WO2025081002A1 - Lipides ionisables pour l'administration d'acides nucléiques - Google Patents

Lipides ionisables pour l'administration d'acides nucléiques Download PDF

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Publication number
WO2025081002A1
WO2025081002A1 PCT/US2024/050999 US2024050999W WO2025081002A1 WO 2025081002 A1 WO2025081002 A1 WO 2025081002A1 US 2024050999 W US2024050999 W US 2024050999W WO 2025081002 A1 WO2025081002 A1 WO 2025081002A1
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linear
alkyl
lipid
independently selected
nanoparticle composition
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English (en)
Inventor
Evan M. LEWOCZKO
William A. SABBERS
Yong-Sik BONG
Neeti ANANTHASWAMY
Renxiang Chen
Zachary DORSEY
Dong Shen
Yiqing Zou
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RNAimmune Inc
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RNAimmune Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • 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
    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/40—Oxygen atoms
    • C07D211/44—Oxygen atoms attached in position 4
    • C07D211/46—Oxygen atoms attached in position 4 having a hydrogen atom as the second substituent in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
    • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain

Definitions

  • This disclosure provides novel compounds, compositions comprising such compounds, and methods to deliver one or more biologically active agents in cells or organs.
  • LNP lipid nanoparticle
  • Lipid nanoparticles are a versatile and successful gene delivery system, notably highlighted by their use in vaccines against COVID-19. Yet some issues exist for traditional LNPs, such as long-term stability, selective biodistribution and better performance to increasing global access to mRNA vaccines, especially in low-resource regions. There exists an unmet need to develop LNP compositions comprising improved ionizable lipids that are stable and there exists a need to develop compounds, compositions, and improve delivery of biologically active substances into cells or organs.
  • Lipid nanoparticle compositions comprising the novel lipids are disclosed.
  • Four-component lipid compositions include a novel lipid as well as additional lipids such as phospholipids, structural lipids, and PEG lipids.
  • Three-component lipid nanoparticle compositions contain a steroidal or structural lipid-containing component, a stabilizing lipid (such as PEGylated lipid-containing component), a novel cationic or ionizable lipid-containing component of the disclosure.
  • Lipid nanoparticle compositions with less than three components, e.g. one component or two components, and lipid nanoparticles with more than four components, e.g. five components or six components, are also contemplated.
  • the novel ionizable lipid compound is a compound of Formula I: (I), or a salt or isomer thereof, wherein, each n is independently selected from 0-8; each Q is independently selected from
  • each X is independently selected from
  • compounds of Formula I may include, but are not limited to:
  • R 3 and R 4 correspond to groups such that “Q” is satisfied.
  • compounds of A-1, A-2, A-5, A-7, A-9 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art
  • the present disclosure provides a method for synthesizing the compound of PMA or 1(a) comprising performing the following reaction:
  • the present disclosure provides a method for synthesizing the compound of EL-396 or 1(b) comprising performing the following reaction:
  • the present disclosure provides a method for synthesizing the compound of EL-382 or 1(c) comprising performing the following reaction:
  • the present disclosure provides a method for synthesizing the compound of EL-360 or 1(d) comprising performing the following reaction:
  • the novel ionizable lipid compound is a compound of Formula II: (II), or a salt or isomer thereof, wherein, each n is independently selected from 0-8; each Q is independently selected from wherein each m is independently selected from 0-6 and o is independently selected from 0-7 and;
  • compounds of Formula II may include, but are not limited to:
  • R 3 and R 4 correspond to groups such that “Q” is satisfied.
  • compounds of B-1, B-2, B-4, B-6, and B-8 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art
  • the present disclosure provides a method for synthesizing the compound of EW-1 or n(a) comprising performing the following reaction:
  • a method for synthesizing the compound of EW-2 or 11(b) comprising performing the following reaction:
  • the present disclosure provides a method for synthesizing the compound of N1 or 11(c) comprising performing the following reaction:
  • the present disclosure provides a method for synthesizing the compound of N4 or 11(d) comprising performing the following reaction:
  • the cationic or ionizable lipid-containing component may comprise compounds of Formula (IA): or a salt or isomer thereof, wherein m is 0-9; n is 0-9; o is 0-12; p is 0-12;
  • Ri is a linear C 1-12 alkyl
  • R 2 is H or a linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or linear C 1-12 alkyl
  • M 1 and M 2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, - OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.
  • compounds of Formula IA may include, for example, the following compounds:
  • the present disclosure provides compounds of Formula (IB): or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12;
  • R is the side chain of an independently selected amino acid
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or linear C 1-12 alkyl
  • R 5 is the side chain of an independently selected amino acid
  • X 1 is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-;
  • X 2 is -C(O)N(H)-, -C(O)O-, -N(H)C(O)-, or -N(H)C(O)-;
  • X 3 is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-;
  • X 4 is -C(O)N(H)-, -C(O)O-, -N(H)C(O)-, or -N(H)C(O)-.
  • R or R 5 comprises the side chain of a Serine (S), Threonine (1), Cysteine (C), Selenocysteine (U), Glycine (G), Alanine (A), Isoleucine (I), Leucine (L), Methionine (M), or Valine (V).
  • S Serine
  • Threonine (1) Cysteine
  • C Selenocysteine
  • U Glycine
  • G Alanine
  • A Isoleucine
  • I Leucine
  • M Methionine
  • V Valine
  • the carbonyl group in Formula IB is bonded to the amino terminus of the amino acid.
  • the carbonyl group in Formula IB is bonded to the carboxy terminus of the amino acid.
  • compounds of Formula IB may include, for example, the following compounds: [00027]
  • the present disclosure provides compounds of Formula (IC): or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-5;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or a linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • R 5 is H or CH 3 ;
  • M 1 and M 2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H; and
  • X is selected from -CH2-, -O-, -S-, or -P(O)(OR)O-.
  • compounds of Formula IC may include, for example, the following compounds:
  • the cationic or ionizable lipid-containing component may comprise compounds of Formula (HA): or a salt or isomer thereof, wherein m is selected from 0-5; n is selected from 0-12; o is selected from 0-12; q is selected from 1-3;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • X is selected from C(R) 2 , N(R), or O, wherein R is independently selected from a methyl and H.
  • compounds of Formula HA may include, for example, the following compound.
  • the present disclosure provides compounds of Formula (IB): or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-6;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or a linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • R 5 is a linear C 1-4 alkyl alcohol
  • R 6 is a linear C 1-4 alkyl alcohol
  • M 1 and M 2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.
  • compounds of Formula HB may include, for example
  • the present disclosure provides compounds of Formula (IIC): or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • R 5 is a linear C 1-4 alkyl alcohol
  • R 6 is a linear C 1-4 alkyl alcohol
  • M 1 and M 2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.
  • compounds of Formula IIC may include, for example
  • the present disclosure provides compounds of Formula (HD): or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 1-7; o is selected from 0-12; p is selected from 0-12;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or a linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • M 1 and M 2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.
  • compounds of Formula IID may include, for example
  • the present disclosure provides compounds of Formula (HE): or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • M 1 and M 2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.
  • compounds of Formula HE may include, for example
  • the present disclosure provides compounds of Formula (IIF): or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • M 1 and Ma are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.
  • compounds of Formula IIF may include, for example
  • the present disclosure includes lipid nanoparticle (LNP) comprising at least one ionizable lipid compound of the disclosure.
  • the LNP further comprises a biologically active agent
  • the biologically active agent is a nucleic acid.
  • the biologically active agent is a RNA molecule.
  • the biologically active agent is siRNA, miRNA, shRNA, tRNA, mRNA, circRNA and mixtures thereof.
  • the LNP further comprises a phospholipid selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-
  • DSPC l,
  • the LNP further comprises a structural lipid selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.
  • the structural component is cholesterol.
  • the LNP further comprises a PEG lipid selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG- modified dialkylglycerol, a PEG-modified myristoyl diglyceride, and mixtures thereof.
  • the PEG lipid is DMG-PEG2000.
  • the components in the LNP have the following relative molar percentages:
  • the present disclosure provides a three-component LNP composition wherein the three components are:
  • the three-component LNP composition contains the three components in the following relative mole percentages:
  • the three-component LNP composition contains the three components in the following relative mole percentages:
  • the three-component LNP composition contains the three components in the following relative mole percentages:
  • the three-component LNP composition contains the three components in the following relative mole percentages:
  • the three-component LNP composition contains the three components in the following relative mole percentages:
  • the cationic or ionizable lipid-containing component may comprise MC3, ALC-0315, ALC-0159, SM-102, DOTAP, or similar, or a cationic and/or ionizable lipid disclosed in WO2017049245 A2 (Benenato), which is incorporated herein by reference.
  • the LNP has an encapsulation efficiency of at least 70% after storage at about -80°C to 25°C for at least 28 days.
  • the LNP has a zeta potential (ZP) of about -30 to +30 mV, -25 to +25 mV, -20 to +20 mV, -15 to +15 mV, -10 to +10 mV, or any range or value in the range of -30 to +30 mV, after storage at about -80°C to 25°C for at least 28 days.
  • the LNP has a polydispersity index (PDI) of less than 0.300 after storage at about -80°C to 25°C for at least 28 days.
  • PDI polydispersity index
  • the LNP has a particle size of less than 220 nm after storage at about -80°C to 25°C for at least 28 days. In some aspects, the LNP has a nitrogen to phosphorus ratio (N:P) range of about 10 to 1.
  • the nanoparticle composition has an encapsulation efficiency of at least 70% when stored at 25°C, 4°C, -20°C, or -80°C for at least 28 days.
  • the nanoparticle composition has a particle size of less than 220 nm when stored at 25°C, 4°C, -20°C, or -80°C for at least 28 days.
  • the nanoparticle composition has a nitrogen to phosphorus ratio (N:P) of 5.6:1.
  • N:P nitrogen to phosphorus ratio
  • “Stability,” “stabilized,” and “stable” in the context of the present disclosure refers to the resistance of nanoparticle compositions and/or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size change, aggregation, change in encapsulation, etc.) under given manufacturing, preparation, transportation, storage and/or in-use conditions, e.g., when stress is applied such as shear force, freeze/thaw stress, etc.
  • the LNP further comprises a cryoprotectant.
  • the cryoprotectant is sucrose.
  • the cryoprotectant has a volume/volume concentration of 5-16% (v/v).
  • the LNP comprises an ionizable lipid of the disclosure, a structural lipid, and a PEG lipid.
  • the pharmaceutical composition comprising the LNP of the disclosure and a pharmaceutically acceptable carrier.
  • the present disclosure includes a method of delivering a biologically active agent to a cell, the method comprising administering to a subject the LNP of the disclosure, said administering comprising contacting the cell with the LNP, whereby the biologically active agent is delivered to the cell.
  • the present disclosure includes a method of delivering a biologically active agent to a cell of a subject, the method comprising administering a pharmaceutical composition of comprising the LNP of the disclosure and a pharmaceutically acceptable carrier to the subject.
  • the present disclosure includes a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the LNP of the disclosure.
  • the present disclosure provides a method of producing a polypeptide of interest in a cell (e.g., a mammalian cell) by contacting the cell with the LNP composition and an mRNA encoding the polypeptide of interest, whereby the mRNA is capable of being translated in the cell to produce the polypeptide.
  • a cell e.g., a mammalian cell
  • an LNP comprising at least one ionizable lipid of the disclosure elicits an in vivo immune response comparable to that of a commercially available LNP, e.g. SMI 02 (Modema, SpikeVax).
