uptake of LNPs by hepatocytes.
  • DNA-LNPs were formulated with ALC-0315 as the ionizable lipid, one of two helper lipids (DSPC or DOPE), one of two PEG-lipids (DMG-PEG2k or DSG-PEG2k), and with or without GalNAc-PEG2k-lipid (structure of GalNAc-PEG-lipid shown in Fig. 2B).
  • LNPs were formulated following the same methods as described in Example 1 but implementing these changes in lipids to create the formulations described in FIG. 2A.
  • npDNA nanoplasmid DNA
  • the size of the resultant LNPs were less than 100 nm, and encapsulation efficiencies were very high (>95%).
  • the 4-week time course of EPO expression from the DNA-LNPs at 1 mg/kg, 0.3 mg/kg, and 0.1 mg/kg are shown in FIG. 2E, 2F, and 2G, respectively, compared to the AAV5.EPO positive control and PBS only negative control.
  • LNPs containing DOPE induced the release of lower levels of cytokines compared to DSPC.
  • LNPs comprising GalNAc were less effective at delivering DNA to hepatocytes as quantified by EPO levels than their unfunctionalized counterpart (Fig. 2C, compare DSPC+PEG-DMG to DSPC+PEG-DSG+GalNAc-PEG-lipid, and DOPE+PEG-DMG to DOPE+PEG-DSG+GalNAc-PEG-lipid).
  • LNPs containing DSG-PEG2k and GalNAc-PEG-lipid resulting in lower cytokines compared to LNPs containing DMG-PEG2k and no GalNAc (Fig. 2D, 2H, 21, 2J, 2K, 2L, 2M).
  • the cytokine levels were sufficiently low that adding DSG-PEG2k and GalNAc-PEG-lipid did not lower them further (Fig. 2D, 2H).
  • the EPO expression levels from all DNA-LNPs compare favorably to those produced by a clinically relevant AAV5 positive control and the gene expression is durable over the entire 4-week time course (Fig 2E-G).
  • EXAMPLE 3 Preparation and analysis of lipid nanoparticle formulations with various phosphatidylcholine (PC) derivatives.
  • DSPC is a derivative of phosphatidylcholine (PC).
  • PC phosphatidylcholine
  • tails PC variants comprising 16-, 18-, 20-, 22-, or 24-carbon tails and 0, 1, or 2 double bonds were tested, again using ALC-0315 as the ionizable lipid and nanoplasmid DNA comprising a hAAT promoter and an EPO transgene expression cassette.
  • LNPs were formulated following the same methods as described in Example 1, to create the formulations described in FIG. 3 A.
  • EXAMPLE 4 Preparation and analysis of lipid nanoparticle formulations with various phosphatidylethanolamine (PE) derivatives.
  • PE phosphatidylethanolamine
  • DOPE is a derivative of phosphatidyethanolamine (PE).
  • PE phosphatidyethanolamine
  • EXAMPLE 5 Preparation and analysis of lipid nanoparticle formulations with various phosphatidylcholine (PC) derivatives containing ring structures in the tails.
  • PC phosphatidylcholine
  • helper lipid can direct the biological properties of the LNP.
  • PC variants PChcPC, PChemsPC, DChemsPC, and OChemsPC which contain ring structures in their tails, and a PC variant comprising a 22-carbon tail with 1 double bond (22-1 PC) were assessed and compared to DSPC (18-0 PC) and DOPE (18-1 PE).
  • LNP formulations used ALC-0315 as the ionizable lipid and nanoplasmid DNA comprising a hAAT promoter (to promote expression in hepatocytes) and an EPO transgene expression cassette. LNPs were formulated as described in the methods to create the formulations presented in Figure 5A.
  • LNPs comprising these PC derivatives were less toxic than the reference LNP comprising DSPC, as evidenced by IL-6 levels 4 hours post-administration that were approximately 5 to 10-fold lower than those elicited by the reference.
  • LNPs comprising PChemsPC, DChemsPC, OChemsPC, and 22: 1 PC were also less toxic than a reference LNP comprising DOPE, eliciting IL-6 levels that were 2 to 5-fold less than this reference (Fig. 5C).
  • EXAMPLE 6 Comparison of LNP formulations with PC and PE helper lipid derivatives.
  • helper lipid transition temperature correlated strongly with cytokine release (Fig. 7B), with LNPs comprising phospholipids having transition temperatures ranging from -36°C through 25°C (16-1(A9-Cis) PC, 16-1 PE, 18-1 PC, 18-1 PE, 16-0.18-1 PC, 18-0/18-1 PE) being the most tolerated.
  • the helper lipid transition temperature did not correlate with EPO levels (Fig. 7A), with LNPs comprising any helper lipids within the -36°C through 25°C transition temperature range yielded comparably improved EPO levels over LNPs comprising helper lipids outside of the range.
  • EXAMPLE 7 Preparation and analysis of lipid nanoparticles co-formulated with both DNA and mRNA along with various atypical helper lipids.
  • LNPs were formulated with mRNA alone (which did not encode for EPO so could serve as a negative control for EPO levels achieved from delivery of the DNA) or co-formulated with DNA and mRNA together as described in the methods to create the formulations presented in Figure 8A.
  • LNPs containing 0.2 mg/kg DNA and 1.5 mg/kg mRNA
  • the serum levels of IL-6 resulting from exposure to the LNPs were quantified 4 hours post-dose, and the serum levels of human EPO protein were measured 3 days post-dose.
  • DNA+mRNA LNPs made with atypical helper lipids achieved levels of gene expression (EPO in serum) that were 10- to 50- fold greater than those achieved with a reference LNP comprising DSPC.
  • EPO in serum levels of gene expression
  • the ALC-0315 LNP comprising 16-1 PE atypical helper lipid produced 50-fold more EPO in serum compared to the reference ALC-0315 LNP comprising DSPC (Fig. 8B).
  • L- 15 LNPs comprising the atypical helper lipids DOPE, 16-1 PE, and 18-0 / 18-1 PE yielded 5, 10 and 25-fold more EPO, respectively, than the reference L-15 LNP comprising DSPC.
  • LNPs comprising other phospholipids having transition temperatures within the -50°C to 50°C range and in some cases more particularly within the -35°C to 25°C range will yield similarly higher expression of their DNA payloads and/or better safety profiles than LNPs comprising helper lipids outside this range such as DSPC.

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Abstract

Novel lipid nanoparticle compositions are provided for the delivery of DNA and/or RNA to cells in vitro and in vivo with different and improved pharmacokinetic profiles as compared to what is typically observed in the art. Also provided are methods for using the compositions in research and as therapeutics.

Description

NOVEL LIPID NANOPARTICLE COMPOSITIONS FOR THE DELIVERY OF
NUCLEIC ACIDS
1. CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/415,229, filed October 11, 2022, and U.S. Provisional Application No. 63/433,398, filed December 16, 2022, the entirety of each of which is hereby incorporated by reference.
2. INTRODUCTION
[0002] There are many instances in which nuclear delivery of a nucleic acid is desired, where such instances include research, diagnostic and therapeutic applications. One example of such a therapeutic application is gene therapy. In the field of gene therapy, viral vectors, such as vectors based on the virus AAV, are commonly employed to deliver genes to the nucleus. However, AAV’s genome is limited in size, so any gene greater than 4.7kB will not be suitable for use AAV vectors, which limits the utility of such vectors for many indications. In addition, viral vectors, such as AAV, induce an antibody response, such that they can only be delivered once, which is not suitable for some indications, such as indications in the liver where the cells are slowly dividing and will lose the transduced genome, thereby requiring redosing. Furthermore, viral vector such as AAV are toxic at the doses that are required for a therapeutic benefit in some indications. Accordingly, what is needed is a new delivery vehicle for delivering nucleic acids, such as DNA, to cells, particularly in patients in need of gene therapy but also in vitro during research.
[0003] That next generation delivery vehicle is a nanoparticle. Of particular interest are lipid nanoparticles (LNPs), given how LNPs have been de-risked by being used in Onpattro and the Covid vaccine. The problem is that the nucleic acid material packaged in a LNP will impact the physiochemical properties of the LNP, which in turn will impact the LNP pharmacokinetic profile, that is, the effect of the LNP on the body. What is needed to make LNPs be a successful delivery vehicle for delivering nucleic acids (e.g., DNA and/or RNA) are novel LNP formulations that are more efficacious and less immunostimulatory.
3. SUMMARY
[0004] Novel lipid nanoparticle (LNP) compositions are provided for the delivery of nucleic acid such as DNA and/or RNA to cells in vitro and in vivo with different and improved pharmacokinetic profiles as compared to what is typically observed in the art. Also provided are methods for using compositions of the invention in research and as therapeutics.
4. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0005] FIGs. 1 A-1C describe studies performed to assess how the mol percentage of ionizable lipid and the type of phospholipid impact efficacy and toxicity of DNA-LNPs. (FIG. 1 A) Formulation details for the test articles. The ionizable lipid in all formulations is ALC-0315, varied to be 40%, 50%, or 60% of the lipid composition. ALC-0315 is an exemplary cationic lipid [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2- hexyl decanoate). The phospholipid is DSPC or DOPE. Plasmid DNA comprising a CBH promoter driving the expression of an EPO transgene was formulated into LNPs. Good encapsulation efficiency and small size were observed of all test articles. (FIGs. IB, 1C) EPO (FIG. IB) and IL-6 (FIG. 1C) serum levels in wild type BALB/c mice were measured 4 hours post-i.v. administration of test articles at a dose of 1 mg/kg.
[0006] FIGs. 2A-2N describe the test articles and results of an experiment to assess the impact of different phospholipids, different PEG lipids, and the inclusion of the livertargeting moiety, GalNAc, on the efficacy and toxicity of DNA-LNPs. (FIG. 2A) Formulation details for the test articles. The ionizable lipid in all formulations is ALC-0315, although it will be appreciated that other ionizable lipids may be used. LNPs in groups 1-3 and 7-9 comprise phospholipid DSPC while LNPs in groups 4-6 and 10-12 comprise phospholipid DOPE. LNPs in groups 1-6 comprise PEG-DMG[2K], while LNPs in groups 7-12 comprise PEG-DSG[2K] and GalNAc-PEG-lipid. Nanoplasmid DNA (npDNA) comprising an expression cassette with a human AAT promoter driving expression of an EPO transgene was formulated into LNPs. (FIG. 2B) The structure of the GalNAc-PEG-lipid used in groups 7-12. (FIGs. 2C, 2D, 2E, 2F, 2G, 21, 2H, 2J, 2K, 2L, 2M) EPO and cytokine levels in serum of wild type mice were measured post-i.v. administration of 1.0 mg/kg, 0.3 mg/kg, and O.lmg/kg test articles. EPO levels in serum were recorded 3 days post-administration (FIG. 2C) and in a time course over 28 days post-administration (l.Omg/kg, FIG. 2E; 0.3 mg/kg, FIG. 2F; 0.1 mg/kg, FIG. 2G). Cytokine levels in serum were recorded 4 hours postadministration (FIGs. 2D, 2H, 21, 2J, 2K, 2L, 2M). Benchmark levels of EPO achieved by other therapeutic modalities are indicated as (i) RETACRIT dose for chronic kidney disease, (ii) AAV5.EPO 2E13 vg injected i.v. and assessed at 4 weeks. [0007] FIGs. 3 A-3I describe the test articles and results of an experiment to assess how varying the length and saturation of the carbon chain of the phospholipid phosphatidylcholine (PC) used as a helper lipid in the DNA-LNP impact the efficacy and toxicity of the DNA- LNP. PC variants comprising 16-, 18-, 20-, 22-, or 24-carbon tails and 0, 1, or 2 double bonds were assessed. (FIG. 3 A) Formulation details for the test articles (X-Y = number of carbons in the tail (X) and saturation of the tail (Y); X-Y / X-Y = asymmetry, with one tail having one structure X-Y and the other chain having a second structure X-Y). The ionizable lipid in all formulations is ALC-0315, although it will be appreciated that other ionizable lipids may be used. Nanoplasmid DNA (npDNA) comprising an expression cassette with a human AAT promoter and EPO transgene was formulated into LNPs. (FIGs. 3B, 3C, 3D, 3E, 3F, 3G, 3H, 31) EPO and cytokine levels in serum of wild type mice were measured post- i.v. administration of test articles. EPO levels were recorded 4 hours (FIG. 3B) and 2 days (FIG. 3C) post-administration, and cytokine levels were recorded 4 hours post-administration (IL-lbeta, FIG. 3D; IL-12, FIG.3E; IFNgamma, FIG. 3F; IL-6, FIG. 3G; KC, FIG. 3H; TNF alpha, FIG. 31).
[0008] FIGs. 4A-4I describe the test articles of an experiment to assess how varying the length and saturation of the carbon chain of the phospholipid phosphatidylethanolamine (PE) used as a helper lipid in the DNA-LNP impact the efficacy and toxicity of the DNA-LNP. PE variants comprising 16-, 18-, 20-, 22-, or 24-carbon tails and 0, 1, or 2 double bonds were assessed. (Figure 4A) Formulation details for the test articles (X-Y = number of carbons in the tail (X) and saturation of the tail (Y); X-Y / X-Y = asymmetry, with one tail having one structure X-Y and the other chain having a second structure X-Y). The ionizable lipid in all formulations is ALC-0315, although it will be appreciated that other ionizable lipids may be used. Nanoplasmid DNA (npDNA) comprising an expression cassette with a human AAT promoter and EPO transgene was formulated into LNPs. (FIGs. 4B, 4C, 4D, 4E, 4F, 4G, 4H, 4J) EPO and cytokine levels in serum of wild type mice were measured post-i.v. administration of test articles. EPO levels were recorded 4 hours (FIG. 4B) and 2 days (FIG. 4C) post-administration, and cytokine levels were recorded 4 hours post-administration (IL- lbeta, FIG. 4D; IL-12, FIG.4E; IFNgamma, FIG. 4F; IL-6, FIG. 4G; KC, FIG. 4H; TNFalpha, FIG. 41).
