EP4619378A2 - Lipides ionisables et compositions de nanoparticules lipidiques pour l'administration d'acides nucléiques - Google Patents

Lipides ionisables et compositions de nanoparticules lipidiques pour l'administration d'acides nucléiques

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Publication number
EP4619378A2
EP4619378A2 EP23892517.6A EP23892517A EP4619378A2 EP 4619378 A2 EP4619378 A2 EP 4619378A2 EP 23892517 A EP23892517 A EP 23892517A EP 4619378 A2 EP4619378 A2 EP 4619378A2
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EP
European Patent Office
Prior art keywords
compound
lipid
alkyl
independently
mol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP23892517.6A
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German (de)
English (en)
Inventor
Amit Sagi
Rob BURKE
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Seawolf Therapeutics Inc
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Seawolf Therapeutics Inc
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Publication of EP4619378A2 publication Critical patent/EP4619378A2/fr
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—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
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/06—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted
    • C07C217/08—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to an acyclic carbon atom
    • 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
    • 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
    • C07C229/24—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 more than one carboxyl group bound to the carbon skeleton, e.g. aspartic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/08—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/10—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings
    • C07D317/14—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D317/28—Radicals substituted by nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/14—All rings being cycloaliphatic
    • C07C2602/24—All rings being cycloaliphatic the ring system containing nine carbon atoms, e.g. perhydroindane

Definitions

  • nucleic acid there are many instances in which delivery of a nucleic acid is desired, where such instances include research, diagnostic and therapeutic applications.
  • An example of such a therapeutic application is gene therapy, which can be used to treat genetic disorders and other conditions. Genetic disorders, although individually rare, collectively represent a significant disease burden, particularly for children, resulting in substantial disability and mortality.
  • viral vectors such as vectors based on AAV
  • AAV vectors are commonly employed to deliver genes into cells.
  • AAV vectors are limited in the size of genetic cargo that can be packaged. Accordingly, any genetic cargo greater than 4.7kB is not suitable for delivery with AAV vectors, which limits the utility of such vectors for many indications.
  • viral vectors, such as AAV induce an antibody response, limiting redosing, which is not suitable for some indications.
  • expression from successfully transduced cells can be reduced or lost with cell division and turnover, requiring redosing - which may not be possible or effective due to immune memory.
  • many subjects have pre-existing immunity to commonly used viral vectors such as AAV, which can limit even initial treatment with an AAV gene therapy.
  • viral vector such as AAV can be toxic at the doses that would be required to achieve therapeutic benefit in some indications.
  • Lipid nanoparticles provide an alternative to viral gene therapy. While lipid nanoparticles have been developed and employed for delivery of many RNA therapeutics, lipid-nanoparticle-delivered RNA has limited therapeutic longevity. DNA delivered by lipid nanoparticles designed for RNA delivery suffers from poor efficiency and significant innate immune response activation in treated subjects. New delivery vehicles for delivering nucleic acids, such as DNA, to cells, stand to significantly advance numerous scientific pursuits, particularly in vivo for patients in need of gene therapy but also in vitro for research applications.
  • novel ionizable lipids having an ionizable headgroup connected to lipid tails via a linear alkyl core.
  • the linear alkyl core can have n carbon atoms, where n-1 carbon atoms in the linear alkyl core are linked to lipid tails.
  • novel lipid nanoparticle (LNP) compositions for the delivery of nucleic acid material to cells in vitro and in vivo with different and improved pharmacokinetic profiles as compared to what is typically observed in the art. Also provided are methods for using compositions of the invention in research and as therapeutics.
  • FIGs. 1 A-C describe studies performed to assess how the structure of the ionizable lipid impacts the efficacy and toxicity of DNA-LNPs.
  • FIG. 1 A Formulation details for the test articles. The ionizable lipid was varied in all formulations.
  • Ionizable cationic lipids tested were ALC-0315 [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2- hexyldecanoate), MC3 (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate, and two ionizable cationic lipids of the present disclosure, L-2 and L-3.
  • the phospholipid in all formulations is DSPC.
  • the nucleic acid cargo used in all formulations is a nanoplasmid DNA (npDNA) comprising a hAAT promoter driving the expression of an EPO transgene. Good encapsulation efficiency and small size were observed of all test articles.
  • npDNA nanoplasmid DNA
  • FIG. IB EPO serum levels in wild type BALB/c mice were measured 4 hours post-i.v. administration of test articles at a dose of 1 mg/kg.
  • FIG. 1C IL-6 cytokine levels in serum were measured 4 hours after i.v. administration of test articles at a dose of 1 mg/kg to wild type BALB/c mice.
  • the cationic lipid comprises a protonatable tertiary amine headgroup, a linear alkyl core, hydrocarbon chains (e.g., as described herein), and ester linkages between the linear alkyl core and hydrocarbon chains. In some embodiments, the cationic lipid comprises the same number of hydrocarbon chains as ester linkages. In some embodiments, the cationic lipid comprises a protonatable tertiary amine headgroup, a linear alkyl core, hydrocarbon chains (e.g., as described herein), and carbonate linkages between the linear alkyl core and hydrocarbon chains. In some embodiments, the cationic lipid comprises the same number of hydrocarbon chains as carbonate linkages. In some embodiments, the cationic lipid comprises 2 or more hydrocarbon chains, such as 3 or more hydrocarbon chains, or 4 or more hydrocarbon chains.
  • Z is an ionizable head group
  • the linear alkyl core W n is: where * depicts the point of attachment to Y and each ** depicts the point of attachment to X; G 1 is H, or a group cyclically linked with Y that together with the carbon atom of W n to which they are attached provide a heterocycle; and G 2 is H or -CH2OH.
  • G 1 is cyclically linked with Y to form a 5-membered heterocycle.
  • G 1 is cyclically linked with Y to form a 6-membered heterocycle.
  • G 1 is H.
  • G 2 is H.
  • G 2 is -CH2OH.
  • linking groups include but are not limited to, alkyl, alkenylene, alkynylene, arylene, alkarylene, aralykylene, amido, ureylene, imide, ether, thioether, carbonate, alkyldioxy, oxyimino, amino, carbonyl, heterocycle (e.g., cyclic acetal) etc.
  • Y is selected from — O — , — C(R 10 )2 — , — OC(O)— , — C(O)O— , — OC(O)O— , — OC(O)NR 10 — , — SC(O)NR 10 — , — C(O)NR 10 — , — NR 10 C(O)— , — S— , —NR 10 —, — NR 10 C(O)O— , and — NR 10 C(O)S— , wherein R 10 is selected from H and C1-6 alkyl.
  • Y is selected from — O — , - OC(O) — , — C(0)0 — , and — OC(O)NR 10 — .
  • Y is — O — .
  • Y is — OC(O) — .
  • Y is — OC(O)NR 10 — , where R 10 is H.
  • Y is — C(R 10 )2 — , where each R 10 is H.
  • Y is — C(O)O — .
  • Y is — OC(O)O — .
  • Y is — SC(O)NR 10 — where each R 10 is H. In some cases, Y is — C(O)NR 10 — where each R 10 is H. In some cases, Y is — NR 10 C(O) — where each R 10 is H. In some cases, Y is — S — . In some cases, Y is — NR 2 — . In some cases, Y is — NR 10 C(O)O — , where each R 10 is H. In some cases, Y is — NR 10 C(O)S — where each R 10 is H.
  • W n comprises a group G 1 adjacent to the point of attachment to linking group Y.