  • Fig. 1 show in vivo transfection efficiency of LNPs formulated using ionizable lipids of the disclosure and control lipid SMI 02 in mouse sera 35 days after two intramuscular injections on mouse models with 3 mice per group.
  • FIG. 2 show in vivo data of end point titer in mouse sera after 35 days following two intramuscular injections in mouse models, with 3 mice per group, of LNPs formulated using ionizable lipids of the disclosure and control lipid SMI 02.
  • FIG. 3 show in vivo data of end point titer in mouse sera after 14 and 35 days following two intramuscular injections of LNPs formulated using ionizable lipids of the disclosure and control lipid SMI 02 encapsulated with mRNA encoding the omicron BA.1 variant of the spike protein of S ARS-CoV-2.
  • FIG. 4 show a comparison of the reciprocal IgG endpoint titer of 4-component LNP formulated with SM-102 compared to 3 -component LNPs formulated using either SM-102 or EL- PMA, demonstrating similar performance between all formulations comprising EL-PMA and SM- 102 ionizable lipids in mouse models.
  • FIG. 5 show a comparison of 4-component LNP formulated with SM-102 compared to 4-component and 3-component LNPs formulated using ionizable lipids of the disclosure - EW1 , EW2, PMA, and N4.
  • Three ionizable lipids of the disclosure - EW2, PMA, and N4 - have similar in vivo performance relative to SM-102.
  • Fig. 6 shows a X H NMR spectrum of an ionizable lipid of the disclosure (EL-396).
  • FIG. 7 shows an HPLC chromatogram of an ionizable lipid of the disclosure (EL-
  • FIG. 8 shows a *H NMR spectrum of an ionizable lipid of the disclosure (EL-382).
  • FIG. 9 shows an HPLC chromatogram of an ionizable lipid of the disclosure (EL-
  • FIG. 10 shows a *H NMR spectrum of an ionizable lipid of the disclosure (EL-360).
  • FIG. 11 shows an HPLC chromatogram of an ionizable lipid of the disclosure (EL-
  • Fig. 12 shows a NMR spectrum of an ionizable lipid of the disclosure (EW-1).
  • Fig. 13 shows an HPLC chromatogram of an ionizable lipid of the disclosure (EW- 1).
  • Fig. 14 shows a NMR spectrum of an ionizable lipid of the disclosure (EW-2).
  • Fig. 15 shows a NMR spectrum of an ionizable lipid of the disclosure (EL-N1).
  • Fig. 16 shows a NMR spectrum of an ionizable lipid of the disclosure (EL-N4).
  • Fig. 17 shows in vitro results from Table 6 and 7 demonstrating that mLNP transfects well after lyophilization compared to before lyophilization.
  • FIG. 18 shows Western Blot data before and after lyophilization (top panel). A graph showing transfection efficiency of mLNP and LNP before and after lyophilization is shown in the bottom panel.
  • Fig. 19 shows IgG binding antibody titers after the first (a) and the second (b) administration with mLNP and LNP.
  • mLNP group 1 showed higher IgG titer before lyophilization.
  • the immunogenicity response of mLNP group 1 could be maintained after lyophilization.
  • Fig. 20 shows that PMA and N4 under “E” condition (mLNP) do not show in vitro expression, but showed excellent in vivo expression.
  • Fig. 21 shows that IgG titer from mouse models indicate excellent expression of the mRNA from the novel lipids under mLNP (E) conditions.
  • Fig. 22 shows that EW-2 and PMA have excellent T-Cell response in mouse models under mLNP conditions.
  • FIG. 23 shows Western blot analysis of HEK293T cells transfected with mRNA encoding Omicron BA.1 formulated various forms lipids in Table 1. After 48 hrs of transfection, Western Blot analysis was performed using anti-SARS-CoV-2 specific RBD antibody to detect the expression of spike protein.
  • Fig. 24 shows a plot of the reciprocal IgG Endpoint titer as a function of dose of various LNP and mLNP formulations.
  • Fig. 25 shows transfection test of PEG content screening data.
  • Fig. 26 shows transfection efficiency of N/P ratio screening (samples dialyzed with 8% sucrose). The transfection efficiency of mLNP was decreased when the N/P ratio was increased.
  • Fig. 27 shows IgG binding antibody titers after the second administration with mLNP and LNP.
  • mLNP produces equivalent immune response with LNP in mice model, which is consistent with the previous results.
  • Fig. 28 shows mLNP sample pre frozen to -45°C (a) and collapse at -30°C.
  • Fig. 29 shows cake appearance after lyophilization.
  • Fig. 30 shows characterization of mLNP after lyophilization. Size (a) and zeta potential (b) of mLNP after 2nd batch lyophilization. Size (c) and zeta potential (d) of mLNP after 12th batch lyophilization.
  • Fig. 31 shows TEM images of SM-102 mLNP before and after lyophilization.
  • Fig. 32 shows TEM images of SM-102 mLNP before and after freezing.
  • Fig. 33 shows stability of RSV-mLNP under four conditions or RSV-LNP as measured by particle size over the course of 1 month at 25°C, 4°C, -20°C, and -80°C.
  • Fig. 34 shows stability of RSV-mLNP under four conditions or RSV-LNP as measured by particle size distribution over the course of 1 month at 25°C, 4°C, -20°C, and -80°C.
  • Fig. 35 shows stability of RSV-mLNP under four conditions or RSV-LNP as measured by zeta potential over the course of 1 month at 25°C, 4°C, -20°C, and -80°C.
  • Fig. 36 shows stability of RSV-mLNP under four conditions or RSV-LNP as measured by encapsulation efficiency over the course of 1 month at 25°C, 4°C, -20°C, and -80°C.
  • Fig. 37 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 8% sucrose as measured by particle size over the course of 1 month.
  • Fig. 38 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 8% sucrose as measured by particle size distribution over the course of 1 month.
  • Fig. 39 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 8% sucrose as measured by zeta potential over the course of 1 month.
  • Fig. 40 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 8% sucrose as measured by encapsulation efficiency over the course of 1 month.
  • Fig. 41 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 10% sucrose as measured by particle size over the course of 1 month.
  • Fig. 42 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 10% sucrose as measured by particle size distribution over the course of 1 month.
  • Fig. 43 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 10% sucrose as measured by zeta potential over the course of 1 month.
  • Fig. 44 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 10% sucrose as measured by encapsulation efficiency over the course of 1 month.
  • Fig. 45 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 12% sucrose as measured by particle size over the course of 1 month.
  • Fig. 46 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 12% sucrose as measured by particle size distribution over the course of 1 month.
  • Fig. 47 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 12% sucrose as measured by zeta potential over the course of 1 month.
  • Fig. 48 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 12% sucrose as measured by encapsulation efficiency over the course of 1 month.
  • Fig. 49 shows Particle size, PDI and encapsulation efficiency change of RSV mLNP after freezing and lyophilization during 6 months.
  • Fig. 50 shows characterization of mLNP with different mRNA. All size of samples was uniform and the PDI was small. The zeta potential was ⁇ 20 mV.
  • Fig. 51 shows characterization of mLNP with different ionizable lipids.
  • Fig. 52 shows characterization of mLNP before and after lyophilization. The size and zeta potential of mLNP was maintained after lyophilization.
  • Fig. 53 shows a titration curve of moisture content of mLNP after lyophilization via C30s+InMotion KF Flex and C30s.
  • Fig. 54 shows biodistribution of mRNA expression (dark spots) in mice differences between LNP and mLNP formulations with SM-102.
  • Fig. 55 shows biodistribution among the lymph nodes, liver, and spleen demonstrating that mLNP has a distinct difference in biodistribution than LNP.
  • Fig. 56 shows biodistribution of both LNP and mLNP can be controlled by choice of ionizable lipid. EL-396 does not deliver to the liver under either LNP or mLNP conditions.
  • Fig. 57 shows Cell uptake of Cy5-LNP and Cy5-mLNP. Flow cytometry was used to determine distribution of Cy5 fluorescence (panel a), percentage of Cy5 positive cells (panel b) and mean fluorescence intensity (panel c).
  • any one of more of the illustrative components of the molecule are optional and the present disclosure includes aspects that contain greater than or fewer than all of the illustrated elements.
  • the disclosure relates to novel ionizable lipid compounds and compositions and methods involving the same.
  • the disclosure also provides methods of delivering a biologically active agent to a cell, and treating a disease or disorder in a mammal in need thereof.
  • a method of producing a polypeptide of interest in a cell involves contacting a nanoparticle composition comprising an mRNA, such as modified mRNA, circular mRNA, or self-amplifying mRNA, with a cell, whereby the mRNA may be translated to produce the polypeptide or regulatory structure of interest
  • the structural formula of the compound represents a certain isomer for convenience in some cases, but the present disclosure includes all isomers, it being understood that not all isomers may have the same level of activity.
  • a crystal polymorphism may be present for the compounds represented by the formula. Any crystal form, crystal form mixture, or anhydride or hydrate thereof is included in the scope of the present disclosure.
  • the term “isomer” means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound.
  • Compounds may include one or more chiral centers and/or double bonds and may thus exist as stereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis/trans isomers).
  • the present disclosure encompasses any and all isomers of the compounds described herein, including stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates. [000126] In the reaction schemes described herein, multiple stereoisomers may be produced.
  • Lipid nanoparticle compositions comprising the novel lipids are disclosed.
  • Four- component lipid compositions include a novel lipid as well as additional lipids such as phospholipids, structural lipids, and PEG lipids.
  • Three-component lipid nanoparticle compositions contain a steroidal or structural lipid-containing component, a stabilizing lipid (such as PEGylated lipid-containing component), a novel cationic or ionizable lipid-containing component of the disclosure.
  • Lipid nanoparticle compositions with less than three components, e.g. one component or two components, and lipid nanoparticles with more than four components, e.g. five components or six components, are also contemplated.
  • a three-component LNP composition (also referred to herein as a “modified LNP” or a “mLNP”) may include one or more biologically active agents. salt or isomer thereof, wherein, each n is independently selected from 0-8; each Q is independently selected from
  • each X is independently selected from
  • compounds of Formula I may include, but are not limited to:
  • R3 and R 4 correspond to groups such that “Q” is satisfied.
  • compounds of A-l, A-2, A-5, A-7, A-9 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art [000132]
  • the present disclosure provides a method for synthesizing the compound of PMA or 1(a) comprising performing the following reaction:
  • the present disclosure provides a method for synthesizing the compound of EL-396 or 1(b) comprising performing the following reaction:
  • the present disclosure provides a method for synthesizing the compound of EL-382 or 1(c) comprising performing the following reaction:
  • the present disclosure provides a method for synthesizing the compound of EL-360 or 1(d) comprising performing the following reaction:
  • the novel ionizable lipid compound is a compound of Formula II: (II), or a salt or isomer thereof, wherein, each n is independently selected from 0-8; each Q is independently selected from wherein each m is independently selected from 0-6 and o is independently selected from 0-7 and; each X is selected from
  • compounds of Formula II may include, but are not limited to:
  • R3 and R 4 correspond to groups such that “Q” is satisfied.
  • compounds of B-l, B-2, B-4, B-6, and B-8 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art
  • the present disclosure provides a method for synthesizing the compound of EW-1 or 11(a) comprising performing the following reaction:
  • a method for synthesizing the compound of EW-2 or 11(b) comprising performing the following reaction:
  • the present disclosure provides a method for synthesizing the compound of N1 or 11(c) comprising performing the following reaction:
  • the present disclosure provides a method for synthesizing the compound of N4 or 11(d) comprising performing the following reaction:
  • the present disclosure discloses novel lipids and lipid nanoparticle compositions comprising such novel lipids.