[0009] FIGs. 5A-5C describe the test articles of an experiment to assess the impact of introducing ring structures into the tails of the phospholipid phosphatidylcholine (PC) used as a helper lipid in the DNA-LNP on the efficacy and toxicity of the DNA-LNP. FIGs. 5A-5C additionally describe the impact of varying the length and saturation of the carbon chain of the phospholipid phosphatidylcholine (PC) used as a helper lipid in the DNA-LNP on the efficacy and toxicity of the DNA-LNP. PC variants PChcPC, PChemsPC, DChemsPC, and OChemsPC, which contain ring structures in their tails, were assessed. Additionally, a PC variant comprising a 22-carbon tail with 1 double bond (22-1 PC) was assessed and compared to DSPC (18-0 PC) and DOPE (18-1 PE). (FIG. 5A) Formulation details for the test articles. The ionizable lipid in all formulations is ALC-0315, although it will be appreciated that other ionizable lipids may be used. Nanoplasmid DNA (npDNA) comprising an expression cassette with a human AAT promoter and EPO transgene was formulated into LNPs. (FIGs. 5B-5C) EPO and IL-6 cytokine levels in serum of wild type mice were measured post-i.v. administration of test articles. EPO levels were recorded 3 days post-administration (FIG 5B), and cytokine levels were recorded 4 hours post-administration (FIG. 5C).
[0010] FIGs. 6A-6B provide a summary of the efficacy and tolerability of LNPs made with some of the best-performing helper lipids. FIG. 6A documents EPO expression 7 days postadministration. FIG. 6B documents IL-6 levels at 4 hours post-administration.
[0011] FIGs. 7A-7B documents phospholipid transition temperature relative to EPO expression 7 days post-administration (FIG, 7A) and IL-6 levels at 4 hours postadministration (FIG. 7B).
[0012] FIGs 8A-8C illustrate that the observations made regarding the superiority of atypical helper lipids for formulating DNA payloads into LNPs also apply to co-formulating DNA+RNA payloads into LNPs. PC and PE variants comprising 16- or 18-carbon tails with 0 or 1 double bond were assessed. (FIG. 8A) Formulation details for the test articles (X-Y = number of carbons in the tail (X) and saturation of the tail (Y); X-Y / X-Y = asymmetry, with one tail having one structure X-Y and the other chain having a second structure X-Y; all formulations have an N/P = 7 and a 50: 10:38:5: 1.5 mol ratio of ionizable lipid:helper lipid:chol:PEG-DMG[2k]). The ionizable lipid used in these formulations is either ALC- 0315 or the novel ionizable lipid L-15. Nanoplasmid DNA (npDNA) comprising an expression cassette with a human AAT promoter and EPO transgene, along with an mRNA payload were co-formulated into LNPs. (FIG 8B-8C) EPO and IL-6 cytokine levels in serum of wild type mice were measured post-i.v. administration of test articles. EPO levels were recorded 3 days post-administration (FIG. 8B), and IL-6 cytokine levels were recorded 4 hours post-administration (FIG. 8C). 5. DETAILED DESCRIPTION
5.1 Lipid Nanoparticle Compositions
[0013] Novel lipid nanoparticle compositions are provided for the delivery of nucleic acid such as DNA to cells in vitro and in vivo with different and improved pharmacokinetic profiles as compared to what is typically observed in the art. Also provided are methods for using the lipid nanoparticle compositions of this disclosure in research and as therapeutics.
[0014] A “lipid nanoparticle” refers to a lipid composition that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., DNA and/or RNA), a protein, a small molecule, and the like to a target site of interest. In the lipid nanoparticle, a nucleic acid agent may be encapsulated in the lipid, thereby protecting the agent from enzymatic degradation.
[0015] In general, the lipid nanoparticle includes several lipid components, including e.g., an ionizable lipid, one or more helper lipid(s) (e.g., non-cationic lipid(s)), and a lipid that prevents aggregation of the nanoparticle (also referred to as a coat lipid or conjugated lipid e.g., a PEG-lipid). In some aspects, this disclosure provides for a lipid nanoparticle (LNP) composition as described herein comprising a nucleic acid, where the nucleic acid is substantially encapsulated by the lipid components of the LNP.
5.2 Ionizable Lipids
[0016] The lipid nanoparticles (LNPs) of this disclosure can include an ionizable lipid. The ionizable lipid is typically employed in the LNP to condense the nucleic acid cargo, e.g., DNA, at low pH and to drive membrane association and fusogenicity. The term “ionizable lipid” refers to a lipid comprising an ionizable group that carries a net charge at a selected pH (e.g., a pH of 6.5 or less), but which can remain neutral at, e.g., a higher pH, such as physiological pH. The pH-sensitivity of such ionizable lipids can be desirable to provide for intracellular delivery of nucleic acid cargo. Ionizable lipids can have less interactions with cell membranes when neutral, and then become charged when internalized into endosomes in a target cell, where the pH is lower than in the extracellular environment. Ionizable lipids which are protonated and, therefore, become positively charged, may promote membrane destabilization and facilitate endosomal escape of the nanoparticle.
[0017] In some embodiments, the ionizable lipid is a cationic lipid. The term “cationic lipid” refers to a lipid that carries a net positive charge at a selected pH (e.g., a pH of 6.5 or less). In some embodiments, the ionizable lipids are cationic lipids including at least one ionizable amino group that is positively charged or becomes protonated at a selected pH, for example at pH of 6.5 or lower. In some embodiments, the cationic lipid includes one or more tertiary amino groups, e.g., a trialkyl amino group.
[0018] In some embodiments, the cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group, hydrocarbon chains (e.g., C8-C20 carbon chains, such as Cis alkyl chains), ether linkages between the head group and hydrocarbon chains, and 0 to 3 double bonds per hydrocarbon chain.
[0019] Non-limiting examples of cationic lipids are described in detail herein. Cationic lipids and related analogs, which are useful in the lipid nanoparticles of the present disclosure, include but are not limited to, those lipids described in U.S. Patent Publication Nos.
20060083780 and 20060240554; U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96/10390, the disclosures of which are herein incorporated by reference in their entirety for all purposes. Additional cationic lipids of interest include, but are not limited to, l,2-distearyloxy-N,N-dimethyl-3- ami nopropane (DSDMA), 1,2 -dilinol eyloxy-N,N -dimethyl -3 -aminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), l,2-dioleyloxy-N,N- dimethyl-3 -aminopropane (DODMA), and heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA).
[0020] Further exemplary ionizable lipids which can be adapted for use in the lipid nanoparticles of the present disclosure are described in International PCT patent application publications W02015/095340, WO2015/199952, W02018/011633, WO2017/049245, WO2015/061467, WO2012/040184, WO2012/000104, WO2015/074085, WO2016/081029, WO20 17/004143, WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541, WO2013/116126, WO2011/153120, WO2012/044638, WO2012/054365, WO2011/090965, W02013/016058, W02012/162210, W02008/042973, W02010/129709, W02010/144740, WO2012/099755, WO2013/049328, WO2013/086322, WO2013/086373, WO2011/071860, W02009/132131, W02010/048536, W02010/088537, WO2010/054401, WO2010/054406, WO20 10/054405, WO2010/054384, WO2012/016184, W02009/086558, WO2010/042877, WO20 11/000106, WO2011/000107, W02005/120152, WO2011/141705, WO2013/126803, W02006/007712, WO2011/038160, WO2005/121348, WO2011/066651, W02009/127060, WO201 1/141704, W02006/069782, WO2012/031043, W02013/006825, WO2013/033563, W02013/089151, WO2017/099823, WO2015/095346, and WO2013/086354, and US patent publications 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, U52013/0116307, US2010/0062967, US2013/0202684, US2014/0141070, US2014/0255472, US2014/0039032, US2018/0028664, U52016/0317458, and US2013/0195920.
5.3 Helper Lipids
[0021] LNPs of this disclosure can also include one or more helper lipids in addition to the ionizable lipid component described herein. In some embodiments, the helper lipid is a neutral lipid. In some embodiments, the neutral lipid is zwitterionic, e.g., has an overall net zero charge. In some embodiments, a helper lipid comprises an internal anionic moiety and internal cationic moiety, e.g., a phosphate and an ammonium.
[0022] In general, the LNPs of this disclosure include a helper lipid component that includes a neutral lipid (e.g., zwitterionic or uncharged) that is a phospholipid.
[0023] In some embodiments, the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG).
[0024] In some embodiments, the phospholipid has a hydrocarbon chain, or “tail” having 12- 24 carbons, e.g. 16-20 carbons, 18-22 carbons, 12-18 carbons. In some embodiments, phospholipid has a carbon tail of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbons. In some embodiments, the phospholipid tail comprises no double bonds, i.e. the bonds are saturated bonds. In some embodiments, the phospholipid tail is unsaturated, that is, it comprises one or more double bonds, e.g. 1, 2, 3, 4 or 5 double bonds. In some embodiments, the phospholipid tail is unsaturated, that is, it comprises one or more triple bonds, e.g. 1, 2, 3, 4 or 5 triple bonds. In some embodiments, the phospholipid tail comprises one or more ring structures. In some embodiments, the one or more ring structures is selected from 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl; 5- to 6- membered aryl; 7- to 10-membered saturated or partially unsaturated bicyclic carbocyclyl; and 7- to 10-membered bicyclic aryl wherein each ring structure is independently substituted with 0-7 RA groups; each RA is independently selected from halogen, or an optionally substituted group selected from C1-12 aliphatic, phenyl, or 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl. In some such instances, the ring structure is a cholesterol or cholesterol derivative. In some embodiments, the phospholipid is symmetric, i.e., all tails of the phospholipid are the same. In other embodiments, the phospholipid is asymmetric, i.e., the phospholipid comprises two or more different hydrocarbon chains.
[0025] In some embodiments, a helper lipid is or comprises symmetric or asymmetric aliphatic phospholipid moieties that are each independently optionally substituted, branched or straight, partially unsaturated or saturated C9-C24 aliphatic.
[0026] In some embodiments, the helper lipid comprises one or more optionally substituted and/or optionally bridged ring structures in the hydrophobic tail. Exemplary helper lipids of this class include:
Figure imgf000010_0001
[0027] In some embodiments, the helper lipid includes a phosphatidylethanolamine (PE). Phosphatidylethanolamines (PE) are a class of phospholipids that incorporate ethanolamine as a headgroup. In some embodiments, the phosphatidylethanolamine selected from the group consisting of phosphatidylethanolamine, dioleoylphosphatidylethanolamine (1,2- dioleyl-sn-glycero-3-phosphoethanolamine) (18: 1(A9-Cis) PE, or DOPE), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine) (DPPE), dimyristoylphosphoethanolamine (l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) (DMPE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), monomethyl- phosphatidyl-ethanolamine (e.g. 16-0-monom ethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), dielaidoyl-phosphatidylethanolamine (DEPE), lysophosphatidylethanolamine, 1,2- dilauroyl-sn-glycero-3 -phosphoethanolamine (DLPE), and l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DiPPE).