  • G 1 is cyclically linked with the linking group Y to provide a heterocycle.
  • G 1 is cyclically linked with Y to provide a 5-membered heterocycle.
  • the 5-membered heterocycle is a cyclic acetal.
  • G 1 is cyclically linked with Y to provide a 6-membered heterocycle.
  • the 6-membered heterocycle is a cyclic acetal.
  • the linking group Y is linked to an ionizable head group Z through an optionally substituted (Ci-Ci2)alkylene L.
  • L is (C2-Ce)alkylene or substituted (C2-Ce)alkylene.
  • L is (C2-C4)alkylene or substituted (C2-C4)alkylene.
  • L is C2- alkylene or substituted C2-alkylene.
  • L is Cs-alkylene or substituted C3- alkylene.
  • L is C4-alkylene or substituted C4-alkylene.
  • L is Cs-alkylene or substituted Cs-alkylene. In certain cases, L is Ce-alkylene or substituted Ce- alkylene. In certain cases, L is -(CH2)2- In certain cases, L is -(CH2)3. In certain cases, L is -(CH2)4- In certain cases, L is -(CH2)5- In certain cases, L is -(CJfcje-
  • -Y-L-Z is of the formula — O(CH2)rZ, where r is 2-6. In some embodiments, -Y-L-Z is of the formula — OC(O)(CH2)rZ, where r is 2-6. In some embodiments, -Y-L-Z is of the formula — OC(O)NH(CH2)rZ, where r is 2-6. In some embodiments, -Y-L-Z is of the formula — CH2(CH2)rZ, where r is 2-6. In some cases, r is 2- 4. In some cases, r is 2. In some cases, r is 3. In some cases, r is 4.
  • the ionizable lipid of formula (I) includes an ionizable head group.
  • the ionizable head group includes a primary, secondary or tertiary amine that may be protonated at physiological pH.
  • the ionizable head group comprises a tertiary amino group.
  • the ionizable head group is of the formula -NR n R 12 , wherein R 11 and R 12 are each independently alkyl or substituted alkyl.
  • R 11 and R 12 are each independently C1-6 alkyl or substituted C1-6 alkyl.
  • R 11 and R 12 are each independently C1-3 alkyl or substituted C1-3 alkyl.
  • R 11 and R 12 are each C1-3 alkyl. In some embodiments, R 11 and R 12 are each methyl. In some embodiments, R 11 and R 12 are each ethyl. In certain cases, both R 11 and R 12 are propyl. In certain cases, both R 11 and R 12 are n-propyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted n-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted secbutyl.
  • both R 11 and R 12 is independently an optionally substituted butyl.
  • each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n- propyl, n-butyl, sec-butyl, tert-butyl, -CH2CH2OH, -CH(CH3)CH2OH, -CH2CH(OH)CH3, and -CH2CH2CH2OH.
  • each R 11 and R 12 is independently selected from an optionally substituted Ci-4 alkyl, C1-3 alkyl, Ci-4 heteroalkyl, and C1-3 heteroalkyl.
  • formula (I) includes 2-5 lipid tails R, that are linked to the core W n , optionally via an additional linking group X (e.g., -(X-R)(n-i>).
  • the ionizable lipid of formula (I) includes a linking group X.
  • the linking group X may be linear, branched, cyclic or a single atom. Examples of such linking groups include but are not limited to, alkyl, alkenylene, alkynylene, arylene, alkarylene, aralykylene, amido, ureylene, imide, ether, thioether, thiocarbamate, carbonate, alkyldioxy, oxyimino, amino, carbonyl etc.
  • each X is independently selected from — (CH2)sOC(O) — , — (CH2)sC(O)O — , — (CH2)sOC(O)O — , — (CH 2 ) S OC(O)NR 10 — , — (CH 2 ) S O— , — (CH 2 ) s SC(O)NR 10 — , — (CH 2 ) S C(O)NR 10 — , — (CH 2 ) s NR 10 C(O)— , — (CH 2 )sS— , — (CH 2 ) S NR 10 — , — (CH 2 ) s NR 10 C(O)O— , and — (CH2)SNR 10 C(O)S — , wherein R 10 is selected from H and C1-6 alkyl and s is 0-6.
  • each X is independently selected from — (CH2)sOC(O) — , — (CH2)sC(O)O — , and — (CH2) S OC(O)O — .
  • each X is — (CH2)sOC(O) — , where s is 0, 1 or 2.
  • each X is — (CH2)sC(O)O — , where s is 0, 1 or 2.
  • each X is — (CH2)sOC(O)O — , where s is 0, 1 or 2.
  • at least one X group is — (CH2)sO — , where s is 0, 1 or 2.
  • At least one X group is — (CH2)SOC(O)NR 10 — , where R 10 is H and s is 0, 1 or 2. In some embodiments, at least one X group is — (CH2)sSC(O)NR 10 — , where R 10 is H. and s is 0, 1, or 2 In some embodiments, at least one X group is — (CH2)sC(O)NR 10 — , where R 10 is H and s is 0, 1 or 2. In some embodiments, at least one X group is — (CH2)sNR 10 C(O) — , where R 10 is H and s is 0, 1 or 2.
  • At least one X group is — (CH2)sS — , where s is 0, 1 or 2. In some embodiments, at least one X group is — (CH2)sNR 10 — , where R 10 is H and s is 0, 1 or 2. In some embodiments, at least one X group is — (CH2)sNR 10 C(O)O — , where R 10 is H and s is 0, 1 or 2. In some embodiments, at least one X group is — (CH2)sNR 10 C(O)S — , where R 10 is H and s is 0, 1 or 2.
  • each X is independently selected from — OC(O)— , — C(O)O— , — OC(O)O— , — O— , — OC(O)NR 10 — , — SC(O)NR 10 — , — C(O)NR 10 — , — NR 10 C(O)— , — S— , —NR 10 —, — NR 10 C(O)O— , and — NR 10 C(O)S— , wherein R 10 is selected from H and Ci-6 alkyl.
  • each X is independently selected from — OC(O) — , — C(O)O — , and — OC(O)O — . In some embodiments, each X is — OC(O) — . In some embodiments, each X is — C(O)O — . In some embodiments, each X is — OC(O)O — . In some embodiments, at least one X group is — O — . In some embodiments, at least one X group is — OC(O)NR 10 — , where R 10 is H. In some embodiments, at least one X group is — SC(O)NR 10 — , where R 10 is H.
  • At least one X group is — C(O)NR 10 — , where R 10 is H. In some embodiments, at least one X group is — NR 10 C(O) — , where R 10 is H. In some embodiments, at least one X group is — S — . In some embodiments, at least one X group is — NR 10 — , where R 10 is H. In some embodiments, at least one X group is — NR 10 C(O)O — , where R 10 is H. In some embodiments, at least one X group is — NR 10 C(O)S — , where R 10 is H.
  • R x and R y is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C10 aliphatic group; r, p, and q is each independently an integer from 0 to 10.
  • R is wherein,
  • R represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R is , where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R is , where each
  • R is , where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R is
  • each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
  • R is each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
  • each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
  • each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain ofR.
  • the compound of formula (I) is of the formula (IIA):
  • Y is selected from — O — , — OC(O) — , and — OC(O)NR 10 — , wherein R 10 H and Ci-6 alkyl.
  • Y is — O — .
  • Y is — OC(O) — .
  • Y is — OC(O)NR 10 — , where R 10 is H.