  • the present disclosure provides compounds of Formula IA: or a salt or isomer thereof, wherein m is 0-9; n is 0-9; o is 0-12; p is 0-12;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or a linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • M 1 and M 2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups wherein R is a H or a methyl group.
  • compounds of Formula IA may include, for example, the following compounds:
  • the present disclosure provides compounds of Formula IB: or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12;
  • R is the side chain of an independently selected amino acid
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • R 5 is the side chain of an independently selected amino acid
  • X 1 is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-;
  • X 2 is -C(O)N(H)-, -C(O)O-, -N(H)C(O)-, or -N(H)C(O)-;
  • X 3 is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-;
  • R or R 5 comprises the side chain of the amino acid, wherein the amino acid is Serine (S), Threonine (T), Cysteine (C), Selenocysteine (U), Glycine (G), Alanine (A), Isoleucine (I), Leucine (L), Methionine (M), or Valine (V).
  • a compound of Formula IB may have the following structure: in which Xi and X2 represent independently an amino acid, wherein the amino acid is Serine (S), Threonine (T), Cysteine (C), Selenocysteine (U), Glycine (G), Alanine (A), Isoleucine (I), Leucine (L), Methionine (M), or Valine (V).
  • the carbonyl group in Formula IB is bonded to the carboxy terminus of the amino acid.
  • Scheme 6a General synthesis for the protection of the amino acid and orientation of the amino acid with the carbonyl group on the fatty acid tail side.
  • R is the selected amino acid side chain
  • R 6 is the protected amino acid side chain
  • X is a compatible functional group with carboxylic acid
  • X 1 is N(H)C(O) or N(H)C(O);
  • X 2 is C(O)N(H) or C(O)O and
  • X3 is a compatible functional group with the amine.
  • R is the selected amino acid side chain
  • R 6 is the protected amino acid side chain
  • X is a compatible functional group with carboxylic acid
  • X 1 is C(O)N(H) or C(O)O;
  • X 2 is N(H)C(O) or N(H)C(O) and;
  • X3 is a compatible functional group with the amine.
  • R 5 is the side chain of an independently selected amino acid side chain and
  • R 7 is the protected side chain of an independently selected amino acid side chain.
  • compounds of Formula IB may include, for example, the following compounds:
  • the present disclosure provides compounds of Formula IC: or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-5;
  • R 1 is a linear C 1-12 alkyl
  • R2 is H or linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • R 5 is H or CH 3 ;
  • M 1 and M 2 are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, -C(O)S- , -SC(O)-, -OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is a H or a methyl group; and X is selected from -CH2-, -O-, -S-, or -P(O)(OR)O-.
  • the present disclosure provides compounds of Formula ILA: or a salt or isomer thereof, wherein m is selected from 0-5; n is selected from 0-12; o is selected from 0-12; q is selected from 1-3;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • X is selected from C(R)i, N(R), or O, wherein R is independently selected from a methyl and H.
  • Scheme 12 General synthesis route for the synthesis of compounds of Formula nA. wherein X 1 , X 2 , and X 3 are either carboxylic acid (RC(O)O) functional groups, or they are isocyanate (RNCO) functional groups, and X is as defined above.
  • Scheme 13 Fatty acid tail synthesis for R 1 and R 2 . The route is the same for R 3 and R 4 .
  • the present disclosure provides compounds of Formula IIB: or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-6;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or a linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • R 5 is a linear C 1-4 alkyl alcohol
  • R 6 is a linear C 1-4 alkyl alcohol
  • M 1 and M 2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.
  • X is a halide and
  • PG is a protecting group such as N-tert-butyloxycarbonyl group.
  • Scheme 17 Fatty acid tail conversion of hydroxy to amine.
  • Scheme 18 Fatty acid tail conversion of hydroxy to carboxylic acid.
  • the present disclosure provides compounds of Formula IIC: or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • R 5 is a linear C 1-4 alkyl alcohol
  • R 6 is a linear C 1-4 alkyl alcohol
  • M 1 and M 2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.
  • the present disclosure provides compounds of Formula IID: or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 1-7; o is selected from 0-12; p is selected from 0-12;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • M 1 and M 2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.
  • X 1 is a functional group (such as an amine, carboxylic acid, isocyante, etc) compatible with X 2 (an amine, carboxylic acid, isocyanate, etc) to give M 1 such that it is -C(O)N(R)-, -N(R)C(O)-, - C(O)S-, -SC(O)-, -OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.
  • R is independently selected from a methyl and H.
  • the present disclosure provides compounds of Formula HE: or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • M 1 and M 2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.
  • the present disclosure provides compounds of Formula IIF : or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;
  • R 1 is a linear C 1-12 alkyl
  • R 2 is H or linear C 1-12 alkyl
  • R 3 is a linear C 1-12 alkyl
  • R 4 is H or a linear C 1-12 alkyl
  • M 1 and M 2 are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.
  • nanoparticle compositions comprise a lipid component including at least one compound according to Formulae I, II, III, IA, IB, IC, IIA, IIB, IIC, IID, IIE, IIF, including lAa-IAc, IBa-Ibe, ICa-ICc, IIAa-IIAb, IIBa, IICa, HDa, HEa-IIEb, and IIFa-IIFb, and any combination thereof. Nanoparticle compositions may also include a variety of other components.
  • the lipid component of a nanoparticle composition may include one or more other lipids in addition to a lipid according to Formula I, II, III, LA, IB, IC, HA, IIB, IIC, IID, IIE, IIF, including lAa-IAc, IBa-Ibe, ICa-ICc, IIAa-IIAb, IIBa, IICa, HDa, HEa-IIEb, and IIFa-IIFb.
  • Three-component Lipid Nanoparticle Compositions [000188] The disclosure includes three-component LNP compositions containing:
  • the largest dimension of a nanoparticle composition is 1 ⁇ m or shorter (e.g., 1 pm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter), e.g., when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method.
  • Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipoplexes.
  • nanoparticle compositions are vesicles including one or more lipid bilayers.
  • a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments.
  • Lipid bilayers may be functionalized and/or crosslinked to one another.
  • Lipid bilayers may include one or more ligands, proteins, or channels.
  • a three component LNP composition of the present disclosure may include one or more cationic and/or ionizable lipids (e.g., lipids that may have a positive or partial positive charge at physiological pH) including, but not limited to, MC3, ALC-0315, ALC-0159, SM-102, DOTAP, Mol-111, Mol-114, MH-094, or a cationic and/or ionizable lipid disclosed in WO2017049245 A2 (Benenato), lipids of Formulae I, II, III, IA, IB, IC, IIA, IIB, IIC, IID, IIE, IIF, including lAa-IAc, IBa-Ibe, ICa-ICc, IIAa-IIAb, IIBa, IICa, HDa, IIEa-IIEb, and IIFa-IIFb, and any combination thereof.
  • cationic and/or ionizable lipids e.g.,
  • a three component LNP composition of the present disclosure may include one or more stabilizing lipids that use steric bulk to minimize or prevent aggregation.
  • a three component LNP composition of the present disclosure may include one or more PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids.
  • a PEG lipid is a lipid modified with polyethylene glycol.
  • a PEG lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG- modified dialkylglycerols, and mixtures thereof.
  • a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • a three component LNP composition of the present disclosure may include one or more structural lipids.
  • Structural lipids can be selected from the group consisting of, but are not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof.
  • the structural lipid is cholesterol.
  • the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.
  • a three component LNP composition of the present disclosure may be free of phospholipids.
  • the three component LNP composition of the present disclosure may be free of any one or more of the following phospholipids: 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl
  • the three-component LNP may be combined in a composition with one or more adjuvants, e.g., Glucopyranosyl Lipid Adjuvant (GLA), CpG oligodeoxynucleotides (e.g., Class A or B), poly(I:C), aluminum hydroxide, Pam3CSK4, saponin extracts (e.g. Quil-A®), and Lipid A.
  • GLA Glucopyranosyl Lipid Adjuvant
  • CpG oligodeoxynucleotides e.g., Class A or B
  • poly(I:C) poly(I:C)
  • aluminum hydroxide e.g., Pam3CSK4, saponin extracts (e.g. Quil-A®), and Lipid A.
  • Quil-A® e.g., Quil-A®
  • the present disclosure includes nanoparticle compositions comprising at least one ionizable lipid compound of the disclosure.
  • the nanoparticle compositions further comprise a biologically active agent.
  • the biologically active agent is a nucleic acid.
  • the biologically active agent is an RNA molecule.
  • the biologically active agent is siRNA, miRNA, shRNA, tRNA, mRNA, circRNA and mixtures thereof.
  • a nanoparticle composition e.g., a LNP, further comprises a phospholipid selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn- glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (DSPC), l
  • the nanoparticle composition further comprises a structural lipid selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.
  • the structural component is cholesterol.
  • a nanoparticle composition further comprises a PEG lipid selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, a PEG-modified myristoyl diglyceride, and mixtures thereof.
  • the PEG lipid is DMG-PEG2000.
  • the components in the nanoparticle composition have the following relative molar percentages:
  • the nanoparticle composition has an encapsulation efficiency of at least 70% after storage at about -80°C to 25°C for at least 28 days.
  • the LNP has a zeta potential of about -30 to +30 mV after storage at about -80°C to 25°C for at least 28 days.
  • the LNP has a PDI of less than 0.300 after storage at about -80°C to 25°C for at least 28 days.
  • the LNP has a particle size of less than 220 nm after storage at about -80°C to 25°C for at least 28 days.
  • the LNP has a nitrogen to phosphorus ratio (N:P) range of about 10 to 1.
  • the nanoparticle composition further comprises a cryoprotectant
  • the cryoprotectant is sucrose.
  • the cryoprotectant has a volume/volume concentration of 5-12% (v/v).
  • the nanoparticle composition comprises an ionizable lipid of the disclosure, a structural lipid, and a PEG lipid.
  • the present disclosure includes a pharmaceutical composition comprising the nanoparticle composition and a pharmaceutically acceptable carrier.
  • the present disclosure includes a method of delivering a biologically active agent to a cell, the method comprising administering to a subject the LNP of the disclosure, said administering comprising contacting the cell with the nanoparticle composition, whereby the biologically active agent is delivered to the cell.
  • the present disclosure includes a method of delivering a biologically active agent to a cell of a subject, the method comprising administering a pharmaceutical composition of comprising the nanoparticle composition of the disclosure and a pharmaceutically acceptable carrier to the subject
  • the present disclosure includes a method of treating a disease or disorder in a subject in need thereof by administering the nanoparticle composition of the disclosure.
  • the present disclosure provides a method of making a composition by lyophilizing a solution comprising a nanoparticle composition and a nucleic acid.
  • the lyophilized sample may be reconstituted in water or an aqueous solution for administering to a subject to deliver the payload.
  • the method comprises freezing a solution comprising a nanoparticle composition and a nucleic acid at a temperature several degree Celsius below the eutectic point of the solution, and applying vacuum to the frozen solution to remove water to produce a sample.
  • the sample may then be placed under vacuum at a warmer temperature (e.g., a temperature around or above room temperature, e.g., 25°C to 35°C, such as 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35°C) to produce a lyophilized sample.
  • a warmer temperature e.g., a temperature around or above room temperature, e.g., 25°C to 35°C, such as 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35°C
  • the lyophilized sample produced according to the present disclosure may be reconstituted very easily in a small amount of water or an aqueous solution.
  • the nanoparticle composition produced by the method may be capable of expressing the payload nucleic acid (e.g., mRNA) at a higher level (e.g., at least 2-fold) compared to the same nanoparticle composition before lyophilization. After reconstitution, the nanoparticle composition produced by the method may remain stable.