[0028] In certain embodiments, the phosphatidylethanolamine is di oleoylphosphatidylethanolamine (also referred to as l ,2-dioleoyl-.s//-glycero-3- phosphoethanolamine, 18: 1(A9-Cis) PE, “18-1 PE”, “18: 1 PE”, or DOPE), having a tail of 18 carbons and one saturated bond as shown below:
Figure imgf000011_0001
[0029] In certain embodiments, the phosphatidylethanolamine is 1,2-dipalmitoleoyl-sn- glycero-3 -phosphoethanolamine (also referred to as “16-1 PE” or “16: 1 PE”), having a tail of 16 carbons and one saturated bond:
Figure imgf000011_0002
[0030] In certain embodiments, the phosphatidylethanolamine is l-stearoyl-2-oleoyl-sn- glycero-3 -phosphoethanolamine (also referred to as “18-0/18-1 PE”, “18:0/18: 1 PE”, or SOPE), an asymmetric lipid having one tail of 18 unsaturated hydrocarbons and a second tail of 18 carbons with one saturated bond:
Figure imgf000011_0003
[0031] In some embodiments, the helper lipid includes a phosphatidylcholine (PC). Phosphatidylcholines (PC) are a class of phospholipids that incorporate choline as a headgroup. In some embodiments, phosphatidylcholine is selected from the group consisting of phosphatidylcholine, di stearoylphosphatidylcholine (l,2-distearoyl-sn-glycero-3- phosphocholine) (DSPC), dioleoylphosphatidylcholine (l,2-dioleoyl-sn-glycero-3- phosphocholine) (18: 1(A9-Cis) PC, or DOPC), dipalmitoylphosphatidylcholine (1,2- dipalmitoyl-sn-glycero-3 -phosphocholine) (DPPC), l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine (16: 1 PC), hydrogenated soy phosphatidylcholine (HSPC), palmitoyloleoylphosphatidylcholine (POPC), l,2-dieicosenoyl-sn-glycero-3 -phosphocholine (“20-1 PC” or “20: 1 PC”), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dimyristoyl phosphatidylcholine (DMPC), dierucoylphosphatidylcholine (DEPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, l,2-dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (DChemsPC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-palmitoyl-2-cholesterylcarbonoyl-sn-glycero-3 -phosphocholine (PChcPC), and l-palmitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC). In certain embodiments, the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) (also referred to as l,2-distearoyl-sw-glycero-3-phosphocholine, “18-0 PC” or “18:0 PC”), having a tail of 18 carbons and no saturated bonds as shown below:
Figure imgf000012_0001
[0032] In certain embodiments, the phosphatidylcholine is dioleoylphosphatidycholine (also referred to as l,2-dioleoyl-sn-glycero-3 -phosphocholine, “18-1 PC”, “18: 1 PC”, or DOPC), in some instances 18: 1(A9-Cis) PC having a tail of 18 carbons and one saturated bond as shown below:
Figure imgf000012_0002
[0033] In certain embodiments, the phosphatidylcholine is l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine (also referred to as “16-1 PC” or “16: 1 PC”), in some embodiments 16: 1(A9- Cis) PC having a tail of 16 carbons and one saturated bond as shown below:
Figure imgf000013_0001
[0034] In certain embodiments, the phosphatidylcholine is an asymmetric lipid, having one tail of 16 carbons and a second tail of 18 carbons. In some such instances, the tail of the phosphatidylcholine having 18 carbons has one saturated bond, e,g, it is l-palmitoyl-2- oleoyl-glycero-3 -phosphocholine (also referred to as “16-0/18-1 PC”, “16:0/18: 1 PC” or POPC) as shown below:
Figure imgf000013_0002
[0035] In certain embodiments, the phosphatidylcholine is 1,2-dicholesterylhemisuccinoyl- sn-glycero-3 -phosphocholine (DChemsPC), as shown below:
Figure imgf000013_0003
[0036] In certain embodiments, the phosphatidylcholine is l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), as shown below:
Figure imgf000013_0004
[0037] In certain embodiments, the phosphatidylcholine is l-palmitoyl-2- cholesterylcarbonoyl-sn-glycero-3-phosphocholine (PChcPC), as shown below:
Figure imgf000014_0001
[0038] In certain embodiments, the phosphatidylcholine is l-palmitoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), as shown below:
Figure imgf000014_0002
[0039] In some embodiments, the helper lipid includes a phosphatidylglycerol selected from the group consisting of phosphatidylglycerol, dioleoylphosphatidylglycerol (1,2-dioleoyl-sn- glycero-3- phospho-(l’-rac-glycerol) (DOPG), dipalmitoylphosphatidylglycerol, (DPPG), dimyristoyl phosphatidylglycerol (DMPG), di stearoylphosphatidylglycerol (DSPG), and palmitoyloleyolphosphatidylglycerol (POPG).
[0040] In some embodiments, the helper lipid includes a phosphatidylserine, e.g. phosphatidyl serine or dioleoylphosphatidylserine (DOPS).
[0041] In some embodiments, the helper lipid includes a lecithin, e.g. lecithin or lysolecithin.
[0042] In some embodiments, the helper lipid includes a sphingomyelin (SM), e.g., egg sphingomyelin (ESM).
[0043] In some embodiments, the helper lipid is cephalin, cardiolipin, phosphatidic acid, cerebrosides, or dicetylphosphate. In some embodiments, the helper lipid is cephalin. In some embodiments, the helper lipid is cardiolipin. In some embodiments, the helper lipid is phosphatidicacid. In some embodiments, the helper lipid is cerebroside. In some embodiments, the helper lipid is ganglioside. In some embodiments, the helper lipid is dicetylphosphate. [0044] In some embodiments, the helper lipid has a transition temperature between about - 50°C and 50°C. As disclosed herein, it has been observed that LNPs comprising phospholipids having phase transition temperatures within about -50°C and 50°C are better tolerated by the organism, promoting more efficacious delivery of the nucleic acid cargo and/or eliciting lower levels of cytokines such as IL-6 than LNPs produced with phospholipids having phase transition temperatures outside of this range. For example, LNPs comprising phospholipids having phase transition temperatures within -50°C and 50°C elicit only about 3, 4, or 5-fold more cytokines than baseline (baseline being defined as untreated or treated with buffer or excipient), and 5- to 10-fold, in some instances 15-fold, in certain cases 25-fold, less cytokines than comparable LNPs formulated with a phospholipid having a transition temperature outside of this range, e.g. DSPC (transition temperature of 55°C). In some embodiments, the helper lipid has a transition temperature between about -50°C and 50°C. In some embodiments, the LNP of the present disclosure comprises a phospholipid having a transition temperature between about -40°C and 30°C. In some embodiments, the LNP of the present disclosure comprises a phospholipid having a transition temperature between about -35°C and 25°C. In some embodiments, the LNP is not 18: 1(A9-Cis) PE (DOPE). Phase transition temperatures of phospholipids are well known in the art and can be determined by referencing various publicly available databases such as the Avanti Polar website and the Encyclopedia of Biophysics, Gordon C.K. Roberts editor, Vol. 1, pages 1841-1854, or by calculation based on chain length and degree of saturation. The phase transition temperatures of exemplary phospholipids are provided in Table 1. In some embodiments, the LNP of the present disclosure comprises a phospholipid that is selected from the group consisting of 16: 1 PC (e.g. A9-Cis), 16:0/22:6 PC, 18: 1 PC (e.g A9-Cis, A6- Cis, A9-trans), 12:0 PC, 16:0/18: 1 PC, 20: 1 PC, 18:0/18: 1 PC, 18: 1/18:0 PC, 22: 1 PC (e.g. A13-Cis), 13:0 PC, 14:0 PC, 16:0/14:0 PC, 18:0/14:0 PC, 16: 1 PE, 18: 1 PE (e.g. A9-Cis), 16:0/18: 1 PE, 18:0/18: 1 PE and 12:0 PE. In certain embodiments, the LNP of the present disclosure comprises a phospholipid that is selected from the group consisting of 16: 1(A9- Cis) PC, 18: 1(A9-Cis) PC, 16:0/18: 1 PC, 20:1 PC, 16: 1 PE, 18: 1(A9-Cis) PE (DOPE), and 18:0/18: 1 PE. In some embodiments, the LNP is not 18: 1(A9-Cis) PE (DOPE). [0045] Table 1. Phospholipid phase transition temperatures
Figure imgf000016_0001
[0046] In some embodiments, the LNP comprising the atypical phospholipid achieves 5-fold more expression or more, e.g. 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold more expression or more, in some instances 12-fold, 15-fold, 20-fold more expression or more, of the delivered nucleic acid cargo one week after delivery of the same dose of a comparable LNP comprising DSPC instead of the atypical phospholipid. For example, the LNP comprising the atypical phospholipid achieves 5-fold more expression or more, e.g. 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold more expression or more, in some instances 12-fold, 15-fold, 20-fold more expression or more, of the delivered nucleic acid cargo one week after delivery of, for example, a 0.3 mg/kg dose i.v. or a 1.0 mg/kg dose i.v. of a comparable LNP comprising DSPC instead of the atypical phospholipid. As another example, the LNP comprising the atypical phospholipid achieves 5-fold more expression or more, e.g. 6-fold, 7-fold, 8-fold, 9- fold, or 10-fold more expression or more, in some instances 12-fold, 15-fold, 20-fold more expression or more, of the delivered nucleic acid cargo one week after delivery of, for example, a 1.0 mg/kg dose i.v. of a comparable LNP comprising DSPC instead of the atypical phospholipid. Changes in expression may be assessed by any method known by the ordinarily skilled artisan, including, for example, measuring RNA levels, for example by RT-PCR, qRT-PCR, Northern blot and the like; or measuring protein levels by, for example, ELISA, Western blot, and the like; or measuring a functional change as the result of a new activity (e.g. increased hematocrit with more EPO expression, an increase in clotting with more clotting factor, improvement in symptoms in a disease, etc.).
[0047] In some embodiments, the LNP comprising the atypical phospholipid elicits 2-fold less cytokines in serum or less, e.g. 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15- fold, 20-fold, 40-fold, 80-fold, in some instances 100-fold less cytokines, 4 hours after delivery of the same dose of a comparable LNP comprising DSPC instead of the atypical phospholipid. In some embodiments, the LNP comprising the atypical phospholipid elicits 2- fold less cytokines in serum or less, e.g. 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 40-fold, 80-fold, in some instances 100-fold less cytokines, 4 hours after delivery of, for example, a 0.3 mg/kg dose i.v. of a comparable LNP comprising DSPC instead of the atypical phospholipid. In some embodiments, the LNP comprising the atypical phospholipid elicits 5-fold less cytokines in serum or less, e.g. e.g. 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 40-fold, 80-fold, in some instances 100-fold less cytokines, 4 hours after delivery of, for example, a 1.0 mg/kg dose i.v. than the comparable LNP comprising DSPC instead of the atypical phospholipid. Changes in expression may be assessed by any method known by the ordinarily skilled artisan, including, for example, measuring RNA levels, for example by RT-PCR, qRT-PCR, Northern blot and the like; or measuring protein levels by, for example, ELISA, Western blot, and the like; or measuring a functional change as the result of a new activity (e.g. increased hematocrit with more EPO expression, an increase in clotting with more clotting factor, improvement in symptoms in a disease, etc.). Changes in cytokines elicited may likewise be measured by any of a number of methods know in the art, including, e.g. measuring levels in serum by, e.g. ELISA.
[0048] In some aspects, the LNP can further comprise a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof. Exemplary cholesterol derivatives are described in International application W02009/127060 and US patent publication US2010/0130588. The component providing membrane integrity, such as a sterol, can comprise 0-50% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20- 50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.
[0049] Accordingly, the neutral lipid component of the LNPs can further include cholesterol or a derivative or analog thereof. A variety of cholesterol analogs and derivatives can be adapted form use in the LNPs of this disclosure. In some embodiments, the helper lipid component includes cholesterol.
[0050] In some embodiments, the LNP includes a neutral lipid component that includes a mixture of phospholipid and cholesterol or a derivative or analog thereof.
[0051] In some embodiments, the LNP includes a neutral lipid component that includes a phosphatidylethanolamine phospholipid and cholesterol or a derivative or analog thereof.
[0052] In some embodiments, the LNP includes a neutral lipid component that includes DOPE phospholipid and cholesterol. In some embodiments, the LNP includes a neutral lipid component that includes DSPC phospholipid and cholesterol. In some embodiments, the LNP includes a neutral lipid component that includes DOPC phospholipid and cholesterol.
5.4 Other Components
[0053] LNPs of this disclosure can also include one or more additional lipid components. Such lipids can be selected to provide for a desirable profile of nanoparticle properties, such as particle stability, delivery efficacy, tolerability and biodistribution.
[0054] In some aspects, the LNP can further comprise a non-cationic lipid. Non-ionic lipids include amphipathic lipids, neutral lipids and anionic lipids. Accordingly, the non-cationic lipid can be a neutral uncharged, zwitterionic, or anionic lipid. Non-cationic lipids are typically employed to enhance fusogenicity. Exemplary non-cationic lipids envisioned for use in the methods and compositions are described in International Application PCT/US2018/050042 published as WO2019051289A1. Exemplary non-cationic lipids are described in International application Publication WO2017/099823 and US patent publication US2018/0028664.
[0055] In some embodiments, the LNP includes one or more lipids capable of reducing aggregation. In general, a lipids capable of reducing aggregation includes at least a hydrocarbon tail or chain linked to a hydrophilic group which is capable of being configured at the surface of the LNP and provide for reduced LNP aggregation. Thus, the lipid capable of reducing aggregation is sometimes referred to as a conjugated lipid or coat lipid.
[0056] A lipid capable of reducing aggregation of particles may comprise a conjugated lipid molecule, such as a polyethylene glycol (PEG). Generally, these are used to inhibit aggregation of lipid nanoparticles and/or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, polyethyleneglycol (PEG)-lipid conjugate, polyoxazoline (POZ)-lipid conjugates, a polyamide (ATTA)-lipid conjugate, a cationic- polymer-lipid conjugates (CPLs), or mixtures thereof. In one embodiment, the LNPs comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate. In certain embodiments, the PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL.
[0057] In some embodiments, the lipid capable of reducing aggregation is a PEG-lipid. A PEG-lipid refers to a lipid having one or more hydrocarbon tail(s) linked to one or more polyethylene glycol (PEG) moiety(ies) via an optional linker.
[0058] It is understood that the PEG moieties may include terminal modification(s) to provide for e.g., conjugation to the lipid tails via the optional linker. The PEG moiety may be terminated in as a hydroxyl group, or an alkyl ether (e.g., a methoxy terminal group). In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid. PEG-lipids of interest include, but are not limited to, a PEG-diacylglycerol (DAG), a PEG di alkyl oxy propyl (DAA), a PEG- phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG- dipalmityloxypropyl (C 16), a PEG-distearyloxypropyl (C 18), or mixtures thereof.
[0059] Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol
(DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG- DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a PEGylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS- DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-l-O-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl- methoxypolyethylene 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 U.S. Pat. Nos. 5,885,613, 6,287,591, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2016/0376224, and US2017/0119904. In some embodiments, a PEG-lipid is a compound disclosed in US2018/0028664. In some embodiments, a PEG-lipid is disclosed in US20150376115 or in US2016/0376224. The PEG-DAA conjugate can be, 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- omegal-methyl-poly(ethylene glycol), PEG- DMB (3,4-Ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some examples, the PEG-lipid can be selected from the group consisting of PEG-DMG, 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000],
[0060] As described above, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamidelipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (CPL) conjugates can be used in place of or in addition to the PEG-lipid. Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymerlipids are described in the International patent application publications WO 1996/010392, WO1998/051278, W02002/087541, W02005/026372, WO2008/147438, W02009/086558, WO20 12/000104, WO2017/117528, WO2017/099823, WO2015/199952, WO2017/004143, WO2015/095346, WO2012/000104, WO2012/000104, and WO2010/006282, US patent application publications 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 US patents U.S. Pat. Nos. 5,885,613, 6,287,591, 6,320,017, and 6,586,559. 5.5 Targeting ligand
[0061] In some embodiments, it may be desirable to limit transfection of the nucleic acids to certain cells or tissues. For example, the liver can be a target organ of interest in part due to its central role in metabolism and production of proteins and accordingly diseases which are caused by defects in liver-specific gene products (e.g., the urea cycle disorders) and may benefit from specific targeting of cells (e.g., hepatocytes).