  • L is (C2-Ce)alkylene or substituted (C2- Ce)alkylene. In certain cases, L is (C2-Ce)alkylene. In certain cases, L is (C2-C4)alkylene. In certain cases, L is -(CH2)2- In certain cases, L is -(CH2)3- In certain cases, L is - (CH 2 )4-.
  • Z is a tertiary amine.
  • Z is -NR n R 12 , wherein R 11 and R 12 are each independently C1-6 alkyl or substituted C1-6 alkyl.
  • R 11 and R 12 are each C1-3 alkyl.
  • R 11 and R 12 are each methyl.
  • R 11 and R 12 are each ethyl.
  • both R 11 and R 12 are propyl.
  • both R 11 and R 12 are n-propyl.
  • both R 11 and R 12 are isopropyl.
  • both R 11 and R 12 are isopropyl.
  • both R 11 and R 12 is independently an optionally substituted butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted n-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted sec-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl. In certain cases, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, -CH2CH2OH, -CH(CH 3 )CH 2 OH, -CH 2 CH(OH)CH 3 , and -CH2CH2CH2OH. In certain cases, each R 11 and R 12 is independently selected from an optionally substituted Ci-4 alkyl, C1-3 alkyl, C 1-4 heteroalkyl, and C1-3 heteroalkyl.
  • each X is independently selected from — OC(O) — , — C(O)O — , and — OC(O)O — .
  • at least one X is — OC(O) — .
  • each X is — OC(O) — .
  • at least one X is — C(O)O — .
  • each X is — C(O)O — .
  • at least one X is — OC(O)O — .
  • each X is — OC(O)O— .
  • each -X-R is of the formula — OC(O)R. In certain embodiments, each -X-R is of the formula — C(O)OR. In certain embodiments, each -X-R is of the formula — OC(O)OR.
  • each R is selected from C5-C20 alkyl, C5- C20 alkenyl, and a C5-C20 alkynyl.
  • each R is C5-C12 alkyl, C5-C12 alkenyl, and a C5-C12 alkynyl.
  • each R is C5-C12 alkyl.
  • each R is C5 alkyl.
  • each R is Ce alkyl.
  • each R is C7 alkyl.
  • each R is Cs alkyl.
  • each R is C9 alkyl.
  • each R is C10 alkyl.
  • each R is C11 alkyl.
  • each R is C12 alkyl.
  • At least one R is a branched hydrocarbon group comprising 8-20 carbon atoms, optionally further comprising one or more cyclic groups (e.g., as described herein).
  • R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • each R is -CH(R 7 )2 and each R 7 is a C5-C 12 alkyl.
  • each R is -CH(R 7 )2 and each R 7 is a C5-C12 alkenyl.
  • At least one R is a linear or branched hydrocarbon group comprising one or more cyclic groups.
  • R is -(CH2)tJ(CH 2 )u, where J is a cyclic group and t and u are each independently 1-10.
  • J is an aryl group.
  • J is phenyl.
  • t is 1 to 5.
  • u is 1 to 5.
  • the compound is of formula (IIIA): UA) wherein:
  • R 11 and R 12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
  • Y is selected from — O — , — OC(O) — , and — OC(O)NR 10 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R 7 )2, and -(CH2)tJ(CH 2 )u, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and t and u are each independently 1-10.
  • formula (IIA) the compound is of formula (IIIA), wherein:
  • R 11 and R 12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
  • Y is selected from — O — , — OC(O) — , and — OC(O)NR 10 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, and -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • Y is selected from — O — , — OC(O) — , and — OC(O)NR 10 — , wherein R 10 is selected from H and C1-6 alkyl.
  • Y is — O — .
  • Y is — OC(O) — .
  • Y is — OC(O)NR 10 — , where R 10 is H.
  • q is 1. In certain cases, q is 2. In certain cases, q is 3. In certain cases, q is 4.
  • R 11 and R 12 are different. In certain cases, at least one of R 11 and R 12 is methyl. In certain cases, R 11 and R 12 are the same. In certain cases, both R 11 and R 12 are methyl. In certain cases, at least one of R 11 and R 12 is ethyl. In certain cases, both R 11 and R 12 are ethyl. In certain cases, both R 11 and R 12 are propyl. In certain cases, both R 11 and R 12 are n-propyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl.
  • both R 11 and R 12 is independently an optionally substituted n-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted sec-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl. In certain cases, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, -CH2CH2OH, -CH(CH 3 )CH 2 OH, -CH 2 CH(OH)CH 3 , and -CH2CH2CH2OH. In certain cases, each R 11 and R 12 is independently selected from an optionally substituted Ci-4 alkyl, Ci- 3 alkyl, C 1-4 heteroalkyl, and Ci- 3 heteroalkyl..
  • each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl. In certain cases, each R is selected from C5-C12 alkyl, C5-C12 alkenyl, and a C5-C12 alkynyl. In certain cases, each R is C5-C12 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is Ce alkyl. In certain cases, each R is C7 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is C9 alkyl. In certain cases, each R is C10 alkyl.
  • each R is C11 alkyl. In certain cases, each R is C12 alkyl. [0060] In certain embodiments of formula (IIIA), at least one R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is a C5-C12 alkyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is a C5-C12 alkenyl.
  • R is -(CH2)tJ(CH2)u, where J is a cyclic group and t and u are each independently 1-10.
  • J is an aryl group.
  • J is phenyl.
  • t and u are each 1 to 5. In some cases, t is 2 and u is 3.
  • each X is independently selected from — OC(O) — , — C(O)O — , and — OC(O)O — .
  • at least one X is — OC(O) — .
  • each X is — OC(O) — .
  • at least one X is — C(O)O — .
  • each X is — C(O)O — .
  • at least one X is — OC(O)O — .
  • each X is — OC(O)O— .
  • R 11 and R 12 are different. In certain cases, at least one of R 11 and R 12 is methyl. In certain cases, R 11 and R 12 are the same. In certain cases, both R 11 and R 12 are methyl. In certain cases, at least one of R 11 and R 12 is ethyl. In certain cases, both R 11 and R 12 are ethyl. In certain cases, both R 11 and R 12 are propyl. In certain cases, both R 11 and R 12 are n-propyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl.
  • both R 11 and R 12 is independently an optionally substituted n-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted sec-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl. In certain cases, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, -CH2CH2OH, -CH(CH 3 )CH 2 OH, -CH 2 CH(OH)CH 3 , and -CH2CH2CH2OH. In certain cases, each R 11 and R 12 is independently selected from an optionally substituted Ci-4 alkyl, Ci- 3 alkyl, C 1-4 heteroalkyl, and Ci- 3 heteroalkyl.
  • R is -(CH2)tJ(CH2)u, where J is a cyclic group and t and u are each independently 1-10.
  • J is an aryl group.
  • J is phenyl.
  • t and u are each 1 to 3. In some cases, t is 2 and u is 3.
  • the compound of formula (I) is of the formula (IIC):
  • Y is selected from — C(R 10 )2 — and —
  • R 10 is selected from H and Ci-6 alkyl.
  • Y is — O — .
  • Y is — C(R 10 )2 — , where R 10 is H.
  • L is (C2-Ce)alkylene or substituted (C2- Ce)alkylene. In certain cases, L is (C2-Ce)alkylene. In certain cases, L is (C2-C4)alkylene. In certain cases, L is -(CH2)2- In certain cases, L is -(CH2)3- In certain cases, L is - (CH 2 )4-.
  • Z is a tertiary amine.