  • the “stabilized” formulations of the disclosure preferably retain at least 80%, 85%,
  • the “stabilized” formulations of the disclosure preferably retain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the encapsulation efficiency of a starting, standard, or reference preparation of the LNP formulation (e.g., mRNA-loaded LNP formulation) under given manufacturing, preparation, transportation, storage and/or in-use conditions.
  • a and an refer to one or more (i.e., at least one) of the grammatical object of the article.
  • a cell encompasses one or more cells.
  • exemplary is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the word exemplary is intended to present concepts in a more concrete fashion. [000220] Furthermore, certain molecules, constructs, compositions, elements, moieties, excipients, disorders, conditions, properties, steps, or the like may be discussed in the context of one specific embodiment or aspect or in a separate paragraph or section of this disclosure.
  • the structural formula of the compound represents a certain isomer for convenience in some cases, but the present disclosure includes all isomers, it being understood that not all isomers may have the same level of activity.
  • a crystal polymorphism may be present for the compounds represented by the formula. Any crystal form, crystal form mixture, or anhydride or hydrate thereof is included in the scope of the present disclosure.
  • the term “isomer” means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound.
  • Compounds may include one or more chiral centers and/or double bonds and may thus exist as stereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis/trans isomers).
  • the present disclosure encompasses any and all isomers of the compounds described herein, including stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates.
  • stereomerically pure forms e.g., geometrically pure, enantiomerically pure, or diastereomerically pure
  • enantiomeric and stereoisomeric mixtures e.g., racemates.
  • the term “compound,” is meant to include all isomers and isotopes of the structure depicted. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium. Further, a compound, salt, or complex of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
  • alkyl or “alkyl group” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted.
  • C1-C24 alkyl means an optionally substituted linear or branched, saturated hydrocarbon including 1-14 carbon atoms.
  • an alkyl group described herein refers to both unsubstituted and substituted alkyl groups.
  • alkenyl or “alkenyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted.
  • C1-C24 alkenyl means an optionally substituted linear or branched hydrocarbon including 1-24 carbon atoms and at least one carbon-carbon double bond.
  • An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds.
  • Cis alkenyl may include one or more double bonds.
  • a Cis alkenyl group including two double bonds may be a linoleyl group.
  • an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.
  • alkynyl or “alkynyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted.
  • the notation “C1-C24 alkynyl” means an optionally substituted linear or branched hydrocarbon including 1-24 carbon atoms and at least one carbon-carbon triple bond.
  • An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds.
  • Cis alkynyl may include one or more carbon-carbon triple bonds.
  • an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.
  • Alkyl, alkenyl, alkynyl, acyl, aryl, heteroaryl and cyclyl (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified.
  • Carbocycle and “carbo cyclic group” refer to an optionally substituted mono- or multi-cyclic system including one or more rings of carbon atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty membered rings.
  • the notation "Ce carbocycle” means a carbocycle including a single ring having 3-6 carbon atoms. Carbocycles may include one or more carbon-carbon double or triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl or aryl groups).
  • carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2-dihy dronaphthyl groups.
  • cycloalkyl as used herein means a non-aromatic carbocycle and may or may not include any double or triple bond.
  • carbocycles described herein refers to both unsubstituted and substituted carbocycle groups, i.e., optionally substituted carbocycles.
  • the term “contacting” means establishing a physical connection between two or more entities.
  • contacting a cell with a nanoparticle composition means that the cell and the nanoparticle composition are made to share a physical connection.
  • Methods of contacting cells with external entities both in vivo and ex vivo are well known in the relevant arts.
  • more than one cell may be contacted by the nanoparticle composition.
  • delivering means providing an entity to a destination.
  • delivering a biologically active agent to a subject may involve administering a LNP including the biologically active agent to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route).
  • Administration of a nanoparticle composition to a subject or cell may involve contacting one or more cells with the nanoparticle composition.
  • encapsulation efficiency refers to the amount of a biologically active agent that becomes part of a nanoparticle composition, relative to the initial total amount of biologically active agent used in the preparation of a nanoparticle composition. For example, if 97 mg of biologically active agent are encapsulated in a nanoparticle composition out of a total 100 mg of therapeutic and/or prophylactic initially provided to the composition, the encapsulation efficiency may be given as 97%. The encapsulation efficiency may be measured, for example, by encapsulating mRNA encoding GFP (green fluorescent protein) sequences in the nanoparticle composition and then measuring the encapsulation efficiency of the RFP sequence using flow cytometry. As used herein, “encapsulation” may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement
  • expression of a nucleic acid sequence refers to translation of an mRNA into a polypeptide or protein and/or post-translational modification of a polypeptide or protein.
  • a “linker” is a moiety connecting two moieties, for example, the connection between two nucleosides of a cap species.
  • a linker may include one or more groups including but not limited to phosphate groups (e.g., phosphates, boranophosphates, thiophosphates, selenophosphates, and phosphorates), alkyl groups, amidates, or glycerols.
  • phosphate groups e.g., phosphates, boranophosphates, thiophosphates, selenophosphates, and phosphorates
  • alkyl groups e.g., phosphates, boranophosphates, thiophosphates, selenophosphates, and phosphorates
  • alkyl groups e.g., phosphates, boranophosphates, thiophosphates, selenophosphates, and phosphorates
  • alkyl groups e.g.,
  • lipid component is that component of a nanoparticle composition that includes one or more lipids.
  • the lipid component may include one or more ionizable, PEGylated, structural, or phospholipids.
  • methods of administration may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject
  • a method of administration may be selected to target delivery (e.g., to specifically deliver) to a specific region or system of a body.
  • RNA may be a modified RNA. That is, an RNA may include one or more nucleobases, nucleosides, nucleotides, or linkers that are non-naturally occurring.
  • a “modified” species may also be referred to herein as an “altered” species. Species may be modified or altered chemically, structurally, or functionally. For example, a modified nucleobase species may include one or more substitutions that are not naturally occurring.
  • the “N:P ratio” is the molar ratio of ionizable (in the physiological pH range) nitrogen atoms in a lipid to phosphate groups in an RNA, e.g., in a nanoparticle composition including a lipid component and an RNA.
  • Nanoparticle composition is a composition comprising one or more lipids and biologically active agents. Nanoparticle compositions are typically sized on the order of micrometers or smaller and may include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition may be a liposome having a lipid bilayer with a diameter of 500 nm or less.
  • LNPs lipid nanoparticles
  • liposomes e.g., lipid vesicles
  • lipoplexes e.g., lipoplexes.
  • a nanoparticle composition may be a liposome having a lipid bilayer with a diameter of 500 nm or less.
  • lipid refers to a group of organic compounds that include, for example, esters of fatty acids that are characterized by being less soluble in water but more soluble in many organic solvents.
  • the term “subject” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
  • “patient” refers to an individual who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or under care by a trained professional for a particular disease or condition.
  • a “PEG lipid” or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component.
  • a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains.
  • a phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations).
  • Particular phospholipids may facilitate fusion to a membrane.
  • a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of a lipid-containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell.
  • structural lipid refers to a compound comprising the following carbon skeleton:
  • cationic lipid and “ionizable lipid” refer to compounds that comprise both a polar (hydrophilic) head-group or moiety and a non-polar (hydrophobic or lipophilic) tail-group or moiety and may have a positive or partial positive charge at physiological pH.
  • polar head-group and non-polar tail-group are bound (e.g., by one or more of hydrogen-bonds, van der Waals' forces, ionic interactions and covalent bonds) to each other (e.g., by an optionally substituted, variably unsaturated Cl -CIO alkyl or alkenyl).
  • the head-group or moiety is hydrophilic (e.g., a hydrophilic head-group comprising an optionally-substituted alkyl amino).
  • hydrophilic is used to indicate in qualitative terms that a functional group is water-preferring, and water-soluble.
  • hydrophobic is used to indicate in qualitative terms that a functional group is wateravoiding, and typically such groups are not water soluble.
  • phrases “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, reasonably suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication.
  • pharmaceutically acceptable excipient refers to any ingredient other than the compounds described herein and having the properties of being substantially nontoxic and non-inflammatory in a patient.
  • Excipients may include, for example: anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration.
  • anti-adherents antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration.
  • excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (com), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-
  • compositions may also include salts of one or more compounds. Salts may be pharmaceutically acceptable salts.
  • pharmaceutically acceptable salts refers to derivatives of the disclosed compounds wherein the parent compound is altered by converting an existing acid or base moiety to its salt form (e.g., by reacting a free base group with a suitable organic acid).
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pe
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • the pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; such as, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile.
  • suitable salts are found in Remington's Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley- VCH, 2008, and Berge et al, Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.
  • polypeptide or “polypeptide of interest” refers to a polymer of amino acid residues typically joined by peptide bonds that can be produced naturally (e.g., isolated or purified) or synthetically.
  • an “RNA” refers to a ribonucleic acid that may be naturally or non- naturally occurring.
  • an RNA may include modified and/or non-naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers.
  • An RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a poly A sequence, and/or a polyadenylation signal.
  • An RNA may have a nucleotide sequence encoding a polypeptide of interest.
  • an RNA may be a messenger RNA (mRNA, such as modified mRNA, circular mRNA, and self-amplifying mRNA), transfer RNA (tRNA), small interference RNA (siRNA), small activating RNA (saRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA)).
  • mRNA messenger RNA
  • tRNA transfer RNA
  • siRNA small interference RNA
  • saRNA small activating RNA
  • aiRNA asymmetrical interfering RNA
  • miRNA microRNA
  • dsRNA Dicer-substrate RNA
  • shRNA small hairpin RNA
  • RNAs may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), small activating RNA (saRNA), transfer RNA (tRNA), mRNA (modified mRNA, circular mRNA, and self-amplifying mRNA), and mixtures thereof.
  • siRNA small interfering RNA
  • aiRNA asymmetrical interfering RNA
  • miRNA microRNA
  • dsRNA Dicer-substrate RNA
  • shRNA small hairpin RNA
  • saRNA small activating RNA
  • tRNA transfer RNA
  • mRNA modified mRNA, circular mRNA, and self-amplifying mRNA
  • Nucleic acids and polynucleotides useful in the disclosure typically include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5 '-terminus of the first region (e.g., a 5'-UTR), a second flanking region located at the 3'-terminus of the first region (e.g., a 3'-UTR), at least one 5'-cap region, and a 3 '-stabilizing region.
  • a nucleic acid or polynucleotide further includes a poly- A region or a Kozak sequence (e.g., in the 5'-UTR).
  • polynucleotides may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide.
  • a polynucleotide or nucleic acid e.g., an mRNA
  • a polynucleotide or nucleic acid may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a poly A sequence, and/or a polyadenylation signal. Any one of the regions of a nucleic acid may include one or more alternative components (e.g., an alternative nucleoside).
  • the 3 '-stabilizing region may contain an alternative nucleoside such as an L-nucleoside, an inverted thymidine, or a 2'-O-methyl nucleoside and/or the coding region, 5'-UTR, 3'-UTR, or cap region may include an alternative nucleoside such as a 5- substituted uridine (e.g., 5-methoxyuridine), a 1 -substituted pseudouridine (e.g., 1-methyl- pseudouridine or 1 -ethyl-pseudouridine), and/or a 5-substituted cytidine (e.g., 5-methyl-cytidine).
  • a 5- substituted uridine e.g., 5-methoxyuridine
  • a 1 -substituted pseudouridine e.g., 1-methyl- pseudouridine or 1 -ethyl-pseudouridine
  • cytidine e.g., 5-
  • biologically active agent refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
  • Therapeutic agents are also referred to as “actives” or “active agents.” Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.