[0062] In some embodiments, the LNP further includes a component including a targeting ligand. The targeting ligand can be selected as desired based on a target sell or tissue to which it is desired to direct the LNPs of this disclosure. In some embodiments, the targeting ligand is a ligand of a cell surface receptor. In some embodiments, the cell surface receptor is asialoglycoprotein receptor (ASGPR). The ASGPR is expressed on the surface of hepatocyte cells. In some embodiments, the targeting ligand is a ligand for ASGPR, such as a N- acetylgalactosamine (GalNAc) containing ligand. A variety of GalNAc containing ligands and ligands, including multivalent GalNAc ligands are available for use in the LNP of this disclosure, including, e.g. those disclosed in WO2021178725, the full disclosure of which is incorporated herein by reference
[0063] In some embodiments, the PEG-lipid is linked to the targeting ligand. In some embodiments, the targeting ligand of interest (e.g., as described herein) is linked to a terminal of the PEG moiety. For example, a trisGalNac ligand conjugated to a PEG-lipid can provide for binding of the LNP to the ASGPR receptor of a target cell and result in endocytosis of the LNP.
5.6 Lipid Nanoparticles
[0064] In some embodiments, the LNPs include an ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group; a phospholipid that is DOPE, cholesterol, and a lipid capable of reducing aggregation that is PEG-DMG.
[0065] In some embodiments, the LNPs include an ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group; a phospholipid that is a phosphatidylethanolamine, (e.g. DOPE), cholesterol, and a lipid capable of reducing aggregation that is PEG-DMG, and/or PEG-DSG-GalNAc. In some embodiments, the LNPs include PEG-DMG. In some embodiments, the LNPs include PEG-DSG-GalNAc. [0066] In some embodiments, the LNPs include an ionizable that is a cationic lipid comprising a tertiary amino ionizable group, a phospholipid that is a phosphatidylcholine (e.g. l,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol and a coat lipid (polyethylene glycol-dimyristolglycerol, PEG-DMG), for example as disclosed by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3): 498-507.
[0067] Generally, the lipid particles are prepared that include a total lipid-to-nucleic acid (e.g., DNA and/or RNA) (mass or weight) ratio of from about 5 : 1 to 50: 1. This is also referred to as the ratio of positively-chargeable polymer amine (N = nitrogen) groups to negatively-charged nucleic acid phosphate (P) groups, or N/P ratio. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) can be in the range of from about 1 : 1 to about 50: 1, from about 7: 1 to about 25: 1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9:1. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) can be in the range of 5 to 10, 11 to 20, or 21 to 30. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) can be in the range of 5 to 10. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) can be in the range of 11 to 20. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) can be in the range of 21 to 30. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) is about 7. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) is about 10. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) is about 14. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) is about 28. The amounts of lipids and nucleic acid (DNA and/or RNA) can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3: 1 (“3”), 4: 1 (“4”), 5: 1 (“5”), 6: 1 (“6”), 7: 1 (“7”), 8: 1 (“8”), 9: 1 (“9”), 10: 1 (“10”), 11 : 1 (“11”), 12:1 (“12”), 13: 1 (“13”), 14: 1 (“14”) or higher. Generally, the lipid particle formulation's overall lipid content can range from about 5 mg/mL to about 30 mg/mL.
[0068] In some embodiments, a lipid nanoparticle has a mean diameter between about 10 and about 1000 nm. In some embodiments, a lipid nanoparticle has a diameter that is less than 300 nm. In some embodiments, a lipid nanoparticle has a diameter between about 10 and about 300 nm. In some embodiments, a lipid nanoparticle has a diameter that is less than 200 nm. In some embodiments, a lipid nanoparticle has a diameter between about 25 and about 200 nm. In some embodiments, a lipid nanoparticle preparation (e.g., composition comprising a plurality of lipid nanoparticles) has a size distribution in which the mean size (e.g., diameter) is about 70 nm to about 200 nm, and more typically the mean size is about 100 nm or less. [0069] In some embodiments, an LNP has a mean diameter of 25 to 250 nm, 25 to 240 nm, 25 to 230 nm, 25 to 220 nm, 25 to 210 nm, 25 to 200 nm, 25 to 190 nm, 25 to 180 nm, 25 to 170 nm, 25 to 160 nm, 25 to 150 nm, 25 to 140 nm, 25 to 130 nm, 25 to 120 nm, 25 to 110 nm, 25 to 100 nm, 25 to 90 nm, 25 to 80 nm, 25 to 70 nm, 25 to 60 nm, or 25 to 50 nm.
[0070] In some embodiments, an LNP has a mean diameter of 60 to 250 nm, 70 to 250 nm, 80 to 250 nm, 90 to 250 nm, 100 to 250 nm, 110 to 250 nm, 120 to 250 nm, 130 to 250 nm, 140 to 250 nm, 150 to 250 nm, 160 to 250 nm, 170 to 250 nm, 180 to 250 nm, 190 to 250 nm, 200 to 250 nm, 210 to 250 nm, 220 to 250 nm, 230 to 250 nm, or 240 to 250 nm
[0071] In some embodiments, an LNP has a mean diameter of 60 to 250 nm, 70 to 240 nm, 80 to 230 nm, 90 to 220 nm, 100 to 210 nm, 110 to 200 nm, 120 to 190 nm, 130 to 180 nm, 140 to 170 nm, or 150 to 160 nm.
[0072] In some embodiments, the structural characteristics of the target tissue may be exploited to direct the distribution of the LNPs to such target tissues. For example, to target hepatocytes a LNP may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; accordingly the LNP can readily penetrate such endothelial fenestrations to reach the target hepatocytes. In some embodiments, a LNP may be sized such that the dimensions of the particles are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues. For example, a LNP may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the LNPs to hepatocytes. In such an embodiment, large LNPs will not easily penetrate the endothelial fenestrations, and would instead be cleared by the macrophage Kupffer cells that line the liver sinusoids. In some embodiments, the size of the LNPs is within the range of about 25 to 250 nm or 25 nm to 100 nm, preferably less than 250 nm, less than 175 nm, less than 150 nm, less than 125 nm, or less than 100 nm.
[0073] Without limitations, ionizable lipid can comprise 20-90% (mol) of the total lipid present in the lipid nanoparticle. For example, ionizable lipid molar content can be 20-70% (mol), 30-60% (mol) 40-60% (mol) or 40-50% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, ionizable lipid comprises from about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle. [0074] The neutral lipid components can comprise 0-30% (mol) of the total lipid present in the lipid nanoparticle. For example, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In various embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges from about 2: 1 to about 8: 1. In some embodiments, the lipid nanoparticles do not comprise any phospholipids.
[0075] In some embodiments, the ratio of ionizable lipid:DOPE:Cholesterol:PEG (as a percentage of total lipid content) is A:B:C:D, wherein: a. A = 40% - 60%, B = 5% - 20%, C = 25% - 50%, and D = 1.5% - 3.0% and wherein A+B+C+D = 100% b. A = 40% - 60%, B = 6% - 20%, C = 35% - 45%, and D = 1.5% - 2.5% and wherein A+B+C+D = 100%; c. A = 40% - 60%, B = 10% - 20%, C = 35% - 45%, and D = 1.5% - 2.5% and wherein A+B+C+D = 100%; d. A = 40% - 49%, B = 10% - 20%, C = 35% - 45%, and D = 1.5% - 2.5% and wherein A+B+C+D = 100%; e. A = 39% - 60%, B = 10% - 25%, C = 20% - 30%, and D = 0% - 3% and wherein A+B+C+D = 100%; f. A = 40% - 60%, B = 10% - 25%, C = 20% - 30%, and D = 0% - 3% and wherein A+B+C+D = 100%; g. A = 45% - 50%, B = 20% - 25%, C = 25% - 30%, and D = 0% - 1% and wherein A+B+C+D = 100% h. A = 40% - 60%, B = 10% - 30%, C = 20% - 45%, and D = 0% - 3% and wherein A+B+C+D = 100%; i. A = 40% - 60%, B = 10% - 30%, C = 25% - 45%, and D = 0% - 3% and wherein A+B+C+D = 100%; j. A = 45% - 55%, B = 10% - 20%, C = 30% - 40%, and D = 1% - 2% and wherein A+B+C+D = 100%; k. A = 45% - 50%, B = 10% - 15%, C = 35% - 40%, and D = 1% - 2% and wherein A+B+C+D = 100%; l. A = 45% - 65%, B = 5% - 20%, C = 20% - 45%, and D = 0% - 3% and wherein A+B+C+D = 100%; m. A = 45%, B = 15%, C = 37.5%, and D = 2.5%; n. A = 57%, B = 12%, C = 28.5%, and D = 2.5% o. A = 50% - 60%, B = 5% - 15%, C = 30% - 45%, and D = 0% - 3% and wherein A+B+C+D = 100%; p. A = 55% - 60%, B = 5% - 15%, C = 30% - 40%, and D = 1% - 2% and wherein A+B+C+D = 100%; or q. A = 55% - 60%, B = 5% - 10%, C = 30% - 35%, and D = 1% - 2% and wherein A+B+C+D = 100%; or .
5.7 Nucleic Acid Cargo
[0076] In many embodiments, a given lipid nanoparticle may include a cargo, or payload, to be delivered to cells. Of particular interest in some embodiments are cargos that comprise a polynucleotide. In some embodiments, the polynucleotide is a DNA. DNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure. For example, the DNA may be circular, e.g. a plasmid, a nanoplasmid, a minicircle, a covalently closed circular DNA, a circular viral genome, and the like. As another example, the DNA may be linear, e.g. a doggybone or other closed-end DNA, a linear viral genome, and the like. As another example, the DNA may be multivalent, e.g. a 3DNA. The DNA may be single stranded or double stranded or a hybrid of single and double stranded. The DNA may be chemically modified. In some embodiments, the polynucleotide is an RNA. RNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure. For example, the RNA may be linear or it may be circular. It may be an mRNA, an siRNA, an shRNA, a guide RNA (gRNA), a microRNA (miRNA), a circular RNA (circRNA). It may be chemically modified. The RNA may be single stranded or double stranded or a hybrid of single and double stranded. The RNA may be chemically modified. In some embodiments, the cargo comprises both DNA and RNA. In some embodiments, DNA and RNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure.
[0077] The one or more additional compounds can be a therapeutic agent. The therapeutic agent can be selected from any class suitable for the therapeutic objective. In other words, the therapeutic agent can be selected from any class suitable for the therapeutic objective. In other words, the therapeutic agent can be selected according to the treatment objective and biological action desired. For example, if the DNA within the LNP is useful for treating cancer, the additional compound can be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate). In another example, if the LNP containing the DNA is useful for treating an infection, the additional compound can be an antimicrobial agent (e.g., an antibiotic or antiviral compound). In yet another example, if the LNP containing the DNA is useful for treating an immune disease or disorder, the additional compound can be a compound that modulates an immune response (e.g., an immunosuppressant, immunostimulatory compound, or compound modulating one or more specific immune pathways). In some embodiments, different cocktails of different lipid nanoparticles containing different compounds, such as a DNA encoding a different protein or a different compound, such as a therapeutic may be used in the compositions and methods of the invention. In some embodiments, the additional compound is an immune modulating agent. For example, the additional compound is an immunosuppressant. In some embodiments, the additional compound is immune stimulatory agent.
5.8 Pharmaceutical Compositions
[0078] Also provided herein is a pharmaceutical composition comprising the lipid nanoparticle-encapsulated nucleic acid (e.g., DNA, RNA, DNA and RNA) and a pharmaceutically acceptable carrier or excipient. In some aspects, the disclosure provides for a lipid nanoparticle formulation further comprising one or more pharmaceutical excipients. In some embodiments, the lipid nanoparticle formulation further comprises sucrose, tris, trehalose and/or glycine.
[0079] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0080] As used herein “pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations.
[0081] “Pharmaceutically acceptable salt” includes both acid and base addition salts. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4- acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-l,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthal ene- 1,5- disulfonic acid, naphthal ene-2-sulfonic acid, 1 -hydroxy -2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, ptoluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.
[0082] A “dosing regimen” (or “therapeutic regimen”), as that term is used herein, is a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
[0083] As will be understood from context, a “reference” compound or composition is one that is sufficiently similar to a particular compound of interest to permit a relevant comparison. In some embodiments, information about a reference compound or composition is obtained simultaneously with information about a particular compound. In some embodiments, comparison of a particular compound of interest with a reference compound or composition establishes identity with, similarity to, or difference of the particular compound or composition of interest relative to the compound.
[0084] In another aspect, the present invention provides pharmaceutical compositions comprising a compound or composition of the present disclosure, in combination with a pharmaceutically acceptable excipient (e.g., carrier). [0085] The pharmaceutical compositions include optical isomers, diastereomers, or pharmaceutically acceptable salts of the composition disclosed herein. A compound or composition may be covalently attached a carrier moiety, as described above. Alternatively, a compound or composition of the pharmaceutical composition is not covalently linked to a carrier moiety.