  • Z is -NR n R 12 , wherein R 11 and R 12 are each independently C1-6 alkyl or substituted C1-6 alkyl.
  • R 11 and R 12 are each C1-3 alkyl.
  • R 11 and R 12 are each methyl.
  • R 11 and R 12 are each ethyl.
  • both R 11 and R 12 are propyl.
  • both R 11 and R 12 are n-propyl.
  • both R 11 and R 12 are isopropyl.
  • both R 11 and R 12 are isopropyl.
  • both R 11 and R 12 is independently an optionally substituted butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted n-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted sec-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl. In certain cases, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, -CH2CH2OH, -CH(CH 3 )CH 2 OH, -CH 2 CH(OH)CH 3 , and -CH2CH2CH2OH. In certain cases, each R 11 and R 12 is independently selected from an optionally substituted Ci-4 alkyl, C1-3 alkyl, C 1-4 heteroalkyl, and C1-3 heteroalkyl.
  • each X is independently selected from — (CH2) S OC(O) — , — (CH2) S C(O)O — , — (CH2) S OC(O)O — , where s is 0-6. In some cases, at least one X is — (CH2)sOC(O) — , where s is 0, 1 or 2. In some cases, each X is — (CH2) S OC(O) — , where s is 0, 1 or 2. In certain embodiments of formula (IIC), at least one X is — (CH2) S C(O)O — , where s is 0, 1 or 2.
  • each X is — (CH2)sC(O)O — , where s is 0, 1 or 2. In certain embodiments of formula (IIC), s is 1 and each X is — CH2C(O)O — . In certain embodiments of formula (IIC), s is 2 and X is — (CH2)2C(O)O — . In certain embodiments of formula (IIC), at least one X is — (CH2)sOC(O)O — , where s is 0, 1 or 2. In certain cases, each X is — (CH2)sOC(O)O — , where s is 0, 1 or 2. In certain embodiments of formula (IIC), s is 0 and X is — OC(O)O — .
  • each -X-R is of the formula — (CH2)SOC(O)R, where s is 0-6. In certain embodiments, each -X-R is of the formula — (CH2)SC(O)OR, where s is 0-6. In certain embodiments, each -X-R is of the formula — (CH2) S OC(O)OR, where s is 0-6.
  • each R is selected from C5-C20 alkyl, C5- C20 alkenyl, and a C5-C20 alkynyl.
  • each R is C5-C12 alkyl, C5-C12 alkenyl, and a C5-C12 alkynyl.
  • each R is C5-C12 alkyl.
  • each R is C5 alkyl.
  • each R is Ce alkyl.
  • each R is C7 alkyl.
  • each R is Cs alkyl.
  • each R is C9 alkyl.
  • each R is C10 alkyl.
  • each R is C11 alkyl.
  • each R is C12 alkyl.
  • At least one R is a branched hydrocarbon group comprising 8-20 carbon atoms, optionally further comprising one or more cyclic groups (e.g., as described herein).
  • R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • each R is -CH(R 7 )2 and each R 7 is a C5-C 12 alkyl.
  • each R is -CH(R 7 )2 and each R 7 is a C5-C12 alkenyl.
  • each R is -CH(R 7 )2 and each R 7 is a C6-C9 alkenyl.
  • At least one R is a linear or branched hydrocarbon group comprising one or more cyclic groups.
  • R is -(CH2)tJ(CH 2 )u, where J is a cyclic group and t and u are each independently 1-10.
  • J is an aryl group.
  • J is phenyl.
  • t is 1 to 5.
  • u is 1 to 5.
  • the compound is of formula (IIIC): wherein:
  • Y is selected from — O — , and — C(R 10 )2 — ; each s is independently 0, 1 or 2;
  • W is — O — or — CH2 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R 7 )2, and - (CH 2 )tJ(CH 2 )u, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and each of t and u are 1-10.
  • the compound is of formula (IIIC), wherein: R 11 and R 12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
  • Y is selected from — O — , and — C(R 10 )2 — ; s is 0 to 2;
  • W is O or CH2; and each R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • Y is selected from — C(R 10 )2 — and — O — , wherein R 10 is selected from H and C1-6 alkyl.
  • Y is — O — .
  • Y is — C(R 10 )2 — , where R 10 is H.
  • q is 1. In certain cases, q is 2. In certain cases, q is 3. In certain cases, q is 4.
  • R 11 and R 12 are different. In certain cases, at least one of R 11 and R 12 is methyl. In certain cases, R 11 and R 12 are the same. In certain cases, both R 11 and R 12 are methyl. In certain cases, at least one of R 11 and R 12 is ethyl. In certain cases, both R 11 and R 12 are ethyl. In certain cases, both R 11 and R 12 are propyl. In certain cases, both R 11 and R 12 are n-propyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl.
  • both R 11 and R 12 is independently an optionally substituted n-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted sec-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl. In certain cases, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, -CH2CH2OH, -CH(CH 3 )CH 2 OH, -CH 2 CH(OH)CH 3 , and -CH2CH2CH2OH. In certain cases, each R 11 and R 12 is independently selected from an optionally substituted Ci-4 alkyl, Ci- 3 alkyl, C 1-4 heteroalkyl, and Ci- 3 heteroalkyl.
  • W is — CH2 — . In certain embodiments of formula (IIIC), W is — O — .
  • s is 0. In certain embodiments, s is 1.
  • W is — CH2 — and s is 0. In certain cases,
  • Cy A and Cy B is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
  • R x and R y is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C10 aliphatic group;
  • r, p, and q is each independently an integer from 0 to 10.
  • R is wherein,
  • Cy A and Cy B is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
  • R x and R y is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, Ci-Ce aliphatic group; r, p, and q is each independently an integer from 0 to 6.
  • the lipid is selected from a compound of TableTable 1 :
  • the lipid nanoparticle compositions can include one or more additional ionizable lipid components in addition to the ionizable lipid of formula (I) (e.g., as described above). Any convenient lipid that carries a net positive charge at or around physiological pH may find use as an additional ionizable lipid in the compositions described herein.
  • Non-limiting examples of cationic lipids are described in detail herein.
  • Cationic lipids and related analogs which are useful in the lipid nanoparticles of the present disclosure, include but are not limited to, those lipids described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96/10390, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • Additional cationic lipids of interest include, but are not limited to, l,2-distearydoxy-N,N- dimethyl-3-aminopropane (DSDMA), l,2-dilinoleyloxy-N,N-dimethyl-3 -aminopropane (DLinDM ), l,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDM ), 1,2- di ol ey I oxy -N,N-d methyl -3 -am nopropane (DODMA), and heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), N,N-dioleyl-N,N- dimethylammonium chloride (“DODAC”); N-(2,3-dioleyloxy)propyl-N,N — N- trieth
  • DC-Chol N-(l-(2,3-dioleyloxy)propyl)-
  • LNPs of this disclosure can also include one or more helper lipid(s), in addition to the ionizable lipid component described herein.
  • the helper lipid is a neutral lipid.
  • the neutral lipid is zwitterionic, e.g., has an overall net zero charge.
  • Neural lipids include, for example, phospholipids, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.
  • the selection of neutral lipids for use in the compositions described herein is generally guided by consideration of, e.g., LNP size and stability of the LNPs in the bloodstream.
  • the LNPs of this disclosure includes a helper lipid component that includes a neutral lipid that is a phospholipid.
  • the neutral lipid component is a lipid having two acyl groups, (i.e., diacylphosphatidylcholine and diacylphosphatidylethanolamine).
  • Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well- known techniques.
  • the neutral lipids include saturated fatty acids with carbon chain lengths in the range of Cio to C30.
  • neutral lipids with mono or diunsaturated fatty acids with carbon chain lengths in the range of Cio to C30 are used. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used.
  • phospholipid has a carbon tail of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbons.
  • the phospholipid tail comprises no double bonds, i.e. the bonds are saturated bonds.
  • the phospholipid tail is unsaturated, that is, it comprises one or more double bonds, e.g., 1, 2, 3, 4 or 5 double bonds.
  • the phospholipid tail is unsaturated, that is, it comprises one or more triple bonds, e.g. 1, 2, 3, 4 or 5 triple bonds.
  • the phospholipid tail comprises one or more ring structures.
  • the one or more ring structures is selected from 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl; 5- to 6- membered aryl; 7- to 10-membered saturated or partially unsaturated bicyclic carbocyclyl; and 7- to 10-membered bicyclic aryl wherein each ring structure is independently substituted with 0-7 R A groups; each R A is independently selected from halogen, or an optionally substituted group selected from C1-12 aliphatic, phenyl, or 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl.
  • the ring structure is a cholesterol or cholesterol derivative.
  • the phospholipid is symmetric, i.e., all tails of the phospholipid are the same. In other embodiments, the phospholipid is asymmetric, i.e., the phospholipid comprises two different hydrocarbon chains.
  • a helper lipid is or comprises symmetric or asymmetric aliphatic phospholipid moieties that are each independently optionally substituted, branched or straight, partially unsaturated or saturated C9-C24 aliphatic.
  • the helper lipid comprises one or more optionally substituted and/or optionally bridged ring structures in the hydrophobic tail.
  • exemplary helper lipids of this class include:
  • the helper lipid includes a phosphatidylethanolamine (PE).
  • PE Phosphatidylethanolamines
  • the phosphatidylethanolamine selected from the group consisting of phosphatidylethanolamine, dioleoylphosphatidylethanolamine (1,2- dioleyl-sn-glycero-3-phosphoethanolamine) (A9-Cis PE, or DOPE), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine) (DPPE), dimyristoylphosphoethanol
  • 16-0-monom ethyl PE dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), di elaidoyl-phosphatidyl ethanolamine (DEPE), lysophosphatidyl ethanolamine, 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), and l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DiPPE).
  • SOPE l-stearoyl-2-oleoyl-phosphatidy ethanolamine
  • DEPE di elaidoyl-phosphatidyl ethanolamine
  • DLPE 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine
  • DiPPE diphytanoyl-sn-glycero-3
  • the phosphatidylethanolamine is dioleoylphosphatidylethanolamine (also referred to as l,2-dioleoyl- w-glycero-3- phosphoethanolamine, or (A9-Cis) PE, or DOPE), having a tail of 18 carbons and one saturated bond (“18-1”) as shown below:
  • the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) (also referred to as 1 ,2-distearoyl-.s//-glycero-3-phosphocholine), having a tail of 18 carbons and no saturated bonds (“18-0”) as shown below:
  • DSPC distearoylphosphatidylcholine
  • the phosphatidylcholine is l,2-dipalmitoyl-sn-glycero-3- phosphocholine (delta9-Cis PC), having a tail of 16 carbons and one saturated bond (“16-1”) as shown below:
  • the phosphatidylcholine is an asymmetric lipid, having one tail of 16 carbons and a second tail of 18 carbons.
  • the tail of the phosphatidylcholine having 18 carbons has one saturated bond, e,g, it is l-palmitoyl-2- oleoyl-glycero-3 -phosphocholine (also referred to as “16-0/18-1 PC”, “16:0/18: 1 PC” or POPC) as shown below:
  • the phosphatidylcholine is 1,2- dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (DChemsPC), as shown below:
  • the phosphatidylcholine is l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), as shown below:
  • the phosphatidylcholine is l-palmitoyl-2- cholesterylcarbonoyl-sn-glycero-3-phosphocholine (PChcPC), as shown below:
  • the phosphatidylcholine is l-palmitoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), as shown below:
  • the helper lipid includes a phosphatidylglycerol selected from the group consisting of phosphatidylglycerol, dioleoylphosphatidylglycerol (1,2- dioleoyl-sn-glycero-3- phospho-(l’-rac-glycerol) (DOPG), dipalmitoylphosphatidylglycerol, (DPPG), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), and palmitoyloleyolphosphatidylglycerol (POPG).
  • the helper lipid includes a phosphatidylserine, e.g. phosphatidyl serine or dioleoylphosphatidylserine (DOPS).
  • DOPS dioleoylphosphatidylserine
  • the helper lipid includes a sphingomyelin (SM), e.g. egg sphingomyelin (ESM).
  • SM sphingomyelin
  • ESM egg sphingomyelin
  • the helper lipid is cephalin, cardiolipin, phosphatidic acid, cerebrosides, or dicetylphosphate.
  • the LNP can further comprise a component, such as a sterol, to provide membrane integrity.
  • a component such as a sterol
  • a sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof.
  • cholesterol derivatives include polar analogues such as 5a-cholestanol, 5P-coprostanol, cholesteryl-(2'-hydroxy)- ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5P-cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the component providing membrane integrity such as a sterol
  • the neutral lipid component of the LNPs can further include cholesterol or a derivative or analog thereof.
  • the helper lipid component includes cholesterol.
  • the LNP includes a neutral lipid component that includes a mixture of one or more phospholipids and cholesterol or a derivative or analog thereof.
  • the LNP includes a neutral lipid component that includes a phosphatidylethanolamine phospholipid and cholesterol or a derivative or analog thereof.
  • LNPs of this disclosure can also include one or more additional lipid components.
  • Such lipids can be selected to provide for a desirable profile of nanoparticle properties, such as particle stability, delivery efficacy, tolerability and biodistribution.
  • the LNP can further comprise a non-cationic lipid.
  • Non-ionic lipids include amphipathic lipids, neutral lipids and anionic lipids. Accordingly, the noncationic lipid can be a neutral uncharged, zwitterionic, or anionic lipid.
  • Non-cationic lipids are typically employed to enhance fusogenicity. Exemplary non-cationic lipids envisioned for use in the methods and compositions are described in International Application PCT/US2018/050042 published as WO2019051289A1. Exemplary non-cationic lipids are described in International application Publication WO2017/099823 and US patent publication US2018/0028664.
  • Non-limiting examples of non-cationic lipids include, nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
  • nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, is
  • the LNP includes one or more lipids capable of reducing aggregation.
  • a lipids capable of reducing aggregation includes at least a hydrocarbon tail or chain linked to a hydrophilic group which is capable of being configured at the surface of the LNP and provide for reduced LNP aggregation.
  • the lipid capable of reducing aggregation is sometimes referred to as a conjugated lipid or coat lipid.
  • Exemplary PEG-lipid conjugates include, but are not limited to, PEG- diacylglycerol (DAG) (such as 1 -(monomethoxy -poly ethyleneglycol)-2, 3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a PEGylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-l-O-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypolyethylene glycol 2000)-l,2-distearoyl-sn
  • PEG-lipid conjugates are described, for example, in U.S. Pat. Nos. 5,885,613, 6,287,591, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2016/0376224, and US2017/0119904.
  • a PEG- lipid is a compound disclosed in US2018/0028664.
  • a PEG-lipid is disclosed in US20150376115 or in US2016/0376224.