  • the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
  • the disease or disorder is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, rare diseases, obesity, diabetes, neurodegenerative diseases, ophthalmic diseases, cardio- and reno-vascular diseases, and metabolic diseases.
  • Lipid nanoparticles with more than four components e.g. five, six, or more components are also included in this disclosure.
  • Lipid nanoparticles with fewer than four components e.g. one, two, or three components are also included in this disclosure.
  • any numerical value within the recited ranges and any combination of ranges and specific numerical values within the claimed ranges are contemplated and supported by the foregoing disclosures, i.e., 5 to 60 mole % of a steroidal or structural lipid-containing component includes any numerical value and range within the range of 5 to 60, e.g., 5, 5.01, 5.02, ...59.97, 59.98, 59.99, 60, 5-10, 5-20, 10-30, 15-25, etc.
  • 0.5 to 20 mole % of a PEGylated lipid- containing component includes any numerical value and range within the range of 0.5 to 20, e.g., 0.5, 0.501, 0.502, ...19.97, 19.98, 19.99, 20, 0.5-10, 0.52-15, 1-14, 5-13, etc.
  • 30 to 70 mole % of a cationic or ionizable lipid-containing component includes any numerical value and range within the range of 30 to 70, e.g., 30, 30.01, 30.02, ...69.97, 69.98, 69.99, 70, 30.5-68, 35- 51, 40-52, 45-63, etc.
  • the three-component LNP composition contains the three components in the following relative mole percentages:
  • the three-component LNP composition contains the three components in the following relative mole percentages:
  • the three-component LNP composition contains the three components in the following relative mole percentages:
  • the three-component LNP composition contains the three components in the following relative mole percentages:
  • the three-component LNP composition contains the three components in the following relative mole percentages:
  • the amount of a biologically active agent in a nanoparticle composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the therapeutic and/or prophylactic.
  • the amount of a RNA useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA.
  • the relative amounts of a biologically active agent and other elements (e.g., lipids) in a nanoparticle composition may also vary.
  • the wt/wt ratio of the lipid component to a biologically active agent in a nanoparticle composition may be from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1.
  • the wt/wt ratio of the lipid component to a biologically active agent may be from about 10: 1 to about 40: 1.
  • the wt/wt ratio is about 20:1.
  • the amount of a biologically active agent in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).
  • the nanoparticle composition has an encapsulation efficiency of at least 80% when stored at 25°C, 4°C, -20°C, or -80°C for at least 28 days.
  • the nanoparticle composition has a zeta potential of 5-15 mV when stored at 25°C, 4°C, -20°C, or - 80°C for at least 28 days.
  • the nanoparticle composition has a PDI of less than 0.2 when stored at 25°C, 4°C, -20°C, or -80°C for at least 28 days.
  • the nanoparticle composition has a particle size of less than 140 nm when stored at 25°C, 4°C, -20°C, or -80°C for at least 28 days.
  • “Stability,” “stabilized,” and “stable” in the context of the present disclosure refers to the resistance of nanoparticle compositions and/or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size change, aggregation, change in encapsulation, etc.) under given manufacturing, preparation, transportation, storage and/or in- use conditions, e.g., when stress is applied such as shear force, freeze/thaw stress, etc.
  • the novel ionizable lipids of the disclosure is characterized using nuclear (or heteronuclear) magnetic resonance (NMR), UV-Vis spectrophotometry, high performance liquid chromatography mass spectrometer (HPLC/MS), and infrared spectrometry, other characterization methods known in the art are also contemplated.
  • NMR nuclear (or heteronuclear) magnetic resonance
  • HPLC/MS high performance liquid chromatography mass spectrometer
  • infrared spectrometry other characterization methods known in the art are also contemplated.
  • the disclosure includes methods used to determine characteristics of the ionizable lipids of the disclosure and LNPs, including the purity, LNP mean size, encapsulation efficiency, biological activity, immunogenicity, therapeutic index, and number of impurities.
  • DLS dynamic light scattering
  • zeta potential including HPLC/MS
  • UV-Vis can be used to determine the concentration of a nucleotide in an LNP
  • MFI micro-flow imaging
  • Other methods known in the art for measuring characteristics such as purity, LNP mean size, encapsulation efficiency, biological activity, immunogenicity, therapeutic index, and number of impurities are also contemplated.
  • the nanoparticle compositions may have a pKa from 5.6 to 7.6.
  • Nanoparticle compositions may be formulated in whole or in part as pharmaceutical compositions.
  • Pharmaceutical compositions may include one or more nanoparticle compositions.
  • a pharmaceutical composition may include one or more nanoparticle compositions including one or more different biologically active agents.
  • Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein.
  • General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington's The Science and Practice of Pharmacy, 21 st Edition, A. R Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006.
  • excipients and accessory ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a nanoparticle composition.
  • An excipient or accessory ingredient may be incompatible with a component of a nanoparticle composition if its combination with the component may result in any undesirable biological effect or otherwise deleterious effect.
  • one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a nanoparticle composition.
  • the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention.
  • a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use in humans and for veterinary use.
  • an excipient is approved by United States Food and Drug Administration.
  • an excipient is pharmaceutical grade.
  • an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • the nanoparticle composition further comprises a cryoprotectant.
  • the cryoprotectant is sucrose.
  • the concentration of the cryoprotectant can be from 4%-32% v/v.
  • the cryoprotectant is from 5-12% (v/v) of the nanoparticle composition.
  • the LNP is cryoprotected with 8% v/v sucrose.
  • any cryoprotectant will suffice (e.g. trehalose).
  • a post formulation sample including a cryoprotectant and a buffer is subjected to dialysis at 2 to 10 °C for 10 - 20 hours.
  • the pharmaceutical composition of the disclosure contain the therapeutic or prophylactic agent at a ratio of 0.05 to 25 mg/ml, 0.1 to 20 mg/ml, 0.2 to 18 mg/ml, 0.5 to 15 mg/ml, 0.7 to 12 mg/ml, 0.9 to 10 mg/ml, 1 to 8 mg/ml, 1.5 to 6 mg/ml, 2 to 5 mg/ml, 2.5 to 4 mg/ml, 0.5 to 3.0 mg/ml, 0.2 to 4.0 mg/ml, 0.4 to 2.0 mg/ml, and any numerical value or range within the range of 0.05 to 25 mg/ml.
  • a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • a pharmaceutical composition may comprise between 0.1% and 100% (wt/wt) of one or more nanoparticle compositions.
  • Nanoparticle compositions and/or pharmaceutical compositions including one or more nanoparticle compositions may be administered to any patient or subject, including those patients or subjects that may benefit from a therapeutic effect provided by the delivery of a biologically active agent to one or more particular cells, tissues, organs, or systems or groups thereof, such as the renal system.
  • a biologically active agent to one or more particular cells, tissues, organs, or systems or groups thereof, such as the renal system.
  • compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
  • Subjects to which administration of the compositions is contemplated include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats.
  • a pharmaceutical composition including one or more nanoparticle compositions may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if desirable or necessary, dividing, shaping, and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient (e.g., nanoparticle composition).
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents.
  • Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid can be used in the preparation of injectables.
  • Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • the present disclosure provides methods of producing a polypeptide of interest in a mammalian cell.
  • Methods of producing polypeptides involve contacting a cell with a nanoparticle composition including an mRNA encoding the polypeptide of interest.
  • the mRNA may be taken up and translated in the cell to produce the polypeptide of interest.
  • the step of contacting a mammalian cell with a nanoparticle composition including an mRNA encoding a polypeptide of interest may be performed in vivo, ex vivo, in culture, or in vitro.
  • the amount of nanoparticle composition contacted with a cell, and/or the amount of mRNA therein, may depend on the type of cell or tissue being contacted, the means of administration, the physiochemical characteristics of the nanoparticle composition and the mRNA (e.g., size, charge, and chemical composition) therein, and other factors.
  • an effective amount of the nanoparticle composition will allow for efficient polypeptide production in the cell.
  • Metrics for efficiency may include polypeptide translation (indicated by polypeptide expression), level of mRNA degradation, and immune response indicators.
  • the step of contacting a nanoparticle composition including an mRNA with a cell may involve or cause transfection.
  • a phospholipid including in the lipid component of a nanoparticle composition may facilitate transfection and/or increase transfection efficiency, for example, by interacting and/or fusing with a cellular or intracellular membrane. Transfection may allow for the translation of the mRNA within the cell.
  • the present disclosure provides methods of delivering a biologically active agent to a mammalian cell or organ.
  • Delivery of a biologically active agent to a cell involves administering a nanoparticle composition including the biologically active agent to a subject, where administration of the composition involves contacting the cell with the composition.
  • a protein, cytotoxic agent, radioactive ion, chemotherapeutic agent, or nucleic acid such as an RNA, e.g., mRNA
  • a biologically active agent is an mRNA
  • a translatable mRNA may be translated in the cell to produce a polypeptide of interest.
  • mRNAs that are substantially not translatable may also be delivered to cells.
  • Substantially non-translatable mRNAs may be useful as vaccines and/or may sequester translational components of a cell to reduce expression of other species in the cell.
  • a nanoparticle composition may target a particular type or class of cells (e.g., cells of a particular organ or system thereof).
  • a nanoparticle composition including a biologically active agent of interest may be specifically delivered to a mammalian liver, kidney, spleen, femur, or lung.
  • Specific delivery to a particular class of cells, an organ, or a system or group thereof implies that a higher proportion of nanoparticle compositions including a biologically active agent are delivered to the destination (e.g., tissue) of interest relative to other destinations, e.g., upon administration of a nanoparticle composition to a mammal.
  • specific delivery may result in a greater than 2 fold, 5 fold, 10 fold, 15 fold, or 20 fold increase in the amount of biologically active agent per 1 g of tissue of the targeted destination (e.g., tissue of interest, such as a liver) as compared to another destination (e.g., the spleen).
  • tissue of interest e.g., tissue of interest, such as a liver
  • another destination e.g., the spleen
  • the tissue of interest is selected from the group consisting of a liver, kidney, a lung, a spleen, a femur, an ocular tissue (e.g., via intraocular, subretinal, or intravitreal injection), vascular endothelium in vessels (e.g., intra-coronary or intra-femoral) or kidney, and tumor tissue (e.g., via intratumoral injection).
  • an mRNA that encodes a protein-binding partner (e.g., an antibody or functional fragment thereof, a scaffold protein, or a peptide) or a receptor on a cell surface may be included in a nanoparticle composition.
  • An mRNA may additionally or instead be used to direct the synthesis and extracellular localization of lipids, carbohydrates, or other biological moieties.
  • other biologically active agents or elements (e.g., lipids or ligands) of a nanoparticle composition may be selected based on their affinity for particular receptors (e.g., low density lipoprotein receptors) such that a nanoparticle composition may more readily interact with a target cell population including the receptors.
  • ligands may include, but are not limited to, members of a specific binding pair, antibodies, monoclonal antibodies, Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and fragments thereof, humanized antibodies and fragments thereof, and multivalent versions thereof; multivalent binding reagents including mono- or bi-specific antibodies such as disulfide stabilized Fv fragments, scFv tandems, diabodies, tribodies, or tetrabodies; and aptamers, receptors, and fusion proteins.
  • a ligand may be a surface-bound antibody, which can permit tuning of cell targeting specificity.
  • each antibody can have a different specificity for a desired target.
  • Such approaches can increase the avidity and specificity of targeting interactions.
  • compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 10 mg/kg, from about 0.001 mg/kg to about 10 mg/kg, from about 0.005 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.05 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 0.0001 mg/kg to about 5 mg/kg, from about 0.001 mg/kg to about 5 mg/kg, from about 0.005 mg/kg to about 5 mg/kg, from about 0.01 mg/kg to about 5 mg/kg, from about 0.05 mg/kg to about 5 mg/kg, from about 0.1 mg/kg to about 5 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about
  • a dose of about 0.001 mg/kg to about 10 mg/kg of a biologically active agent (e.g., mRNA) of a nanoparticle composition may be administered.
  • a dose of about 0.005 mg/kg to about 2.5 mg/kg of a biologically active agent may be administered.
  • a dose of about 0.1 mg/kg to about 1 mg/kg may be administered.
  • a dose of about 0.05 mg/kg to about 0.25 mg/kg may be administered.
  • a dose may be administered one or more times per day, in the same or a different amount, to obtain a desired level of mRNA expression and/or therapeutic, diagnostic, prophylactic, or imaging effect.
  • the desired dosage may be delivered, for example, three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
  • the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • a single dose may be administered, for example, prior to or after a surgical procedure or in the instance of an acute disease, disorder, or condition.
  • Nanoparticle compositions including one or more biologically active agents may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents.
  • combination with it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure.
  • one or more nanoparticle compositions including one or more different biologically active agents may be administered in combination.
  • Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • the present disclosure encompasses the delivery of compositions, or imaging, diagnostic, or prophylactic compositions thereof in combination with agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
  • one or more biologically active or imaging agents may be administered together in a single composition or administered separately in different compositions.
  • agents utilized in combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some aspects, the levels utilized in combination may be lower than those utilized individually.
  • the particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, a composition useful for treating cancer may be administered concurrently with a chemotherapeutic agent), or they may achieve different effects (e.g., control of any adverse effects, such as infusion related reactions).
  • the ionizable lipids of the present disclosure were synthesized and formulated into LNPs with cholesterol, DSPC and DMG-PEG2k and characterized. Physical chemical data of EL- 396 and EL-PMA and EL-N4 relative to SM-102 for both 4-component systems (SM-102, EL- 396, EL-N4, and EL-PMA) and 3 -component systems (SM-102-E, EL-396-E, EL-N4-E, and EL- PMA-E) are shown in Table 1.
  • the three-component or four-component LNP compositions were prepared for testing by creating 3x or 4x molarity stock solutions in 200 proof ethanol for each lipid to be used in the formulation. These stock solutions were diluted or combined to give the desired final molar ratio and total lipid concentration for each experiment.
  • the mRNA was prepared from GMP-grade stock solutions of mRNA. The aqueous stock solution using either sodium citrate or sodium acetate master stock solutions at select pH values. The buffered, aqueous stock solution for the formulation was prepared by adding the appropriate master buffer stock solution to an aqueous aliquot and then adding the stock solution of mRNA to give the desired final mRNA concentration, buffer, and pH value.
  • the lipids ethanolic solution and the mRNA aqueous solutions were mixed by microfluidic mixing by PNI Ignite. Solutions were then dialyzed into the desired final product buffer using Thermofisher dialysis cassettes (MWCO lOkDa, PES).
  • the final product buffer was generally prepared by adding the appropriate cryoprotectant, e.g. sucrose, by weight into the corresponding weight of water to create the desired weight percent of cryoprotectant.
  • the final product buffer was sterilized by sterile filtration through a 0.2um PES bulk filter.
  • a three-component LNP composition of the present disclosure was made containing 25 mM SM-102 in ethanol, 19 mM cholesterol in ethanol, and 0.75 mM DMG- PEG2000. Equal volumes of each lipid and of a blank ethanol were combined to give lipid mole ratios as follows: SM-102 (55.9%), cholesterol (42.4%), and DMG-PEG2000 (1.7%). The 0.13 mg/g mRNA in 25 mM sodium acetate pH 6.0 was prepared.
  • the LNP was formulated with a mRNA aqueous solution to lipid ethanolic solution ratio of 3:1 using microfludics to give unimodal peaks.
  • the sample was then dialyzed using 10 kDa MWCO cassettes at 4 °C against 20 mM Tris-HCl, 8% sucrose to produce the final three- component LNP composition.
  • the sample was concentrated to an mRNA concentration over 0.2 mg/mL by UV and filter-sterilization was performed. Surprisingly, the encapsulation was found to be comparable to current 4-component LNP systems.
  • a control (four-component LNP) composition was formulated using SM-102 (50%), cholesterol (38.5%), DSPC (10%), and DMG-PEG2000 (1.5%) in ethanol mixed by microfluidics with mRNA (0.13 mg/mL) in 25 mM sodium acetate pH 6.0 and dialyzed into 20 mM Tris-HCl pH 7.4, 8% sucrose (“RL-007”).
  • the LNPs were filter-sterilized and concentrated to give an mRNA concentration over 0.2 mg/mL by UV.
  • the mLNP of the present disclosure possesses good appearance, physicochemical properties, in vitro cell uptake and expression.
  • Fig. 18 show the in vitro results from Table 9.
  • the Western Blot data indicates that before and after lyophilization is good. Further, the transfection efficiency of mLNP was decreased observably when the PEG content increasing to 3.4%. The transfection of mLNP and LNP before and after lyophilization could be maintained.
  • the mLNPs of the disclosure were also tested in vivo.
  • Table 10 shows the results of a mouse immunogenicity study design of lipid ratio screening.
  • the in vivo study was performed by formulating the mLNP by microfluidic mixing.
  • the mRNA was prepared in a 25 mM sodium acetate buffer, pH 5.0.
  • the Lipids were prepared in ethanol at the relative molar ratios given in Table 10.
  • Three formulations were performed, Ml, M3, and LI (Table 10). After formulation, these samples were dialyzed against 20mM Tris-HCl, pH 7.4, 8%w/w sucrose.
  • the mRNA concentrations were tested by the Invitrogen Ribogreen Assay.
  • [000303 J Fig. 19 show IgG binding antibody titers after the first (a) and the second (b) administration with mLNP and LNP.
  • the mice from Table 10 were injected and bled 14 days after after injection.
  • the second immunization was administered 7 days later.
  • the second bleeding was at day 35 from first injection.
  • the second bleeding was 14 days after second immunization.
  • Female BALB/c mice, aged 6 to 8 weeks, were randomly grouped into sets of five mice each. They received intra-muscular vaccinations with SARS-CoV-2 Omicron BA.1 mRNA, which was formulated using groups from Table 10. Each mouse underwent two rounds of immunization, spaced 21 days apart.
  • the plate was incubated with 100 ⁇ l of serially diluted sera for 2 hours at RT. After 2 hours incubation, the plate was washed 5 times with PBS- T and incubated with HRP-conjugated secondary antibody for 1 hour at RT. Finally, the plate was washed 5 times with PBS-T and incubated with 100 ⁇ l of lx TMB substrate for 10-20 minutes. The reaction was stopped by adding 100 ⁇ l of 1N-HC1 and read at 450nm using the Cytation?.
  • each “condition” represents a LNP composition comprising different lipid ratios.
  • Condition “E” represents a 3 component mLNP of the disclosure.
  • the samples were formulated by microfluidic mixing.
  • the mRNA was prepared in a 25 mM sodium acetate buffer, pH 5.0.
  • the lipids were prepared in 200 proof ethanol at the relative molar ratios given in Table 11. After formulation, these samples were dialyzed against 20mM Tris-HCl, pH 7.4, 8%w/w sucrose. After dialysis, the samples were concentrated by centrifuge. The mRNA concentrations were tested by the Invitrogen Ribogreen Assay.
  • ionizable lipids Using the conditions and formulation technique from Table 11, ionizable lipids (SM-102, EW1, EW2, PMA, and N4) were tested at various conditions. Size and PDI were measured on Malvern Zetasizer. Concentration and encapsulation efficiency were measured using the Invitrogen Ribogreen Assay.
  • Fig. 20 show that PMA and N4 under “E” condition (mLNP) do not show in vitro expression, but showed excellent in vivo expression.
  • the Western blot analysis was performed using lysates of transfected HEK293T cells were prepared in 200 ⁇ l RIP A buffer (Thermo Scientific) containing proteinase inhibitor cocktail per well in a 6-well plate. The plate was incubated on ice for 30 min with a gentle mixing. The cell lysates were transferred to 1.5 ml tube and sonicated briefly, followed by centrifugation for 15 minutes at maximum speed. The BCA assays were performed for quantifying the concentration of proteins.
  • the cell lysates were (10-20 ⁇ g per lane) inactivated by heat (95°C for 5 min) were separated in an SDS-PAGE gel (4-12% NuPAGE, Invitrogen) with MES Running buffer for 30 minutes at 200 V and transferred onto PVDF membrane using iBlot2 system (Invitrogen). After blocking with 5% milk/TBST (lx Trisbuffered saline containing 0.1% Tween-20), membranes were incubated with primary antibody in 5% milk/TBST at 4°C overnight After washing 3 times with TBST, the secondary antibodies conjugated with HRP were added to the membrane in 5% milk/TBST for 1 hour at room temperature.
  • Fig. 21 shows that IgG titer from mouse models indicate excellent expression of the mRNA from the novel lipids under mLNP (E) conditions.
  • the in vivo data from Fig. 21 was collected from female BALB/c mice, aged 6 to 8 weeks. They received intra-muscular vaccinations with SARS-CoV-2 Omicron BA.1 mRNA, which was formulated using groups from Table 12.
  • EW-2 and PMA have excellent T-Cell response in mouse models under mLNP conditions. Further, mLNP conditions improve the T-cell response of EW-2 from poor to excellent under EW-2-E.
  • Spleens from mice were collected and processed into single-cell suspensions in RPMI1640 media supplemented with 10% heat-inactivated fetal calf serum and penicillin/streptomycin (R10 media). Red blood cells were lysed using RBC lysis buffer (KD Medical) and resuspended in R10 media to stop the lysis. Splenocytes were counted and 200,000 cells were added to the 96 well plates of the Mouse JFN-g ELISpot PLUS (ALP) kit (Mabtech).
  • ALP Mouse JFN-g ELISpot PLUS
  • Fig. 23 shows that EL-396 can produce an in vitro response while EL-PMA does not produce an in vitro response under both 4-component and mLNP conditions.
  • the Western blot analysis was performed using lysates of transfected HEK293T cells were prepared in 200 ⁇ RlIP A buffer (Thermo Scientific) containing proteinase inhibitor cocktail per well in a 6-well plate. The plate was incubated on ice for 30 min with a gentle mixing. The cell lysates were transferred to 1.5 ml tube and sonicated briefly, followed by centrifugation for 15 minutes at maximum speed.
  • the BCA assays were performed for quantifying the concentration of proteins.
  • the cell lysates were (10-20 ⁇ pger lane) inactivated by heat (95°C for 5 min) were separated in an SDS-PAGE gel (4-12% NuPAGE, Invitrogen) with MES Running buffer for 30 minutes at 200 V and transferred onto PVDF membrane using iBlot2 system (Invitrogen). After blocking with 5% milk/TBST (lx Tris-buffered saline containing 0.1% Tween-20), membranes were incubated with primary antibody in 5% milk/TBST at 4°C overnight. After washing 3 times with TBST, the secondary antibodies conjugated with HRP were added to the membrane in 5% milk/TBST for 1 hour at room temperature.
  • Fig. 24 shows that EL-396 and PMA perform well in both 4-component and mLNP conditions which is in contrast to in vitro data which shows that only EL-396 can express the spike protein encoded by the payload delivered using the mLNP of the disclosure.