[0086] A “pharmaceutically acceptable carrier”, as used herein refers to pharmaceutical excipients, for example, pharmaceutically, physiologically, acceptable organic or inorganic carrier substances suitable for enteral or parenteral application that do not deleteriously react with the active agent. Suitable pharmaceutically acceptable carriers include water, salt solutions (such as Ringer’s solution), alcohols, oils, gelatins, and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
[0087] The compounds or compositions of the present disclosure can be administered alone or can be coadministered to the subject. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). The preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation).
[0088] In some embodiments, a compound or composition as described herein can be incorporated into a pharmaceutical composition for administration by methods known to those skilled in the art and described herein for provided compounds or compositions.
D. Formulations
[0089] Compounds or compositions of the present invention can be prepared and administered in a wide variety of oral, parenteral, and topical dosage forms. Thus, the compounds or compositions of the present invention can be administered by injection (e.g., intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally). In some embodiments, compounds or composition of the present disclosure are administered orally. Also, the compounds or compositions described herein can be administered by inhalation, for example, intranasally. Additionally, the compounds or
- l- compositions of the present disclosure can be administered transdermally. It is also envisioned that multiple routes of administration (e.g., intramuscular, oral, transdermal) can be used to administer compounds or compositions of the present disclosure. Accrordingly, the present disclosure also provides pharmaceutical compositions comprising pharmaceutically acceptable carrier or excipient and one or more compounds or compositions of the disclosure.
[0090] For preparing pharmaceutical compositions from the compounds of the present disclosure, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances that may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
[0091] In powders, the carrier is finely divided solid in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
E. Effective Dosages
[0092] Pharmaceutical compositions provided by the present disclosure include compositions wherein the active ingredient, i.e. the nucleic acid, is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. For example, when administered in methods to treat phenylketonuria (PKU), such compositions will contain an amount of active ingredient effective to achieve the desired result of increasing the amount of phenylalanine hydroxylase made by cells that have been contacted with a pharmaceutical composition of the present disclosure, which in turn will increase the amount of phenylalanine that gets processed and will decrease the amount of phenylalanine that accumulates in tissues.
[0093] The dosage and frequency (single or multiple doses) of compound or composition administered can vary depending upon a variety of factors, including route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of the symptoms of the disease being treated (e.g., the disease responsive treatment; and complications from any disease or treatment regimen. Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of the invention.
[0094] For any provided compound or test agent, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of active compound(s), i.e. nucleic acid, that are capable of increasing the amount of gene product, e.g. RNA or protein, that is encoded by the nucleic acid
[0095] Therapeutically effective amounts for use in humans may be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals to treat the disease. Additionally or alternatively, a dose for humans can be formulated to achieve a concentration of protein typically found in individuals that are unaffected by disease, or that has been found to be effective in treating individuals having the disease, e.g. during protein or enzyme replacement therapy. The dosage in humans can be adjusted by methods well understood by the ordinarily skilled artisan as they pertain to the disease being treated, including but not limited to monitoring the amount of gene product, e.g. RNA or protein, that is expressed in the contacted tissue, monitoring the amount of metabolite of the disease, monitoring the progression of the disease, and so on following administration of the pharmaceutical composition and adjusting the dosage upwards or downwards, as described above.
[0096] Dosages may be varied depending upon the requirements of the patient and the compound being employed. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects.
[0097] In one aspect, compositions provided herein display one or more improved pharmacokinetic (PK) properties (e.g., Cmax, tmax, Cmin, tl/2, AUC, CL, bioavailability, etc.) or one or more improved pharmacodynamic (PD) properties (e.g. cytokines secreted, or changes in expression level of one or more RNAs or proteins in the body) when compared to a reference composition. In some embodiments, a reference composition is a viral gene therapy known in the art. In some embodiments, a reference composition is an enzyme replacement therapy. In some embodiments, a reference composition is an LNP comprising a specific lipid that differs from the composition provided herein. In certain embodiments, the reference composition is an LNP comprising a phospholipid that differs from the composition provided herein. In some such embodiments, the reference composition is an LNP comprising DSPC. In other such embodiments, the reference composition is an LNP comprising DOPE.
[0098] In some embodiments, a compound of the disclosure or a pharmaceutical composition comprising the same is provided as a unit dose.
5.9 Methods of Preparation
[0099] Any convenient methods can be used to prepare the LNPs of this disclosure. The LNP compositions can be prepared by high energy mixing of ethanolic lipids with aqueous DNA at low pH which protonates the ionizable lipid and provides favorable energetics for DNA / lipid association and nucleation of particles. The particles can be further stabilized through aqueous dilution and removal of the organic solvent. The particles can be concentrated to the desired level.
5.10 Methods of Use
[0100] As illustrated in the working examples and figures herein, the LNPs and LNP pharmaceutical composition of the present disclosure, when formulated with nucleic acids, are less toxic in vivo as compared to a reference LNP, for example an industry standard LNP comprising DSPC (e.g. comprising 50% ionizable lipid ALC-0315, 10% DSPC, 38.5% cholesterol, and 1.5% PEG lipid) administered at the same dose, e.g. at least 2-fold less toxic, e.g. 3-fold, 4-fold or 5-fold less toxic, in some instances 10-fold, 20-fold, or 50-fold less toxic, in certain instances 100-fold less toxic. By “less toxic”, it is meant eliciting a reduced amount of one or more cytokines upon administration to an organism.
[0101] At the same time, the LNPs and LNP pharmaceutical composition of the present disclosure have been observed to be equally or more efficacious at delivering their nucleic acid cargo to the target cell of interest as that same reference LNP administered at the same dose, e.g. having 2-fold the efficacy or more, e.g. 3-fold, 4-fold or 5-fold the efficacy or more, in some instances 10-fold, 20-fold or 50-fold the efficacy, in certain instances 100-fold more efficacious or more. By “more efficacious”, it is meant able to deliver more nucleic acid cargo to the cell, resulting in an increase in the amount of mRNA transcribed from that nucleic acid cargo or an increase in the amount of protein translated, for example a 2-fold increase or more, e.g. a 3-fold, 4-fold, 5-fold increase, e.g. 10-fold, 20-fold, 50-fold increase, in some instances a 100-fold increase or more. [0102] For example, in some embodiments, the LNPs and the LNP pharmaceutical compositions of the present disclosure achieve 5-fold more expression or more, e.g. 6-fold, 7- fold, 8-fold, 9-fold, or 10-fold more expression or more, in some instances 12-fold or 15-fold more expression or more, of the delivered nucleic acid cargo one week after delivery of, for example, a 0.3 mg/kg dose i.v. than a reference LNP, e.g. a comparable LNP comprising DSPC instead of the phospholipid of the present disclosure. Changes in expression may be assessed by any method known by the ordinarily skilled artisan, including, for example, measuring RNA levels, for example by RT-PCR, qRT-PCR, Northern blot and the like; or measuring protein levels by, for example, ELISA, Western blot, and the like; or measuring a functional change as the result of a new activity (e.g. increased hematocrit with more EPO expression, an increase in clotting with more clotting factor, improvement in symptoms in a disease, etc.). As another example, the LNPs and the LNP pharmaceutical compositions of the present disclosure may elicit 4-fold less cytokines in serum or less, e.g. 5-fold, 6-fold, 7- fold less cytokines, 4 hours after delivery of a 0.3 mg/kg dose i.v. or 5-fold less cytokines in serum or less, e.g. 5-fold, 7-fold, 10-fold, and in some instance 15-fold less cytokines or less, 4 hours after delivery of a 1.0 mg/kg dose i.v. than the reference LNP, e.g. the comparable LNP comprising DSPC instead of the phospholipid of the present disclosure. Changes in cytokines may be measured by any of a number of methods know in the art, including for example measuring levels in serum by, e.g. ELISA.
[0103] Put another way, the LNPs of the present disclosure demonstrate an improved pharmacokinetics (PK) profile that broadens the therapeutic index of the composition. By a therapeutic index, or therapeutic ratio, it is meant the range of doses at which a medication is effective without unacceptable adverse events, calculated as the ratio that compares the blood concentration at which a drug becomes toxic and the concentration at which the drug is effective. This improvement over the art makes them more amenable to delivering nucleic acids to cells in vitro and in vivo, and accordingly they find many uses in many applications, including in the delivery of nucleic acids to cells for research and for therapeutic applications.
[0104] In performing such methods, the cells are typically contacted with the composition, e.g. LNP or pharmaceutical composition thereof, in amount effective to deliver the agent into the cytoplasm of the cell. In some embodiments, the contacting is in vitro. In other embodiments, the contacting is in vivo. In some embodiments, the method further comprises measuring the amount of protein produced. [0105] The present disclosure further provides methods of treating or preventing diseases in a subject in need thereof wherein an effective amount of the therapeutic compositions described herein is administered to the subject. The route of administration will vary, naturally, with the location and nature of the disease being treated, and may include, for example intradermal, transdermal, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration. The encapsulated polynucleotide compositions described herein are useful in the treatment of any of any indication in which it is beneficial to deliver a therapeutic cargo into the target cell.
[0106] The present disclosure further provides methods of immunizing a subject against a disease wherein an effective amount of a therapeutic composition described herein is administered to the subject. The route of administration will vary, naturally, with the location and nature of immunization agent, and may include, for example intradermal, transdermal, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration.
[0107] The present disclosure further provides a particle of the disclosure, a vector of the disclosure, a recombinant DNA of the disclosure, or compositions thereof, for use as a medicament. In some embodiments, the medicament is for expressing a protein in a cell. In some embodiments, the expressing of a protein is for the treatment of a disease in which the cell is deficient for the protein. In some embodiments, the expressing of a protein is for the treatment of a disease in which another cell is deficient for the protein. In some embodiments, the medicament is for the treatment of a cancer. In some embodiments, the medicament is for immunization against a disease.
5.11 Utility
[0108] The subject methods and compositions, e.g., as described above, can be used in any application where delivery of a cargo nucleic acid is desired. Applications of interest include both research and therapeutic applications. Applications of interest include, but are not limited to: research applications, diagnostic applications and therapeutic applications. In some instances, cargo nucleic acids that may be introduced into a nucleus via methods of the invention include those encoding research proteins, diagnostic proteins and therapeutic proteins. [0109] Research proteins are proteins whose activity finds use in a research protocol. As such, research proteins are proteins that are employed in an experimental procedure. The research protein may be any protein that has such utility, where in some instances the research protein is a protein domain that is also provided in research protocols by expressing it in a cell from an encoding vector. Examples of specific types of research proteins include, but are not limited to: transcription modulators of inducible expression systems, members of signal production systems, e.g., enzymes and substrates thereof, hormones, prohormones, proteases, enzyme activity modulators, perturbimers and peptide aptamers, antibodies, modulators of protein-protein interactions, genomic modification proteins, such as CRE recombinase, meganucleases, Zinc-finger nucleases, CRISPR/Cas-9 nuclease, TAL effector nucleases, etc., cellular reprogramming proteins, such as Oct 3/4, Sox2, Klf4, c-Myc, Nanog, Lin-28, etc., and the like.
[0110] Diagnostic proteins are proteins whose activity finds use in a diagnostic protocol. As such, diagnostic proteins are proteins that are employed in a diagnostic procedure. The diagnostic protein may be any protein that has such utility. Examples of specific types of diagnostic proteins include, but are not limited to: members of signal production systems, e.g., enzymes and substrates thereof, labeled binding members, e.g., labeled antibodies and binding fragments thereof, peptide aptamers and the like.
[OHl] Proteins of interest further include therapeutic proteins. Therapeutic proteins of interest include without limitation, hormones and growth and differentiation factors, fibrinolytic proteins, transcription factors, and enzymes.
[0112] Target cells to which nucleic acids may be delivered in accordance with embodiments of this disclosure may vary widely. Target cells of interest include, but are not limited to: cell lines, HeLa, HEK, CHO, 293 and the like, Mouse embryonic stem cells, human stem cells, mesenchymal stem cells, primary cells, tissue samples and the like. Some non-limiting examples of a mammalian cell include, without limitation, a mouse cell, a rat cell, hamster cell, a rodent cell, and a nonhuman primate cell. In some embodiments, the target cell is a human cell. It should also be appreciated that the target cell may be of any cell type. For example, the target cell may be a stem cell, which may include embryonic stem cells, induced pluripotent stem cells (iPS cells), fetal stem cells, cord blood stem cells, or adult stem cells (i.e., tissue specific stem cells). In other cases, the target cell may be any differentiated cell type found in a subject. Cells of interest include both dividing cells and non-dividing cells. Examples of specific target cells of interest include, but are not limited to: hepatocytes, stellate cells, T lymphocytes, B lymphocytes, NK cells, skeletal muscle cells, cardiomyocytes, neurons, astrocytes, oligodendrocytes, dendritic cells, skin cells, etc.
[0113] In some instances, the application of interest is a therapeutic application, for example, in the treatment of a disease. For example, the compositions and methods of the present application may be used to deliver a nucleic acid sequence to the nucleus of a cell to complement a genetic deficiency. As one nonlimiting example, compositions of the present application may be used in the treatment of a genetic deficiency that impacts the function of hepatocytes, or in the treatment of a genetic deficiency elsewhere in the body that can be remedied by leveraging hepatocytes as a biofactory to secrete the deficient protein.
5.12 Definitions
[0114] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”
[0115] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
[0116] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
[0117] The terms "individual," "subject" and "host" are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired. In some aspects, the subject is a mammal. In some aspects, the subject is a human being. In some aspects, the subject is a patient. In some aspects, the subject is a human patient. In some aspects, the subject can have or is suspected of having a disorder or health condition associated with a gene-of-interest (GOI). In some aspects, the subject is a human who is diagnosed with a risk of disorder or health condition associated with a GOI at the time of diagnosis or later. In some cases, the diagnosis with a risk of disorder or health condition associated with a GOI can be determined based on the presence of one or more mutations in the endogenous GOI or genomic sequence near the GOI in the genome that may affect the expression of GOI.