  • the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl.
  • the PEG-lipid can be one or more of PEG-DMG, PEG- dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG- cholesterol (l-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl- omegal-methyl-poly(ethylene glycol), PEG-DMB (3,4-Ditetradecoxylbenzyl-[omega]- methyl-poly(ethylene glycol) ether), and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(poly ethylene glycol
  • the PEG-lipid can be selected from the group consisting of PEG-DMG, l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], PEG-DSG.
  • lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid.
  • PEG-lipid conjugates polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (CPL) conjugates can be used in place of or in addition to the PEG-lipid.
  • POZ polyoxazoline
  • CPL cationic-polymer lipid
  • conjugated lipids i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids are described in the International patent application publications WO 1996/010392, WO1998/051278, W02002/087541, W02005/026372, WO2008/147438, W02009/086558, WO2012/000104, WO2017/117528, WO2017/099823, WO2015/199952, WO20 17/004143, WO2015/095346, WO2012/000104, WO2012/000104, and WO20 10/006282, US patent application publications US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2013/0303587, US2018/0028664, US2015/0376115, US2016/0376224, US2016/0317458, US2013/0303587, US2013/0303587, and
  • the liver can be a target organ of interest in part due to its central role in metabolism and production of proteins and accordingly diseases which are caused by defects in liver-specific gene products (e.g., the urea cycle disorders) and may benefit from specific targeting of cells (e.g., hepatocytes).
  • the LNP further includes a component including a targeting ligand.
  • the targeting ligand can be selected as desired based on a target sell or tissue to which it is desired to direct the LNPs of this disclosure.
  • the targeting ligand is a ligand of a cell surface receptor.
  • the cell surface receptor is asialoglycoprotein receptor (ASGPR). The ASGPR is expressed on the surface of hepatocyte cells.
  • the targeting ligand is a ligand for ASGPR, such as a N- acetylgalactosamine (GalNAc) containing ligand.
  • GalNAc N- acetylgalactosamine
  • a variety of GalNAc containing ligands and ligands, including multivalent GalNAc ligands are available for use in the LNP of this disclosure, including, e.g. those disclosed in WO2021178725, the full disclosure of which is incorporated herein by reference
  • the PEG-lipid is linked to the targeting ligand.
  • the targeting ligand of interest e.g., as described herein
  • a trisGalNac ligand conjugated to a PEG-lipid can provide for binding of the LNP to the ASGPR receptor of a target cell and result in endocytosis of the LNP.
  • the LNPs include an ionizable lipid of Formula (I) (e.g., as described herein); a nucleic acid cargo (e.g., as described herein); an additional ionizable lipid (e.g., as described herein); a phospholipid (e.g., as described herein), cholesterol (e.g., as described herein); and a lipid capable of reducing aggregation (e.g., as described herein).
  • an ionizable lipid of Formula (I) e.g., as described herein
  • a nucleic acid cargo e.g., as described herein
  • an additional ionizable lipid e.g., as described herein
  • a phospholipid e.g., as described herein
  • cholesterol e.g., as described herein
  • a lipid capable of reducing aggregation e.g., as described herein.
  • the nucleic acid cargo comprises DNA, e.g., an oligonucleotide, a plasmid DNA, a doggybone DNA, a minicircle DNA, a covalently closed circular DNA, a ceDNA, or a chemically modified derivative thereof.
  • the nucleic acid consists essentially of DNA.
  • the nucleic acid cargo comprises RNA, e.g., an siRNA, a gRNA, an mRNA, a circular RNA, or a chemically modified derivative thereof.
  • the nucleic acid consists essentially of RNA.
  • the nucleic acid cargo includes DNA, e.g,. an oligonucleotide, a plasmid DNA, a doggybone DNA, a minicircle DNA, a covalently closed circular DNA, a ceDNA, or a chemically modified derivative thereof, and further includes RNA, e.g., an siRNA, a gRNA, an mRNA, a circular RNA, and the like, or a chemically modified derivative thereof.
  • DNA e.g,. an oligonucleotide, a plasmid DNA, a doggybone DNA, a minicircle DNA, a covalently closed circular DNA, a ceDNA, or a chemically modified derivative thereof
  • RNA e.g., an siRNA, a gRNA, an mRNA, a circular RNA, and the like, or a chemically modified derivative thereof.
  • the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG), and derivatives thereof.
  • the phospholipid is phosphatidylethanolamine (PE).
  • the phospholipid is a phosphatidylcholine (PC).
  • the phospholipid includes hydrocarbon chains each independently having 12-24 carbons. In some cases, the hydrocarbon chains each independently have 16-20 carbons. In certain cases, the hydrocarbon chains are saturated.
  • the hydrocarbon chains are unsaturated. In certain cases, the hydrocarbon chains each independently comprise 1-4 double bonds. In certain cases, the phospholipid comprises two different hydrocarbon chains. In certain embodiments of the LNP, the phospholipid includes dioleoylphosphatidylethanolamine (DOPE, 18: 1 PE). In certain cases, the phospholipid includes l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In certain cases, the phospholipid includes l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • DOPE dioleoylphosphatidylethanolamine
  • DOPC l,2-dioleoyl-sn-glycero-3-phosphocholine
  • DSPC l,2-distearoyl-sn-glycero-3-phosphocholine
  • the phospholipid includes l,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine (delta9-Cis PC). In certain cases, the phospholipid includes l-stearoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (SOPE). In certain cases, the phospholipid includes a mixture of di oleoylphosphatidylethanolamine (DOPE, 18-1) and di oleoylphosphatidy choline (DOPC, 18-1).
  • DOPE di oleoylphosphatidylethanolamine
  • DOPC di oleoylphosphatidy choline
  • the lipid capable of reducing aggregation is a PEG-lipid.
  • the PEG lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1, 2-distearoyl-rac-glycero-3- methylpolyoxyethylene 2000 (PEG-DSG[2K]).
  • the LNP further comprises a targeting ligand (e.g., as described herein).
  • the targeting ligand comprises GalNac.
  • the targeting ligand is linked to the ligand capable of reducing aggregation.
  • the lipid capable of reducing aggregation linked to the targeting ligand is PEG- l,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).
  • the LNPs include an ionizable lipid of Formula (I) (e.g., as described herein); a phospholipid that is DOPE, cholesterol, and a lipid capable of reducing aggregation that is PEG-DMG.
  • the LNPs include an ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group; a phospholipid that is a phosphatidylethanolamine, (e.g. DOPE), cholesterol, and a lipid capable of reducing aggregation that is PEG-DMG, and/or PEG-DSG-GalNAc or PEG-DSPE-GalNac.
  • a ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group
  • DOPE phosphatidylethanolamine
  • the LNPs include an ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group, a phospholipid that is a phosphatidylcholine (e.g. l,2-distearoyl-sn-glycero-3 -phosphocholine, DSPC), cholesterol and a coat lipid (polyethylene glycol-dimyristolglycerol, PEG-DMG), for example as disclosed by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3): 498-507.
  • the N/P ratio can be in the range of from about 1 : 1 to about 50: 1, from about 7: 1 to about 25:1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1.
  • the amounts of lipids and DNA can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3 :1 (“3”), 4:1 (“4”), 5: 1 (“5”), 6: 1 (“6”), 7: 1 (“7”), 8: 1 (“8”), 9: 1 (“9”), 10: 1 (“10”), 11 : 1 (“11”), 12: 1 (“12”), 13: 1 (“13”), 14: 1 (“14”) or higher.