  • the in vivo data from Fig. 24 was collected from female BALB/c mice, aged 6 to 8 weeks. They received intra-muscular vaccinations with SARS-CoV-2 Omicron BA.1 mRNA, which was formulated using 6 formulations.
  • the LNP conditions were at molar ratios of ionizable lipid (SM-102, EL-396, or PMA), DSPC, cholesterol, and DMG-PEG2000 of 50: 10:38.5: 1.5.
  • the mLNP formulation condition was for the ionizable lipid (SM-102, EL-396, or PMA), cholesterol, and DMG-PEG2000 at 59.8:38.5: 1.7.
  • the OmicronBA.1 mRNA was prepared in 25mM sodium acetate pH 5.0. After formulation each of the six formulations was dialyzed into 20mM Tris-HCl, 8% sucrose. The sample concentrations were determined by Invitrogen Ribogreen Assay.
  • the doses (0.5, 1, 2.5, and 5 ug) for the in vivo study were prepared by dilution into PBS.
  • SM-102 and EL-396 each mouse underwent two rounds of immunization, spaced 21 days apart. Blood samples were obtained from all mice prior to each immunization at 14 and 35 days after the initial immunization, using submandibular bleeding.
  • PMA each mouse underwent one round of immunization. Blood samples were obtained 14 days after the immunization, using submandibular bleeding. After collection, the blood was allowed to clot overnight at 4°C, followed by serum extraction through centrifugation at 500 g for 15 minutes at 22°C. These serum samples were then stored at 4°C until they were ready for further analysis.
  • the plate was washed 5 times with PBS-T and incubated with 100 ⁇ olf lx TMB substrate for 10-20 minutes.
  • the reaction was stopped by adding 100 ⁇ l of 1N-HC1 and read at 450nm using the Cytation7.
  • the sterol component of the 3 component mLNP of the disclosure was also studied. Table 13 below show that animal derived and plant derived cholesterol have little gap in the physical characterization. Cholesterol was the only registered sterol lipid at DMF and CDE. Of the sterol lipids tested, mLNPs comprising cholesterol showed the most optimal physiochemical parameters.
  • the formulations were performed using SM-102, a sterol lipid according to Table 13, and DMG-PEG2000 at molar ratios of 59.8, 38.5, and 1.7 respectively. After microfluidic formulation with mRNA, the samples were dialyzed against 20mM Tris-HCl, pH 7.4 and concentrated by centrifuge. The particle size and PDI and zeta potential were determined by Malvern Zetasizer using DTS1070 folded cuvettes. The encapsulation efficiency was determined by Invitrogen Ribogreen Assay.
  • the PEG lipid component of the 3 component mLNP of the disclosure was also studied. Table 14 below show that all mLNP groups tested had uniform size, DSG-PEG2000 had lower transfection efficiency and was not registered at DMF or CDE. Of the PEG lipids tested, mLNPs comprising DMG-PEG2000 showed the most optimal physiochemical.
  • the LNP formulations were performed using SM-102, DSPC, cholesterol, and DMG-PEG2000 at molar ratios of 50:10:38.5:1.5.
  • the mLNP formulations were prepared using SM-102, cholesterol, and DMG-PEG2000 at molar ratios according to Table 14.
  • the samples were dialyzed against 20mM Tris-HCl, pH 7.4 and concentrated by centrifuge.
  • the particle size and PDI and zeta potential were determined by Malvern Zetasizer using DTS1070 folded cuvettes.
  • the encapsulation efficiency was determined by Invitrogen Ribogreen Assay.
  • the group with 1% PEG had largest size. This size is above the 220nm sterile filtration limit, indicating that more than 1% DMG-PEG2000 is needed to control the size to a level for sterile filtration.
  • the same experiments were performed on a second batch of mLNPs. Each condition was tested before and after lyophilization.
  • the LNP formulation was prepared using SM-102,
  • DSPC DSPC, cholesterol, and DMG-PEG2000 at molar ratios of 50:10:38.5:1.5.
  • the mLNP formulations were prepared using SM-102, cholesterol, and DMG-PEG2000 at molar ratios according to Table
  • FIG. 25 shows Western blot data of the formulations in Table 15 under the various conditions. After lyophilization column show three entries compared to the before lyophilization column because three reconstitution conditions were evaluated: one, 8% sucrose; two, 8% sucrose and 4% mannitol; and, three, 8% sucrose and 4% trehalose.
  • the Western blot analysis was performed using lysates of transfected HEK293T cells were prepared in 200 pl RIP A buffer (Thermo Scientific) containing proteinase inhibitor cocktail per well in a 6-well plate.
  • the plate was incubated on ice for 30 min with a gentle mixing.
  • the cell lysates were transferred to 1.5 ml tube and sonicated briefly, followed by centrifugation for 15 minutes at maximum speed.
  • the BCA assays were performed for quantifying the concentration of proteins.
  • the cell lysates were (10-20 ⁇ g per lane) inactivated by heat (95°C for 5 min) were separated in an SDS-PAGE gel (4-12% NuPAGE, Invitrogen) with MES Running buffer for 30 minutes at 200 V and transferred onto PVDF membrane using iBlot2 system (Invitrogen).
  • the mRNA was diluted in 25mM sodium acetate, pH 5.0 and the lipids (SM-102, cholesterol, and DMG-PEG2000) were diluted in 200 proof ethanol.
  • the microfluidic mixing was performed at a flow rate ration of 3:1 of aqueous to organic phase. The total flow rate was 8mL/min.
  • the samples were dialyzed against 20mM Tris- HC1, pH 7.4, 8% sucrose.
  • the size, PDI, and zeta potential were measured using Malvern Zetasizer using DTS1070 folded cuvettes.
  • the encapsulation efficiency was calculated using the Ribogreen Assay kit from Invitrogen.
  • lysates of transfected HEK293T cells were prepared in 200 RI ⁇ Pl A buffer (Thermo Scientific) containing proteinase inhibitor cocktail per well in a 6-well plate. The plate was incubated on ice for 30 min with a gentle mixing. The cell lysates were transferred to 1.5 ml tube and sonicated briefly, followed by centrifugation for 15 minutes at maximum speed. The BCA assays were performed for quantifying the concentration of proteins.
  • the cell lysates were (10-20 per lan ⁇ eg) inactivated by heat (95°C for 5 min) were separated in an SDS-PAGE gel (4-12% NuPAGE, Invitrogen) with MES Running buffer for 30 minutes at 200 V and transferred onto PVDF membrane using iBlot2 system (Invitrogen). After blocking with 5% milk/TBST (lx Tris-buffered saline containing 0.1% Tween-20), membranes were incubated with primary antibody in 5% milk/TBST at 4°C overnight. After washing 3 times with TBST, the secondary antibodies conjugated with HRP were added to the membrane in 5% milk/TBST for 1 hour at room temperature. Chemiluminescence (Clarity Western ECL substrate, Bio-Rad) image acquisition and quantitation of band intensities were performed using an Azure ChemidocTM Imaging System (Azure Biosystem 400).
  • the N/P ratio of the same mLNP compositions were studied using a higher amount of cryoprotectant.
  • Table 17 and 18 show that the higher cryoprotectant did not change the physical chemical data significantly.
  • the zeta potential of mLNP with N/P ratio 3:1 is positive charge, which is different with 8% and 10% sucrose groups. Increasing the content of sucrose could protect the microstructure of mLNP better. Each data point was acceptable.
  • the formulations for Table 17 and 18 were prepared on microfluidic mixer. The mRNA was diluted in 25mM sodium acetate, pH 5.0 and the lipids (SM-102, cholesterol, and DMG-PEG2000) were diluted in 200 proof ethanol.
  • the microfluidic mixing was performed at a flow rate ration of 3: 1 of aqueous to organic phase. The total flow rate was 8mL/min.
  • the samples were dialyzed against 20mM Tris-HCl, pH 7.4, and a cryoprotectant.
  • the cryoprotectant for Table 17 was 10% sucrose and the cryoprotectant for Table 18 was 12%.
  • the size, PDI, and zeta potential were measured using Malvern Zetasizer using DTS1070 folded cuvettes.
  • the encapsulation efficiency was calculated using the Ribogreen Assay kit from Invitrogen.
  • N/P ratio 4.6 and 6.6 had better control after lyophilization.
  • the lyophilized LNP or mLNP structures were better protected in 12% sucrose buffer (EE%).
  • the formulations for Table 19 was prepared on microfluidic mixer.
  • the mRNA was diluted in 25mM sodium acetate, pH 5.0 and the lipids (SM-102, cholesterol, and DMG-PEG2000) were diluted in 200 proof ethanol.
  • the microfluidic mixing was performed at a flow rate ratio of 3:1 of aqueous to organic phase. The total flow rate was 8mL/min.
  • the samples were dialyzed against 20mM Tris-HCl, pH 7.4, and 8% serose. Each formulation was aliquoted and a portion was lyophilized.
  • the size, PDI, and zeta potential were measured using Malvern Zetasizer using DTS1070 folded cuvettes.
  • the encapsulation efficiency was calculated using the Ribogreen Assay kit from Invitrogen.
  • the in vitro analysis was performed (Fig. 26) and expression appeared to increase as the N/P ratio was lowered.
  • the Western blot analysis in Fig. 26 was performed using lysates of transfected HEK293T cells were prepared in 200 R ⁇ lIP A buffer (Thermo Scientific) containing proteinase inhibitor cocktail per well in a 6-well plate. The plate was incubated on ice for 30 min with a gentle mixing.
  • the cell lysates were transferred to 1.5 ml tube and sonicated briefly, followed by centrifugation for 15 minutes at maximum speed.
  • the BCA assays were performed for quantifying the concentration of proteins.
  • the cell lysates were (10-20 per ⁇ lagne) inactivated by heat (95°C for 5 min) were separated in an SDS-PAGE gel (4-12% NuPAGE, Invitrogen) with MES Running buffer for 30 minutes at 200 V and transferred onto PVDF membrane using iBlot2 system (Invitrogen). After blocking with 5% milk/TBST (lx Tris-buffered saline containing 0.1% Tween- 20), membranes were incubated with primary antibody in 5% milk/TBST at 4°C overnight.
  • 5% milk/TBST lx Tris-buffered saline containing 0.1% Tween- 20
  • FIG. 27 show the data. IgG binding antibody titers after the second administration with mLNP and LNP.
  • the in vivo data from Fig. 27 was collected from female BALB/c mice, aged 6 to 8 weeks. They received intra-muscular vaccinations with SARS-CoV-2 Delta mRNA, which was formulated using groups from Table 19. Each mouse underwent two rounds of immunization, spaced 21 days apart. Blood samples were obtained from all mice prior to each immunization at 14 and 35 days after the initial immunization, using submandibular bleeding.
  • the plate was washed 5 times with PBS- T and incubated with HRP-conjugated secondary antibody for 1 hour at RT. Finally, the plate was washed 5 times with PBS-T and incubated with 100 of ⁇ llx TMB substrate for 10-20 minutes. The reaction was stopped by adding 100 of 1 ⁇ Nl -HC1 and read at 450nm using the Cytation?. The reciprocal serum endpoint titer was calculated by serial dilution.
  • mLNP produces equivalent immune response with LNP in mice model, which is consistent with the previous results. N/P ratio of mLNP did not greatly affect the in vivo effect.
  • the 1 st batch lyophilization condition was cool to -5°C (5min), hold at -5°C (lh), cool to -45°C (30min), hold at -45°C (9h), warm to -35 (50min), hold at -35°C (36h), warm to 30°C (6.25h), hold at 30°C (4h), cool to 2°C (15min), and hold at 2°C (48h).