[0118] The term "treatment" used referring to a disease or condition means that at least an amelioration of the symptoms associated with the condition afflicting an individual is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom, associated with the condition (e.g., hemophilia A) being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or eliminated entirely such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus, treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease.
[0119] The terms "effective amount," "pharmaceutically effective amount," or "therapeutically effective amount" as used herein mean a sufficient amount of the composition to provide the desired utility when administered to a subject having a particular condition. The term "therapeutically effective amount" therefore refers to an amount of therapeutic cells or a composition having therapeutic cells that is sufficient to promote a particular effect when administered to a subject in need of treatment. An effective amount would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using routine experimentation.
[0120] The term "pharmaceutically acceptable excipient" as used herein refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive or diluent for administration of a compound(s) of interest to a subject. "Pharmaceutically acceptable excipient" can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.
[0121] Compounds of this invention include those described generally above, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.
[0122] The abbreviations used herein have their conventional meaning without the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.
[0123] The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocyclyl”, “cycloaliphatic”, or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocyclyl” or “cycloalkyl”) refers to a monocyclic C3-C7 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
[0124] The term “unsaturated”, as used herein, means that a moiety has one or more units of unsaturation. [0125] The term “alkylene” refers to a bivalent alkyl group. An “alkylene chain” is a polymethylene group, i.e., -(CH2)n-, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
[0126] As used herein, the term “cyclopropylenyl” refers to a bivalent cyclopropyl group of the following structure:
Figure imgf000040_0001
.
[0127] The term “halogen” means F, Cl, Br, or I.
[0128] As used herein, the term “bridged bicyclic” refers to any bicyclic ring system, i.e., carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, a bridged bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include those groups set forth below where each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group is optionally substituted with one or more substituents as set forth for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include:
Figure imgf000041_0001
[0129] The term “aryl” refers to monocyclic and bicyclic ring systems having a total of five to ten ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term “aryl” may be used interchangeably with the term “aryl ring”. In some embodiments, and 8- to 10-membered bicyclic aryl group is an optionally substituted naphthyl ring. In certain embodiments of the present invention, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl”, as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.
[0130] As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.
[0131] As used herein and unless otherwise specified, the suffix “-ene” is used to describe a bivalent group. Thus, any of the terms above can be modified with the suffix “-ene” to describe a bivalent version of that moiety. For example, a bivalent carbocycle is “ carb ocyclyl ene”, a bivalent aryl ring is “arylene”, a bivalent benzene ring is “phenylene”, a bivalent heterocycle is “heterocyclylene”, a bivalent heteroaryl ring is “heteroarylene”, a bivalent alkyl chain is “alkylene”, a bivalent alkenyl chain is “alkylene”, a bivalent alkynyl chain is “alkynylene”, and so forth.
[0132] As described herein, compounds of the invention may, when specified, contain “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either explicit or implicit from the structure (e.g.,
Figure imgf000042_0002
refers to at least
Figure imgf000042_0001
Figure imgf000042_0003
). In addition, in a polycyclic ring system, substituents may, unless otherwise indicated, replace a hydrogen on any individual ring (e.g.,
Figure imgf000042_0005
refers to at least
Figure imgf000042_0004
Figure imgf000042_0006
Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their purification, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
[0133] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH2)o-4R°; -(CH2)o-40R°; -0(CH2)o-4R°; - 0(CH2)O-4C(0)OR°; -0(CH2)O-40R°; -(CH2)O-4CH(OR°)2; -(CH2)O-4SR°; -(CH2)o-4Ph, which may be substituted with R°; -(CH2)o-40(CH2)o-iPh, which may be substituted with R°, - CH=CHPh, which may be substituted with R°; -(CH2)o-40(CH2)o-i-pyridyl which may be substituted with R°; -NO2; -CN; -N3; -(CH2)o-4N(R°)2; -(CH2)o-4N(R0)C(0)R°; - N(R°)C(S)R°; -(CH2)O-4N(R0)C(0)N(R°)2; -N(RO)C(S)N(R°)2; -(CH2)O-4N(R0)C(S)N(R°)2; - (CH2)O-4N(R0)C(0)OR°; -N(R°)N(R°)C(O)R°; -N(RO)N(RO)C(O)N(RO)2; - N(R°)N(R°)C(O)OR°; -(CH2)o-4C(0)R°; -C(S)R°; -(CH2)o-4C(0)OR°; -(CH2)o-4C(0)SR°; - (CH2)o-4C(0)OSi(R°)3; -(CH2)o-4OC(0)R°; -OC(0)(CH2)o-4SR°; -SC(S)SR°; -(CH2)o- 4SC(O)R°; -(CH2)O-4C(0)N(R°)2; -C(S)N(RO)2; -C(S)SR°; -SC(S)SR°; -(CH2)O- 4OC(O)N(RO)2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)RO; -C(NOR°)R°; -(CH2)O- 4SSRO; -(CH2)O-4S(0)2R°; -(CH2)O-4S(0)2OR°; -(CH2)O-4OS(0)2R°; -S(O)2NRO; -(CH2)O- 4S(O)RO; -N(RO)S(O)2N(R°)2; -N(RO)S(O)2R°; -N(OR°)R°; -C(NH)N(RO)2; -P(ORO)2; - P(O)(R°)2; -OP(O)(RO)2; -OP(O)(ORO)2; -SiR°3; -(Ci-4 straight or branched alkylene)O- N(R°)2; or -(Ci-4 straight or branched alkylene)C(O)O-N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, Ci-6 aliphatic, -CH2Ph, - 0(CH2)o-iPh, -CH2-(5- to 6-membered heteroaryl ring), or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atoms(s), form a 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, which may be substituted as defined below.
[0134] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen; -(CH2)o- 2R*; -(haloR*), -(CH2)o-2OH; -(CH2)o-2ORe; -(CH2)o-2CH(OR’)2; -O(haloR’); -CN; -N3; - (CH2)O-2C(0)R*; -(CH2)O-2C(0)OH; -(CH2)O-2C(0)OR’; -(CH2)O-2SR’; -(CH2)O-2SH; -(CH2)O- 2NH2; -(CH2)O-2NHR*; -(CH2)O-2NR’2; -NO2, -SiR*3; -OSiR’3; -C(O)SR’; -(Ci-4 straight or branched alkylene)C(O)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from Ci-4 aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 5- to 6-memebered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.
[0135] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0; =S; =NNR# 2; =NNHC(0)R# 2;
=NNHC(0)0R# 2; =NNHS(O)2R# 2; =NR#; =N0R#; -O(C(R# 2))2-3O-; or -S(C(R# 2))2-3S-; wherein each independent occurrence of R# is selected from hydrogen, Ci-6 aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR#2)2-3O-, wherein each independent occurrence of R#is selected from hydrogen, Ci-6 aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
[0136] Suitable substituents on the aliphatic group of R# include halogen, -R*, -(haloR*), - OH, -OR*, -O(haloR’), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci-4 aliphatic, -CH2PI1, -0(CH2)o-iPh, or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
[0137] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -R', -NR’2, -C(O)Rf, -C(O)ORf, -C(O)C(O)Rf, -C(O)CH2C(O)Rf, -S(O)2Rf, - S(O)2NRf2, -C(S)NRf2, -C(NH)NRf2, or -N(Rf)S(O)2Rf2; wherein each RUs independently hydrogen, Cl -6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the definition above, two independent occurrences or R1', taken together with their intervening atom(s) form an unsubstituted 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
[0138] Suitable substituents on the aliphatic group of R' are independently halogen, -R*, - (haloR*), -OH, -OR*, -O(haloR’), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci-4 aliphatic, -CH2PI1, -0(CH2)o-iPh, or a 5- to 6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
[0139] As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.
[0140] In certain embodiments, the neutral forms of the compounds are regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. In some embodiments, the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.
[0141] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are with the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention. In some embodiments, compounds of the present disclosure are provided as a single enantiomer or single diastereomer. Single enantiomer refers to an enantiomeric excess 80% or more, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.
[0142] The term “oxo”, as used herein, means an oxygen that is double bonded to a carbon atom thereby forming a carbonyl.
[0143] The following example(s) is/are offered by way of illustration and not by way of limitation. ENUMERATED EMBODIMENTS
Aspects of this disclosure include the following numbered embodiments:
Embodiment 1. A lipid nanoparticle comprising: a. a nucleic acid, b. an ionizable lipid, c. a phospholipid that is not DSPC, d. cholesterol, and e. a lipid capable of reducing aggregation.
Embodiment 2. The lipid nanoparticle of embodiment 1, wherein the nucleic acid comprises DNA.
Embodiment 3. The lipid nanoparticle of embodiment 2, wherein the nucleic acid consists essentially of DNA.
Embodiment 4. The lipid nanoparticle of embodiment 2, wherein the nucleic acid further comprises RNA.
Embodiment 5. The lipid nanoparticle of embodiment 4, wherein the RNA is selected from mRNA, gRNA, and siRNA.
Embodiment 6. The lipid nanoparticle of embodiment 1, wherein the nucleic acid consists essentially of RNA.
Embodiment 7. The lipid nanoparticle of embodiment 6, wherein the RNA is selected from mRNA, gRNA, and siRNA.
Embodiment 8. The lipid nanoparticle of any one of embodiments 1-7, wherein the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG), and derivatives thereof.
Embodiment 9. The lipid nanoparticle of any one of embodiments 1-8, wherein the phospholipid comprises hydrocarbon chains each independently having 12-24 carbons.
Embodiment 10. The lipid nanoparticle of embodiment 9, wherein the phospholipid comprises hydrocarbon chains each independently having 16-20 carbons.
Embodiment 11. The lipid nanoparticle of embodiment 9 or 10, wherein the hydrocarbon chains are saturated.
Embodiment 12. The lipid nanoparticle of embodiment 9 or 10, wherein one or more of the hydrocarbon chains are unsaturated.
Embodiment 13. The lipid nanoparticle of embodiment 12, wherein the hydrocarbon chains each independently comprise 1-4 double bonds.
Embodiment 14. The lipid nanoparticle of any one of embodiments 6-13, wherein the phospholipid comprises two different hydrocarbon chains. Embodiment 15. The lipid nanoparticle of embodiment 8, wherein the phospholipid has a phase transition temperature of -40 °C and 30 °C.
Embodiment 16. The lipid nanoparticle of any one of embodiments 6-15, wherein the phospholipid is a phosphatidylethanolamine.
Embodiment 17. The lipid nanoparticle of embodiment 16, wherein the phosphatidylethanolamine is selected from the group consisting of 1 -stearoyl -2 -oleoyl-sn- glycero-3 -phosphoethanolamine (18:0/18: 1 PE), l,2-dipalmitoleoyl-sn-glycero-3- phosphoethanolamine (16: 1 PE), and dioleoylphosphatidylethanolamine (18:1(A9-Cis) PE, or DOPE).
Embodiment 18. The lipid nanoparticle of embodiment 17, wherein the phosphatidylethanolamine is l-stearoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (18:0/18: 1 PE).
Embodiment 19. The lipid nanoparticle of embodiment 17, wherein the phosphatidylethanolamine is l,2-dipalmitoleoyl-sn-glycero-3 -phosphoethanolamine (16: 1 PE).
Embodiment 20. The lipid nanoparticle of any one of embodiments 6-15, wherein the phospholipid is a phosphatidylcholine.
Embodiment 21. The lipid nanoparticle of embodiment 20, wherein the phosphatidylcholine is selected from the group consisting of l,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (16: 1(A9-Cis) PC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (18: l(A9-cis) PC), and 1- palmitoyl-2-oleoyl-glycero-3-phosphocholine (16:0/18: 1 PC).
Embodiment 22. The lipid nanoparticle of embodiment 21, wherein the phosphatidylcholine is 1 -palmitoyl -2 -oleoyl -glycero-3-phosphocholine (16:0/18: 1 PC).
Embodiment 23. The lipid nanoparticle of embodiment 21, wherein the phosphatidylcholine is
1.2-dipalmitoleoyl-sn-glycero-3-phosphocholine (16: 1(A9-Cis) PC).
Embodiment 24. The lipid nanoparticle of embodiment 21, wherein the phosphatidylcholine is
1.2-dioleoyl-sn-glycero-3 -phosphocholine (18:1(A9-Cis) PC).
Embodiment 25. The lipid nanoparticle of any one of embodiments 1-24 wherein the lipid capable of reducing aggregation is a PEG-lipid.
Embodiment 26. The lipid nanoparticle of embodiment 25, wherein the PEG-lipid is 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1, 2- distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).
Embodiment 27. The lipid nanoparticle of any one of embodiments 1-26, further comprising a targeting ligand.
Embodiment 28. The lipid nanoparticle of embodiment 27, wherein the targeting ligand comprises GalNAc. Embodiment 29. The lipid nanoparticle of embodiment 27 or 28, wherein the targeting ligand is linked to the lipid capable of reducing aggregation.
Embodiment 30. The lipid nanoparticle of embodiment 29, wherein the lipid capable of reducing aggregation is PEG- l,2-distearoyl-rac-glycero-3 -methylpoly oxyethylene 2000 (PEG-DSG[2K]).
Embodiment 31. The lipid nanoparticle of any one of embodiments 1-30, wherein the N/P ratio
(ratio of moles of the amine groups of cationic lipids to those of the phosphate ones of DNA) is from 5 to 30.
Embodiment 32. The lipid nanoparticle of embodiment 31, wherein the N/P ratio is 5-10
Embodiment 33. The lipid nanoparticle of embodiment 32, wherein the N/P ratio is 7.
Embodiment 34. The lipid nanoparticle of embodiment 31, wherein the N/P ratio is 10.