  • N/P ratio 3 :1 (“3”), 4:1 (“4”), 5: 1 (“5”), 6: 1 (“6”), 7: 1 (“7”), 8: 1 (“8”), 9: 1 (“9”), 10: 1 (“10”), 11 : 1 (“11”), 12: 1 (“12”), 13: 1 (“13”), 14: 1 (“14”) or higher.
  • the lipid particle formulation's overall lipid content can range from about 5 mg/mL to about 30 mg/mL.
  • the N/P ratio is from 5 to 30. In certain cases, the N/P ratio is 7. In certain cases, the N/P ratio is 14. In certain cases, the N/P ratio is 28.
  • a lipid nanoparticle preparation (e.g., composition comprising a plurality of lipid nanoparticles) has a size distribution in which the mean size (e.g., diameter) is about 70 nm to about 200 nm, and more typically the mean size is about 100 nm or less.
  • the mean size e.g., diameter
  • an LNP has a mean diameter of 25 to 250 nm, 25 to 240 nm, 25 to 230 nm, 25 to 220 nm, 25 to 210 nm, 25 to 200 nm, 25 to 190 nm, 25 to 180 nm, 25 to 170 nm, 25 to 160 nm, 25 to 150 nm, 25 to 140 nm, 25 to 130 nm, 25 to 120 nm, 25 to 110 nm, 25 to 100 nm, 25 to 90 nm, 25 to 80 nm, 25 to 70 nm, 25 to 60 nm, or 25 to 50 nm.
  • an LNP has a mean diameter of 60 to 250 nm, 70 to 250 nm, 80 to 250 nm, 90 to 250 nm, 100 to 250 nm, 110 to 250 nm, 120 to 250 nm, 130 to 250 nm, 140 to 250 nm, 150 to 250 nm, 160 to 250 nm, 170 to 250 nm, 180 to 250 nm, 190 to 250 nm, 200 to 250 nm, 210 to 250 nm, 220 to 250 nm, 230 to 250 nm, or 240 to 250 nm
  • an LNP has a mean diameter of 60 to 250 nm, 70 to 240 nm, 80 to 230 nm, 90 to 220 nm, 100 to 210 nm, 110 to 200 nm, 120 to 190 nm, 130 to 180 nm, 140 to 170 nm, or 150 to 160 nm.
  • the structural characteristics of the target tissue may be exploited to direct the distribution of the LNPs to such target tissues.
  • a LNP may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; accordingly, the LNP can readily penetrate such endothelial fenestrations to reach the target hepatocytes.
  • a LNP may be sized such that the dimensions of the particles are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues.
  • a LNP may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the LNPs to hepatocytes.
  • large LNPs will not easily penetrate the endothelial fenestrations, and would instead be cleared by the macrophage Kupffer cells that line the liver sinusoids.
  • the size of the LNPs is within the range of about 25 to 250 nm or 25nm to lOOnm, preferably less than 250 nm, less than 175 nm, less than 150 nm, less than 125 nm, or less than 100 nm.
  • ionizable lipid can comprise 20-90% (mol) of the total lipid present in the lipid nanoparticle.
  • ionizable lipid molar content can be 20-70% (mol), 30-60% (mol) or 40-50% (mol) of the total lipid present in the lipid nanoparticle.
  • ionizable lipid comprises from about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle.
  • the ionizable lipid comprises from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, from about 50 mol % to about 60 mol %, from about 55 mol % to about 65 mol %, or from about 55 mol % to about 70 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the cationic lipid comprises 40 mol %, 41 mol %, 42 mol %, 43 mol %, 44 mol %, 45 mol %, 46 mol %, 47 mol %, 48 mol %, 49 mol %, 50 mol %, 51 mol %, 52 mol %, 53 mol %, 54 mol %, 55 mol %, 56 mol %, 57 mol%, 58 mol% or 60 mol% (or any fraction thereof) of the total lipid present in the particle.
  • the neutral lipid components can comprise 10-60% (mol) of the total lipid present in the lipid nanoparticle.
  • the non-cationic lipid content is 10-50% (mol) or 20- 55% (mol) of the total lipid present in the lipid nanoparticle.
  • the noncationic lipid comprises from about 10 mol % to about 60 mol %, from about 20 mol % to about 55 mol %, from about 20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, from about 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %, from about 30 mol % to about 50 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 35 mol % to about 45 mol %, from about 37 mol % to about 42 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the non-cationic lipid comprises 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %, 43 mol %, 44 mol %, 45 mol %, 46%, 47%, 48%, 49%, or 50% (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the lipid particles contain a mixture of phospholipid and cholesterol or a cholesterol derivative
  • the mixture may comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the particle.
  • the mixture of phospholipid and cholesterol or a cholesterol derivative comprises up to 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %, 43 mol %, 44 mol %, 45 mol %, 46%, 47%, 48%, 49%, or 50% (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the LNP comprises a phospholipid component in the mixture in an amount of from about 2 mol % to about 20 mol %, from about 2 mol % to about 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol % to about 15 mol %, or from about 4 mol % to about 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the phospholipid component in the mixture comprises from about 5 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the LNP includes a cholesterol component in the mixture in an amount of from about 25 mol % to about 45 mol %, from about 25 mol % to about 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 27 mol % to about 37 mol %, from about 25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the cholesterol component in the mixture comprises from about 25 mol % to about 35 mol %, from about 27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, from about 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %, from about 31 mol % to about 33 mol %, or 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, 35 mol %, 36%, 37%, 38%, or 39% (or any fraction thereof or range therein) of the total lipid present in the particle.
  • mol percentage of components described herein in the LNP is a target amount, and that the actual amount of each lipid component present in the formulation may vary, for example, by ⁇ 5 mol %.
  • the LNP includes a lipid capable of reducing aggregation (e.g., a PEG-lipid conjugate) in an amount of about 1.5% to about 4%, for example about 1.5% to about 3%, about 2% to about 3%, about 2.5% to about 3%, about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 1.5% to about 1.75%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, about 2% to about 2.25% (or any fraction thereof or range therein) of the total lipid present in the particle.
  • a lipid capable of reducing aggregation e.g., a PEG-lipid conjugate
  • the lipid capable of reducing aggregation is present at 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3% or any fraction thereof or range therein) of the total lipid present in the particle.
  • the molar ratio of ionizable lipid to the neutral lipid ranges from about 2: 1 to about 8: 1.
  • the lipid nanoparticles do not comprise any phospholipids.
  • the LNP comprises: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 6 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
  • the LNP comprises: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 10 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
  • the LNP comprises: a) an ionizable lipid at 40 to 49 mol % of the total lipid present; b) a phospholipid at 10 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
  • A+B+C+D 100%; d.
  • A+B+C+D 100% g.
  • A 40% - 60%
  • B 10% - 30%
  • C 20% - 45%
  • D 0% - 3%
  • A+B+C+D 100%; k.
  • a given lipid nanoparticle may include a cargo, or payload, to be delivered to cells.
  • cargos that comprise a polynucleotide.
  • the polynucleotide is a DNA.
  • DNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure.
  • the DNA may be circular, e.g., a plasmid, a nanoplasmid, a mini circle, a covalently closed circular DNA, a circular viral genome, and the like.
  • the DNA may be linear, e.g., a doggybone or other closed-end DNA, a linear viral genome, and the like.
  • the DNA may be multivalent, e.g., a 3DNA.
  • the DNA may be single stranded or double stranded or a hybrid of single and double stranded.