  • the 2 nd batch lyophilization condition was cool to -5°C (5min), hold at -5°C (lh), cool to -50°C (30min), hold at -50°C (9h), warm to -45 (50min), hold at -45°C (36h), warm to 25°C (6.25h), hold at 25°C (4h), cool to 2°C (15min), and hold at 2°C (48h).
  • the 12 th batch lyophiliation condition was cool to -5°C (40min), hold at -5°C (30min), cool to -45°C (lOOmin), hold at -45°C (9h), warm to -30 (90min), hold at -30°C (36h), warm to 25°C (4.5h), and hold at 25°C (4h).
  • Fig. 30 shows characterization of mLNP after lyophilization. Size (a) and zeta potential (b) of mLNP after 2 nd batch lyophilization. Size (c) and zeta potential (d) of mLNP after 12 th batch lyophilization.
  • the best lyophilization cycle began with a freezing step at -45°C for 9 hours, followed by primary drying at -30 °C and 0.1 ⁇ 0.2 mbar for 36 hours, and finishing with secondary drying at 25 °C and 0.1 -0.2 mbar for 4 hours.
  • the freezing rate is 1 °C per min.
  • Ramp rate from primary to secondary drying is 0.2 °C per min.
  • the size was 126.3 nm, the PDI was 0.07081, the zeta potential was -6.159 and the encapsulation was 86%.
  • the moisture content of mLNP after lyophilization was controlled in 2.0%, which was met the quality requirements.
  • the encapsulated mRNA length ranges from around 4000 nb to 900 nb.
  • the robustness of the mLNP for encapsulation of a wide range of mRNA sizes was established with different mRNAs (Delta, RSV, Flue, RAS, NYESO1, VEE-RAS, and a bivalent SARS-CoV2 Delta + BA.1.
  • Each mRNA was formulated at an N/P ratio of 5.6.
  • the lipids used were SM-102, cholesterol, and DMG- PEG2000 at molar ratio of 59.8:38.5:1.7.
  • the mixing was performed by microfluidic mixing at a flow rate ratio of 3:1 and a total flow rate of 8mL/min.
  • Particle sample (4 pL, 25 ng/pL of mRNA) in buffer solution was dropped on a 200-mesh copper grid coated with a continuous carbon film and excess solution was removed after 60 s of waiting by blotting with a wipe. Then 4 pL of negative staining solution, phosphotungstic acid as a 1% aqueous solution was dropped on the TEM grid and removed by blotting after 30 s, and the grid is dried at room temperature. The dried grid was mounted on a JEOL single tilt holder equipped in the TEM column. The specimen was cooled down by liquid-nitrogen. Imaging on a JEOL 2100 PEG microscope was conducted using a minimum dose method that is essential to avoid sample damage under the electron beam.
  • the microscope was operated at 200 kV and with a magnification in the ranges of 10,000-60,000 for assessing particle size and distribution. All images were recorded on a Gatan 2kx2k UltraScan CCD camera. The image was analysed by DigitalMicrograph 3.
  • FIG. 31 shows TEM images of LNP and mLNP before and after lyophilization. Images of LNP before (a) and after (b) lyophilization. Images of mLNP before (c) and after (d) lyophilization.
  • the microstructure of LNP and mLNP is relatively uniform. There are some bulges on the particle surface.
  • the microstructure of mLNP and LNP is different. A layer is on the surface of mLNP.
  • LNP after lyophilization is not uniform and there are more bulges on the particle surface.
  • mLNP after lyophilization is more uniform than LNP. It is circular in its shape. The mLNP showed great stability after lyophilization.
  • Fig. 32 shows TEM images of SM-102 mLNP before and after freezing.
  • the microstructure of SM-102 mLNP is relatively uniform. There were no particulate larger than 500 nm in these samples. Some small black spots in the mLNP, which might be the concavity of surface. The mLNP showed great stability after freezing.
  • Figs. 33-36 show characterization of RSV mLNP and LNP after lyophilization after 1 month storage. Size did not change too much at 4 and -80 °C. At -20 °C, change of mLNP was more than LNP. Zeta potential was a little bit higher than before at 4, -20 , -80 °C.
  • Table 25 Characterization of Delta mLNP and LNP after lyophilization after 1 month storage.
  • Fig. 37 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 8% sucrose as measured by particle size over the course of 1 month.
  • Fig. 38 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 8% sucrose as measured by particle size distribution over the course of 1 month.
  • Fig. 39 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 8% sucrose as measured by zeta potential over the course of 1 month.
  • Fig. 40 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 8% sucrose as measured by encapsulation efficiency over the course of 1 month.
  • Fig. 41 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 10% sucrose as measured by particle size over the course of 1 month.
  • Fig. 42 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 10% sucrose as measured by particle size distribution over the course of 1 month.
  • Fig. 43 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 10% sucrose as measured by zeta potential over the course of 1 month.
  • Fig. 44 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 10% sucrose as measured by encapsulation efficiency over the course of 1 month.
  • Fig. 45 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 12% sucrose as measured by particle size over the course of 1 month.
  • Fig. 46 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 12% sucrose as measured by particle size distribution over the course of 1 month.
  • Fig. 47 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 12% sucrose as measured by zeta potential over the course of 1 month.
  • Fig. 48 shows stability of Delta-mLNP at five N/P ratios when dialyzed with 12% sucrose as measured by encapsulation efficiency over the course of 1 month.
  • Fig. 49 shows Particle size, PDI and encapsulation efficiency change of RSV mLNP after freezing and lyophilization during 6 months.
  • Fig. 50 shows characterization of mLNP with different mRNA. All size of samples was uniform and the PDI was small. The zeta potential was ⁇ 20 mV.
  • Fig. 51 shows Characterization of mLNP with different ionizable lipids. All size of samples was uniform and the PDI was small. The zeta potential was ⁇ 20 mV.
  • Fig. 52 shows Characterization of mLNP before and after lyophilization. The size and zeta potential of mLNP was maintained after lyophilization. The zeta potential was ⁇ 20 mV.
  • Fig. 53 shows a titration curve of moisture content. The moisture content of mLNP after lyophilization via C30s+InMotion KF Flex and C30s.
  • Fig. 53 shows a titration curve of moisture content of mLNP after lyophilization via C30s+InMotion KF Flex and C30s.
  • mice were intramuscularly injected (50 p.1 in both hind legs) with LNPs formulated with Flue mRNA at a dose of 0.1 mg kg-1. The biodistribution of different preparations at 6, 10, 24, 48, and 72 hours was observed. At every time point, mice were intraperitoneal injected with D-luciferin (150 mg kg-1). After 4 min, the whole of mice was imaged using imaged using an IVIS Lumina system. After that, organs (hearts, lungs, liver, kidneys, spleens and lymph nodes) were dissected and imaged. Luminescence was quantified as average luminescence of total organ areas (ps-1 cm-2 si ⁇ l) using Living image 4.4.
  • Fig. 54 shows biodistribution of mRNA expression (dark spots) in mice differences between LNP and mLNP formulations with SM-102.
  • Fig. 55 shows biodistribution among the lymph nodes, liver, and spleen demonstrating that mLNP has a distinct difference in biodistribution than LNP.
  • Robust luciferase signal was observed both at the injection sites and the liver between 6 and 10 hours after intramuscular administration, which gradually declined within 72 hours.
  • the biodistribution of LNP or mLNP with different lipids was different. Compared to mLNP, LNP had a better secondary lymphoid organ draining ability.
  • Fig. 56 shows biodistribution of both LNP and mLNP can be controlled by choice of ionizable lipid.
  • EL-396 does not deliver to the liver under either LNP or mLNP conditions.
  • DC2.5 cells were treated with Cy5-labeled Delta mRNA LNP or mLNP to quantify the percentage of transfection by flow cytometry.
  • Cells were seeded at 7 x 10 5 cells per well in a 6-well plate. After 24 h, cells were treated with LNPs (500 ng and 1 Cy5-l ⁇ agbeled Delta mRNA) and 1 mL of fresh DMEM (10% FBS was added). Cells were washed with PBS, trypsinized for 3 min at 37 °C, neutralized with 1 mL of DMEM, centrifuged at 300g for 5 min, washed with cold PBS twice, and diluted in 300 pL of cold PBS. Finally, cells were kept on ice until they were analyzed by a flow cytometer. Fluorescence intensity was quantified by FlowJo VI 0.
  • Fig. 57 shows Cell uptake of Cy5-LNP and Cy5-mLNP. Flow cytometry was used to determine distribution of Cy5 fluorescence (a), percentage of Cy5 positive cells (b) and mean fluorescence intensity (c). The cell uptake of mLNP was higher than that of LNP.

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Abstract

L'invention divulgue de nouveaux lipides ionisables, des compositions et des méthodes d'utilisation des nouveaux lipides et des compositions. Les compositions de nanoparticules lipidiques comprennent un nouveau lipide ainsi que des lipides supplémentaires tels que des phospholipides, des lipides structuraux et des lipides PEG. Les compositions de nanoparticules lipidiques comprenant en outre des agents biologiquement actifs tels que l'ARNm sont utiles dans l'administration d'agents thérapeutiques, de diagnostics et/ou de prophylactiques à des cellules ou des organes.
PCT/US2024/050999 2023-10-11 2024-10-11 Lipides ionisables pour l'administration d'acides nucléiques Pending WO2025081002A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2026003582A2 (fr) 2024-06-27 2026-01-02 Axelyf ehf. Lipides et nanoparticules lipidiques

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WO2006138380A2 (fr) * 2005-06-15 2006-12-28 Massachusetts Institute Of Technology Lipides contenant des amines et utilisations
WO2011075656A1 (fr) * 2009-12-18 2011-06-23 The University Of British Columbia Procédés et compositions pour l'administration d'acides nucléiques
WO2015095340A1 (fr) * 2013-12-19 2015-06-25 Novartis Ag Lipides et compositions lipidiques pour le largage d'agents actifs
WO2017049245A2 (fr) 2015-09-17 2017-03-23 Modernatx, Inc. Composés et compositions pour l'administration intracellulaire d'agents thérapeutiques
WO2021231929A1 (fr) * 2020-05-15 2021-11-18 Modernatx, Inc. Formulations d'arn pour une distribution de volume élevé

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Publication number Priority date Publication date Assignee Title
WO2006138380A2 (fr) * 2005-06-15 2006-12-28 Massachusetts Institute Of Technology Lipides contenant des amines et utilisations
WO2011075656A1 (fr) * 2009-12-18 2011-06-23 The University Of British Columbia Procédés et compositions pour l'administration d'acides nucléiques
WO2015095340A1 (fr) * 2013-12-19 2015-06-25 Novartis Ag Lipides et compositions lipidiques pour le largage d'agents actifs
WO2017049245A2 (fr) 2015-09-17 2017-03-23 Modernatx, Inc. Composés et compositions pour l'administration intracellulaire d'agents thérapeutiques
WO2021231929A1 (fr) * 2020-05-15 2021-11-18 Modernatx, Inc. Formulations d'arn pour une distribution de volume élevé

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"Remington's Pharmaceutical Sciences", 1985, MACK PUBLISHING COMPANY, pages: 1418
"Remington's The Science and Practice of Pharmacy", 2006, LIPPINCOTT, WILLIAMS & WILKINS
BERGE ET AL., JOURNAL OF PHARMACEUTICAL SCIENCE, vol. 66, 1977, pages 1 - 19

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2026003582A2 (fr) 2024-06-27 2026-01-02 Axelyf ehf. Lipides et nanoparticules lipidiques

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