Embodiment 35. The lipid nanoparticle of embodiment 31, wherein the N/P ratio is 11-20.
Embodiment 36. The lipid nanoparticle of embodiment 35, wherein the N/P ratio is 14.
Embodiment 37. The lipid nanoparticle of embodiment 31, wherein the N/P ratio is 21-30.
Embodiment 38. The lipid nanoparticle of embodiment 37, wherein the N/P ratio is 28.
Embodiment 39. The lipid nanoparticle of any one of embodiments 1-38, comprising: a. an ionizable lipid at 40 to 60 mol % of the total lipid present; b. a phospholipid at 5 to 20 mol % of the total lipid present; c. cholesterol at 25 to 50 mol % of the total lipid present; and d. a lipid capable of reducing aggregation at 1.5 to 3.0 mol % of the total lipid present. Embodiment 40. The lipid nanoparticle of any one of embodiments 1-38, comprising: a. an ionizable lipid at 40 to 60 mol % of the total lipid present; b. a phospholipid at 6 to 20 mol % of the total lipid present; c. cholesterol at 35 to 45 mol % of the total lipid present; and d. a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present. Embodiment 41. The lipid nanoparticle of any one of embodiments 1-38, comprising: a. an ionizable lipid at 40 to 60 mol % of the total lipid present; b. a phospholipid at 10 to 20 mol % of the total lipid present; c. cholesterol at 35 to 45 mol % of the total lipid present; and d. a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present. Embodiment 42. The lipid nanoparticle of any one of embodiments 1-38, comprising: a. an ionizable lipid at 40 to 49 mol % of the total lipid present; b. a phospholipid at 10 to 20 mol % of the total lipid present; c. cholesterol at 35 to 45 mol % of the total lipid present; and d. a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present. Embodiment 43. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 44. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 45. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 46. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 47. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 48. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 49. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 50. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 51. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 52. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 53. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 54. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 55. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 56. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 57. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 58. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 59. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 60. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 61. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 62. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 63. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 64. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 65. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 66. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 67. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 68. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 69. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 70. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc. Embodiment 71. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 72. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 73. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 74. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 75. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 76. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 77. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. The nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 78. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG- GalNAc.
Embodiment 79. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 80. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. The nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 81. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 82. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DMG.
Embodiment 83. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE or 18:0/18:1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 84. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. The nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 16: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 85. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0/18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 86. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18:0 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 87. The lipid nanoparticle of any one of embodiments 29-38, wherein: a. the nucleic acid is DNA and RNA; b. the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; c. the phospholipid is 18: 1 PE; and d. the lipid capable of reducing aggregation is PEG-DSG-GalNAc.
Embodiment 88. The lipid nanoparticle of any one of embodiments 1-87, wherein the nucleic acid encodes for a therapeutic agent.
Embodiment 89. A pharmaceutical composition comprising a lipid nanoparticle of any one of embodiments 1-88 and a pharmaceutically acceptable excipient, carrier, or diluent.
Embodiment 90. A method for delivering a nucleic acid into a cell, the method comprising contacting the cell with a lipid nanoparticle of any one of embodiments 1 to 88 or the pharmaceutical according to embodiment 89.
Embodiment 91. The method according to embodiment 90, wherein the cell is in vitro.
Embodiment 92. The method according to embodiment 90, wherein the cell is in vivo.
Embodiment 93. The method according to embodiment 90, wherein at least 8-fold more nucleic acid is delivered to the cell than a lipid nanoparticle comprising a DSPC phospholipid.
Embodiment 94. The method according to embodiment 90, wherein at least 5 -fold less cytokines are elicited than a lipid nanoparticle comprising the same nucleic acid, the same ionizable lipid, a DSPC phospholipid, the same cholesterol, and the same lipid capable of reducing aggregation.
Embodiment 95. A method for delivering a nucleic acid for in vivo production of target protein, the method comprising: administering systemically to a subject in need thereof a pharmaceutical composition of embodiment 89, wherein the nucleic acid encodes a target protein and is encapsulated within the lipid nanoparticles, and the administering of the pharmaceutical composition results in the prolonged stable expression of the target protein. 7. EXAMPLES
[0144] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
[0145] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for methods referred to in, or related to, this disclosure are available from commercial vendors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and the like, as well as repositories such as e.g., Addgene, Inc., American Type Culture Collection (ATCC), and the like.
Materials and Methods
[0146] LNP formulation. LNPs encapsulating nucleic acid payloads were prepared by mixing an organic solution of lipids with an aqueous solution of nucleic acid (e.g., DNA only, mRNA only, or DNA/mRNA mixtures) as described in Prud’homme et al. (J Pharm Sci. 2018). Briefly, the lipidic excipients mixture (ionizable lipid, helper lipid, cholesterol, PEG-lipid and potentially other targeting moieties) is dissolved in an organic solvent. An aqueous solution of the nucleic acid is prepared in a low pH buffer of range pH 3.0 - 4.0. The lipid mixture is then mixed with the aqueous nucleic acid solution at a flow ratio of 1 :3 (VW) using a commercially available mixer device. The resulting solution is immediately diluted with a buffer pH range of 5.0-6.5. The diluted LNP is subjected to dialysis purification against a secondary buffer with the pH range of 7.0-8.0. The LNP solution is concentrated by using 100,000 MWCO Amicon Ultra centrifuge tubes (Millipore Sigma) followed by filtration through 0.2 pm PES sterilizing-grade filter. Particle size is determined by dynamic light scattering (Horiba nanoPartica SZ-100). Encapsulation efficiency is calculated by using Quant-it RiboGreen assay kit.
[0147] EPO and cytokine detection in serum. Blood is collected via a retro-orbital bleed into serum separator tubes and processed to serum. The serum samples may be stored at -80C from collection until analysis. The serum levels of human EPO protein driven by expression from the DNA payload are quantified using the U-PLEX Human EPO Assay from MSD according to the manufacturer’s instructions. The serum levels of mouse cytokines resulting from exposure to DNA-LNPs were quantified using the Mouse Prolnflammatory 7-Plex Tissue Culture Kit from MSD according to the manufacturer’s instructions.
EXAMPLE 1: Preparation and analysis of lipid nanoparticle formulations with varying mol% of ionizable lipid and different helper lipids
[0148] DNA payloads were formulated into lipid nanoparticles (LNPs) comprising the ALC- 0315 ionizable lipid at several mol ratios and one of two helper lipids (DSPC or DOPE), as described above and detailed in FIG. 1 A. Wild type female BALB/c mice (approximately 7- 8 weeks old) were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight. The DNA-LNPs were administered at 1 mg/kg based on the weight of the DNA payload. Four hours after dosing, blood was collected via a retro-orbital bleed and EPO levels in serum determined as presented in in FIG. IB. The serum levels of mouse cytokines resulting from exposure to DNA-LNPs were quantified, with the results of IL-6 as a representative cytokine shown in FIG. 1C. Varying the ionizable lipid mol% and type of helper lipid only had a minor effect on the EPO expression levels, with most groups within a 2- to 3-fold range. However, the ionizable lipid mol% and type of helper lipid had a very strong effect on cytokine release, with lower mol% of ionizable lipid resulting in lower cytokines and DOPE resulting in significantly less cytokines (approx. 10-fold to 15-fold less) compared to DSPC.
EXAMPLE 2: Preparation and analysis of lipid nanoparticle formulations with different helper phospholipids, different PEG-lipids, and with or without GalNAc-PEG-lipid [0149] The sugar moiety N-Acetylgalactosamine (GalNAc) has been shown to increase uptake of LNPs by hepatocytes. To determine if GalNAc on the surface of an LNP could increase the efficacy of LNPs to deliver DNA to hepatocytes, DNA-LNPs were formulated with ALC-0315 as the ionizable lipid, one of two helper lipids (DSPC or DOPE), one of two PEG-lipids (DMG-PEG2k or DSG-PEG2k), and with or without GalNAc-PEG2k-lipid (structure of GalNAc-PEG-lipid shown in Fig. 2B). LNPs were formulated following the same methods as described in Example 1 but implementing these changes in lipids to create the formulations described in FIG. 2A. Because nanoplasmids are less immunostimulatory than plasmids, the plasmid DNA used in the prior study was replaced with a nanoplasmid DNA (npDNA) comprising an expression cassette with a human AAT promoter driving expression of an EPO transgene. The size of the resultant LNPs were less than 100 nm, and encapsulation efficiencies were very high (>95%).
[0150] Adult female BALB/c mice were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight. The DNA-LNPs were administered at 1, 0.3, or 0.1 mg/kg based on the weight of the DNA payload. AAV5 expressing EPO was administered at 2E13 vg/kg as a positive control. At several time points after dosing, blood was collected via a retro-orbital bleed. Three days post-dose, the serum levels of human EPO protein driven by expression from the DNA payload were quantified, (Fig. 2C). Four hours post-dose, the serum levels of mouse cytokines resulting from exposure to DNA-LNPs were quantified (Fig. 2D). The 4-week time course of EPO expression from the DNA-LNPs at 1 mg/kg, 0.3 mg/kg, and 0.1 mg/kg are shown in FIG. 2E, 2F, and 2G, respectively, compared to the AAV5.EPO positive control and PBS only negative control.
[0151] As observed previously, LNPs containing DOPE induced the release of lower levels of cytokines compared to DSPC. Surprisingly, LNPs comprising GalNAc were less effective at delivering DNA to hepatocytes as quantified by EPO levels than their unfunctionalized counterpart (Fig. 2C, compare DSPC+PEG-DMG to DSPC+PEG-DSG+GalNAc-PEG-lipid, and DOPE+PEG-DMG to DOPE+PEG-DSG+GalNAc-PEG-lipid). In formulations comprising the DSPC helper lipid, LNPs containing DSG-PEG2k and GalNAc-PEG-lipid resulting in lower cytokines compared to LNPs containing DMG-PEG2k and no GalNAc (Fig. 2D, 2H, 21, 2J, 2K, 2L, 2M). In formulations comprising the DOPE helper lipid, the cytokine levels were sufficiently low that adding DSG-PEG2k and GalNAc-PEG-lipid did not lower them further (Fig. 2D, 2H). The EPO expression levels from all DNA-LNPs compare favorably to those produced by a clinically relevant AAV5 positive control and the gene expression is durable over the entire 4-week time course (Fig 2E-G).
EXAMPLE 3: Preparation and analysis of lipid nanoparticle formulations with various phosphatidylcholine (PC) derivatives.
[0152] DSPC is a derivative of phosphatidylcholine (PC). To determine if all variants of phosphatidylcholine behaved the same way in directing the biological properties of the LNP, we assessed the impact of the length and saturation of the phosphatidylcholine carbon chains, or “tails”. PC variants comprising 16-, 18-, 20-, 22-, or 24-carbon tails and 0, 1, or 2 double bonds were tested, again using ALC-0315 as the ionizable lipid and nanoplasmid DNA comprising a hAAT promoter and an EPO transgene expression cassette. LNPs were formulated following the same methods as described in Example 1, to create the formulations described in FIG. 3 A.
[0153] Adult BALB/c mice were dosed with 0.3 or 1 mg/kg DNA-LNP by tail vein injection. The serum levels of mouse cytokines resulting from exposure to DNA-LNPs were quantified 4 hours post-dose, and the serum levels of human EPO protein were measured 4 hours and 2 days post-dose. LNPs comprising PC variants with unsaturated tails performed better than those with saturated tails at delivering DNA, as measured by EPO levels in serum (compare 16-1, 18-1, 18-2, or 20-1 versus DSPC (18-0) or 20-0) (Fig. 3C). Amongst the monounsaturated lipids, shorter carbon chains appeared to provide better efficacy at delivering DNA than longer carbon chains (compare 16-1 versus 18-1 versus 20-1) (Fig. 3C). All unsaturated PC derivatives had a better toxicity profile than the saturated PCs (DSPC and 20-0), as evidenced by less cytokine release 4 hours post-administration (Fig. 3D, 3E, 3F, 3G, 3H, 31).
EXAMPLE 4: Preparation and analysis of lipid nanoparticle formulations with various phosphatidylethanolamine (PE) derivatives.
[0154] DOPE is a derivative of phosphatidyethanolamine (PE). To determine if all variants of phosphatidyethanolamine behaved the same way in directing the biological properties of the LNP, we assessed the impact of the length and saturation of the hydrocarbon tails. PE variants comprising 16-, 18-, 20-, 22-, or 24-carbon tails and 0, 1, or 2 double bonds were tested, again using ALC-0315 as the ionizable lipid and nanoplasmid DNA comprising a hAAT promoter - EPO transgene expression cassette. LNPs were formulated following the same methods as described in Example 1, to create the formulations described in FIG. 4 A.
[0155] Adult BALB/c mice were dosed with 0.3 or 1 mg/kg DNA-LNP by tail vein injection. The serum levels of mouse cytokines resulting from exposure to DNA-LNPs were quantified 4 hours post-dose, and the serum levels of human EPO protein were measured 4 hours and 2 days post-dose. LNPs comprising PE variants with unsaturated tails containing 1 or 2 double bonds (including DOPE, which is 18-1) performed better than those with unsaturated tails containing 4 double bonds and better than DSPC, as measured by EPO levels in serum (compare 16-1, 18-1, 18-2 versus 20-4 or DSPC) (Fig. 4C). Additionally, the hemi BMP formulation produced higher EPO expression compared to the 18-1/BDP formulation (Fig. 4C). Most unsaturated PE derivatives had a better toxicity profile than DSPC, as evidenced by less cytokine release 4 hours post-administration (FIGs.. (Fig. 4D, 4E, 4F, 4G, 4H, 41).
EXAMPLE 5: Preparation and analysis of lipid nanoparticle formulations with various phosphatidylcholine (PC) derivatives containing ring structures in the tails.
[0156] To further understand how the structure of the helper lipid can direct the biological properties of the LNP, we assessed the impact of the presence of ring structures, length, and saturation of the phosphatidylcholine carbon chains, or “tails”. PC variants PChcPC, PChemsPC, DChemsPC, and OChemsPC, which contain ring structures in their tails, and a PC variant comprising a 22-carbon tail with 1 double bond (22-1 PC) were assessed and compared to DSPC (18-0 PC) and DOPE (18-1 PE). Again, the LNP formulations used ALC-0315 as the ionizable lipid and nanoplasmid DNA comprising a hAAT promoter (to promote expression in hepatocytes) and an EPO transgene expression cassette. LNPs were formulated as described in the methods to create the formulations presented in Figure 5A.
[0157] Adult BALB/c mice were dosed with 0.3 or 1 mg/kg DNA-LNP by tail vein injection. The serum levels of IL-6 resulting from exposure to DNA-LNPs were quantified 4 hours post-dose, and the serum levels of human EPO protein were measured 3 days post-dose. LNPs comprising PC variants delivered DNA to hepatocytes at 1 mg/ml doses as well as or better than a reference LNP comprising DSPC, as illustrated by the equal or greater levels of EPO that were observed in serum relative to levels achieved from the reference LNP (Fig. 5B). Importantly, LNPs comprising these PC derivatives were less toxic than the reference LNP comprising DSPC, as evidenced by IL-6 levels 4 hours post-administration that were approximately 5 to 10-fold lower than those elicited by the reference. Interestingly, LNPs comprising PChemsPC, DChemsPC, OChemsPC, and 22: 1 PC were also less toxic than a reference LNP comprising DOPE, eliciting IL-6 levels that were 2 to 5-fold less than this reference (Fig. 5C).
EXAMPLE 6: Comparison of LNP formulations with PC and PE helper lipid derivatives.
[0158] Adult BALB/c mice were dosed with 0.3 or 1 mg/kg DNA-LNP by tail vein injection. The serum levels of mouse cytokines resulting from exposure to DNA-LNPs were quantified 4 hours post-dose, and the serum levels of human EPO protein were measured 7 days postdose.
[0159] Similar to the EPO levels at 4 h and 2 d post-dose, several LNP formulations comprising atypical helper lipids produced higher EPO levels at 7 d post-dose, which was especially dramatic at the 0.3 mg/kg dose level (Fig. 6A and Table 2). As observed previously, the mono-unsaturated and di-unsaturated helper lipid variants (including the asymmetric variants) elicited lower cytokine release compared to the fully saturated (18-0 or 20-0) and poly-unsaturated (20-4) helper lipid variants (Fig. 6B, and Table 3). Interestingly, across all the PC and PE helper lipid derivatives, it was observed that the helper lipid transition temperature correlated strongly with cytokine release (Fig. 7B), with LNPs comprising phospholipids having transition temperatures ranging from -36°C through 25°C (16-1(A9-Cis) PC, 16-1 PE, 18-1 PC, 18-1 PE, 16-0.18-1 PC, 18-0/18-1 PE) being the most tolerated. In contrast, across all the PC and PE helper lipid derivatives, the helper lipid transition temperature did not correlate with EPO levels (Fig. 7A), with LNPs comprising any helper lipids within the -36°C through 25°C transition temperature range yielded comparably improved EPO levels over LNPs comprising helper lipids outside of the range.
Table 2. Average EPO levels detected in serum 7 days after dosing at 1 mg per kg (mpk) or 0.3 mpk (n > 3).
Figure imgf000061_0001
Figure imgf000062_0001
Table 3. Average IL-6 levels detected in serum 4 hours after dosing at Impk or 0.3 mpk (n > 3).
Figure imgf000062_0002
EXAMPLE 7: Preparation and analysis of lipid nanoparticles co-formulated with both DNA and mRNA along with various atypical helper lipids.
[0160] To understand if the observations made regarding the superiority of atypical helper lipids for formulating DNA payloads into LNPs also apply to co-formulating DNA+RNA payloads into LNPs, PC and PE variants comprising 16- or 18-carbon tails with 0 or 1 double bond were assessed in the context of co-formulated LNPs. The LNP formulations used ALC- 0315 or the novel lipid L-15 as the ionizable lipid, a nanoplasmid DNA comprising a hAAT promoter and an EPO transgene expression cassette, and an mRNA. LNPs were formulated with mRNA alone (which did not encode for EPO so could serve as a negative control for EPO levels achieved from delivery of the DNA) or co-formulated with DNA and mRNA together as described in the methods to create the formulations presented in Figure 8A. [0161] Adult BALB/c mice were dosed with LNPs (containing 0.2 mg/kg DNA and 1.5 mg/kg mRNA) by tail vein injection. The serum levels of IL-6 resulting from exposure to the LNPs were quantified 4 hours post-dose, and the serum levels of human EPO protein were measured 3 days post-dose. As with DNA alone payloads, DNA+mRNA LNPs made with atypical helper lipids achieved levels of gene expression (EPO in serum) that were 10- to 50- fold greater than those achieved with a reference LNP comprising DSPC. For example, the ALC-0315 LNP comprising 16-1 PE atypical helper lipid produced 50-fold more EPO in serum compared to the reference ALC-0315 LNP comprising DSPC (Fig. 8B). Likewise, L- 15 LNPs comprising the atypical helper lipids DOPE, 16-1 PE, and 18-0 / 18-1 PE yielded 5, 10 and 25-fold more EPO, respectively, than the reference L-15 LNP comprising DSPC. All LNPs elicited cytokine response that was comparable to or lower than the response elicited from the reference LNP comprising DSPC, as exemplified by IL-6 levels assessed 4 hours post administration (Fig. 8B and 8C). It is expected that reducing the dose of these LNPs will result in comparable levels of EPO achieved as the reference LNP and 5- to 10-fold less cytokines elicited than the reference LNP. It is also expected that LNPs comprising other phospholipids having transition temperatures within the -50°C to 50°C range and in some cases more particularly within the -35°C to 25°C range will yield similarly higher expression of their DNA payloads and/or better safety profiles than LNPs comprising helper lipids outside this range such as DSPC.
8. EQUIVALENTS AND INCORPORATION BY REFERENCE
[0162] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
[0163] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.
[0164] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter.
All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.
[0165] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.
[0166] All references, issued patents and patent applications mentioned or cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

WHAT IS CLAIMED IS:
1. A lipid nanoparticle comprising: a) a nucleic acid, b) an ionizable lipid, c) a phospholipid that is not DSPC, d) cholesterol, and e) a lipid capable of reducing aggregation.
2. The lipid nanoparticle of claim 1, wherein the nucleic acid comprises DNA.
3. The lipid nanoparticle of claim 2, wherein the nucleic acid consists essentially of DNA.
4. The lipid nanoparticle of claim 2, wherein the nucleic acid further comprises RNA.
5. The lipid nanoparticle of claim 4, wherein the RNA is selected from mRNA, gRNA, and siRNA.
6. The lipid nanoparticle of claim 1, wherein the nucleic acid consists essentially of RNA.
7. The lipid nanoparticle of claim 6, wherein the RNA is selected from mRNA, gRNA, and siRNA.
8. The lipid nanoparticle of any one of claims 1-7, wherein the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidyl serine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG), and derivatives thereof.
9. The lipid nanoparticle of any one of claims 1-8, wherein the phospholipid comprises hydrocarbon chains each independently having 12-24 carbons.
10. The lipid nanoparticle of claim 9, wherein the phospholipid comprises hydrocarbon chains each independently having 16-20 carbons.
11. The lipid nanoparticle of claim 9 or 10, wherein the hydrocarbon chains are saturated.
12. The lipid nanoparticle of claim 9 or 10, wherein one or more of the hydrocarbon chains are unsaturated.
13. The lipid nanoparticle of claim 12, wherein the hydrocarbon chains each independently comprise 1-4 double bonds.
14. The lipid nanoparticle of any one of claims 6-13, wherein the phospholipid comprises two different hydrocarbon chains.
15. The lipid nanoparticle of claim 8, wherein the phospholipid has a phase transition temperature of -40 °C and 30 °C.
16. The lipid nanoparticle of any one of claims 6-15, wherein the phospholipid is a phosphatidylethanolamine.
17. The lipid nanoparticle of claim 16, wherein the phosphatidylethanolamine is selected from the group consisting of l-stearoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (18:0/18:1 PE), l,2-dipalmitoleoyl-sn-glycero-3 -phosphoethanolamine (16:1 PE), and dioleoylphosphatidylethanolamine (18:1(A9-Cis) PE, or DOPE).
18. The lipid nanoparticle of claim 17, wherein the phosphatidylethanolamine is l-stearoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (18:0/18:1 PE).
19. The lipid nanoparticle of claim 17, wherein the phosphatidylethanolamine is l,2-dipalmitoleoyl-sn-glycero-3 -phosphoethanolamine (16:1 PE).
20. The lipid nanoparticle of any one of claims 6-14, wherein the phospholipid is a phosphatidylcholine.
21. The lipid nanoparticle of claim 20, wherein the phosphatidylcholine is selected from the group consisting of l,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine (16:1(A9-Cis) PC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (18:l(A9-cis) PC), and l-palmitoyl-2-oleoyl- glycero-3 -phosphocholine (16:0/18:1 PC).
22. The lipid nanoparticle of claim 21, wherein the phosphatidylcholine is 1 - palmitoyl-2-oleoyl-glycero-3-phosphocholine (16:0/18:1 PC).
23. The lipid nanoparticle of claim 21, wherein the phosphatidylcholine is 1,2- dipalmitoleoyl-sn-glycero-3-phosphocholine (16:1(A9-Cis) PC).
24. The lipid nanoparticle of claim 21, wherein the phosphatidylcholine is 1,2- dioleoyl-sn-glycero-3-phosphocholine (18:1(A9-Cis) PC).
25. The lipid nanoparticle of any one of claims 1-24 wherein the lipid capable of reducing aggregation is a PEG-lipid.
26. The lipid nanoparticle of claim 25, wherein the PEG-lipid is 1,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-l,2-distearoyl- rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).
27. The lipid nanoparticle of any one of claims 1-26, further comprising a targeting ligand.
28. The lipid nanoparticle of claim 27, wherein the targeting ligand comprises GalNAc.
29. The lipid nanoparticle of claim 27 or 28, wherein the targeting ligand is linked to the lipid capable of reducing aggregation.
30. The lipid nanoparticle of claim 29, wherein the lipid capable of reducing aggregation is PEG-1, 2-distearoyl-rac-glycero-3-methylpolyoxy ethylene 2000 (PEG- DSG[2K]).
31. The lipid nanoparticle of any one of claims 1-30, wherein the N/P ratio (ratio of moles of the amine groups of cationic lipids to those of the phosphate ones of DNA) is from 5 to 30.
32. The lipid nanoparticle of claim 31, wherein the N/P ratio is 5-10.
33. The lipid nanoparticle of claim 32, wherein the N/P ratio is 7.
34. The lipid nanoparticle of claim 31, wherein the N/P ratio is 10.
35. The lipid nanoparticle of claim 31, wherein the N/P ratio is 11-20.
36. The lipid nanoparticle of claim 35, wherein the N/P ratio is 14.
37. The lipid nanoparticle of claim 31, wherein the N/P ratio is 21-30.
38. The lipid nanoparticle of claim 37, wherein the N/P ratio is 28.
39. The lipid nanoparticle of any one of claims 1-38, comprising: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 5 to 20 mol % of the total lipid present; c) cholesterol at 25 to 50 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 3.0 mol % of the total lipid present.
40. The lipid nanoparticle of any one of claims 1-38, comprising: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 6 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
41. The lipid nanoparticle of any one of claims 1-38, comprising: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 10 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
42. The lipid nanoparticle of any one of claims 1-38, comprising: a) an ionizable lipid at 40 to 49 mol % of the total lipid present; b) a phospholipid at 10 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
43. The lipid nanoparticle of any one of claims 29-38, wherein: the nucleic acid is DNA; the ionizable lipid is a cationic lipid comprising a tertiary amino ionizable group; the phospholipid is 16: 1 PE or 18:0/18: 1 PE; and the lipid capable of reducing aggregation is selected from PEG-DMG and PEG-DSG-GalNAc.
44. The lipid nanoparticle of any one of claims 1-43, wherein the nucleic acid encodes for a therapeutic agent.
45. A pharmaceutical composition comprising a lipid nanoparticle of any one of claims 1-44 and a pharmaceutically acceptable excipient, carrier, or diluent.
46. A method for delivering a nucleic acid into a cell, the method comprising contacting the cell with a lipid nanoparticle of any one of claims 1 to 44 or the pharmaceutical composition according to claim 45.
47. The method according to claim 46, wherein the cell is in vitro.
48. The method according to claim 46, wherein the cell is in vivo.
49. The method according to claim 46, wherein at least 8-fold more nucleic acid is delivered to the cell than a lipid nanoparticle comprising a DSPC phospholipid.
50. The method according to claim 46, wherein at least 5-fold less cytokines are elicited than a lipid nanoparticle comprising the same nucleic acid, the same ionizable lipid, a DSPC phospholipid, the same cholesterol, and the same lipid capable of reducing aggregation.
51. A method for delivering a nucleic acid for in vivo production of target protein, the method comprising: administering systemically to a subject in need thereof a pharmaceutical composition of claim 45, wherein the nucleic acid encodes a target protein and is encapsulated within the lipid nanoparticles, and the administering of the pharmaceutical composition results in the prolonged stable expression of the target protein.
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