  • the DNA may be chemically modified.
  • the polynucleotide is an RNA.
  • RNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure.
  • the RNA may be linear or it may be circular. It may be an mRNA, an siRNA, an shRNA, a guide RNA (gRNA), a microRNA (miRNA), a circular RNA (circRNA). It may be chemically modified.
  • the one or more additional compounds can be a therapeutic agent.
  • the therapeutic agent can be selected from any class suitable for the therapeutic objective.
  • the therapeutic agent can be selected from any class suitable for the therapeutic objective.
  • the therapeutic agent can be selected according to the treatment objective and biological action desired.
  • the additional compound can be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate).
  • the additional compound can be an antimicrobial agent (e.g., an antibiotic or antiviral compound).
  • the additional compound can be a compound that modulates an immune response (e.g., an immunosuppressant, immunostimulatory compound, or compound modulating one or more specific immune pathways).
  • an immunosuppressant e.g., an immunosuppressant, immunostimulatory compound, or compound modulating one or more specific immune pathways.
  • different cocktails of different lipid nanoparticles containing different compounds, such as a DNA encoding a different protein or a different compound, such as a therapeutic may be used in the compositions and methods of the invention.
  • the additional compound is an immune modulating agent.
  • the additional compound is an immunosuppressant.
  • the additional compound is immune stimulatory agent.
  • lipid nanoparticle-encapsulated nucleic acid e.g., DNA
  • a pharmaceutically acceptable carrier or excipient e.g., water
  • the disclosure provides for a lipid nanoparticle formulation further comprising one or more pharmaceutical excipients.
  • the lipid nanoparticle formulation further comprises sucrose, tris, trehalose and/or glycine.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
  • Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water
  • “Pharmaceutically acceptable salt” includes both acid and base addition salts.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4- acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-l,2-disulfonic acid, ethanesulfonic acid, 2-hydroxye
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methyl
  • the LNPs and LNP pharmaceutical composition of the present disclosure have been observed to be efficacious at delivering their nucleic acid cargo to the target cell of interest, including where LNPs and LNP pharmaceutical composition of the present disclosure are equally or more efficacious at delivering their nucleic acid cargo to the target cell of interest as that same industry standard LNP administered at the same dose, e.g. having 2-fold the efficacy or more, e.g. 3-fold, 4-fold or 5-fold the efficacy or more, in some instances 10-fold, 20-fold or 50-fold the efficacy, in certain instances 100-fold more efficacious or more.
  • more efficacious it is meant able to deliver more nucleic acid cargo to the cell, resulting in an increase in the amount of mRNA transcribed from that nucleic acid cargo or an increase in the amount of protein translated, for example a 2-fold increase or more, e.g. a 3-fold, 4-fold, 5-fold increase, e.g. 10-fold, 20-fold, 50-fold increase, in some instances a 100-fold increase or more.
  • the LNPs of the present disclosure demonstrate an improved pharmacokinetics (PK) profile that broadens the therapeutic index of the composition.
  • PK pharmacokinetics
  • a therapeutic index or therapeutic ratio, it is meant the range of doses at which a medication is effective without unacceptable adverse events, calculated as the ratio that compares the blood concentration at which a drug becomes toxic and the concentration at which the drug is effective.
  • This improvement over the art makes them more amenable to delivering nucleic acids, including DNA, to cells in vitro and in vivo, and accordingly they find many uses in many applications, including in the delivery of nucleic acids, including DNA, to cells for research and for therapeutic applications.
  • the cells are typically contacted with the composition, e.g., LNP or pharmaceutical composition thereof, in amount effective to deliver the agent into the cytoplasm of the cell.
  • the contacting is in vitro. In other embodiments, the contacting is in vivo. In some embodiments, the method further comprises measuring the amount of protein produced.
  • the present disclosure further provides methods of treating or preventing diseases in a subject in need thereof wherein an effective amount of the therapeutic compositions described herein is administered to the subject.
  • the route of administration will vary, naturally, with the location and nature of the disease being treated, and may include, for example intradermal, transdermal, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration.
  • the encapsulated polynucleotide compositions described herein are useful in the treatment of any of any indication in which it is beneficial to deliver a therapeutic cargo into the target cell.
  • the present disclosure further provides methods of immunizing a subject against a disease wherein an effective amount of a therapeutic composition described herein is administered to the subject.
  • the route of administration will vary, naturally, with the location and nature of immunization agent, and may include, for example intradermal, transdermal, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration.
  • the present disclosure further provides a particle of the disclosure, a vector of the disclosure, a recombinant DNA of the disclosure, or compositions thereof, for use as a medicament.
  • the medicament is for expressing a protein in a cell.
  • the expressing of a protein is for the treatment of a disease in which the cell is deficient for the protein.
  • the expressing of a protein is for the treatment of a disease in which another cell is deficient for the protein.
  • the medicament is for the treatment of a cancer.
  • the medicament is for immunization against a disease. 4.12 Utility
  • the subject methods and compositions can be used in any application where delivery of a cargo nucleic acid is desired.
  • Applications of interest include both research and therapeutic applications.
  • Applications of interest include, but are not limited to: research applications, diagnostic applications and therapeutic applications.
  • cargo nucleic acids that may be introduced into a cell, and subsequently a nucleus, via methods of the invention include those encoding research proteins, diagnostic proteins and therapeutic proteins.
  • Research proteins are proteins whose activity finds use in a research protocol. As such, research proteins are proteins that are employed in an experimental procedure.
  • the research protein may be any protein that has such utility, where in some instances the research protein is a protein domain that is also provided in research protocols by expressing it in a cell from an encoding vector.
  • transcription modulators of inducible expression systems include, but are not limited to: transcription modulators of inducible expression systems, members of signal production systems, e.g., enzymes and substrates thereof, hormones, prohormones, proteases, enzyme activity modulators, perturbimers and peptide aptamers, antibodies, modulators of protein-protein interactions, genomic modification proteins, such as CRE recombinase, meganucleases, Zinc-finger nucleases, CRISPR/Cas-9 nuclease, TAL effector nucleases, etc., cellular reprogramming proteins, such as Oct 3/4, Sox2, Klf4, c-Myc, Nanog, Lin-28, etc., and the like.
  • transcription modulators of inducible expression systems include, but are not limited to: transcription modulators of inducible expression systems, members of signal production systems, e.g., enzymes and substrates thereof, hormones, prohormones, proteases, enzyme activity modulators, perturbimers and
  • Diagnostic proteins are proteins whose activity finds use in a diagnostic protocol. As such, diagnostic proteins are proteins that are employed in a diagnostic procedure.
  • the diagnostic protein may be any protein that has such utility. Examples of specific types of diagnostic proteins include, but are not limited to: members of signal production systems, e.g., enzymes and substrates thereof, labeled binding members, e.g., labeled antibodies and binding fragments thereof, peptide aptamers and the like.
  • Proteins of interest further include therapeutic proteins.
  • Therapeutic proteins of interest include without limitation, hormones and growth and differentiation factors, fibrinolytic proteins, transcription factors, and enzymes.
  • Target cells to which nucleic acids may be delivered in accordance with embodiments of this disclosure may vary widely.
  • Target cells of interest include, but are not limited to: cell lines, HeLa, HEK, CHO, 293 and the like, Mouse embryonic stem cells, human stem cells, mesenchymal stem cells, primary cells, tissue samples and the like.
  • Some non-limiting examples of a mammalian cell include, without limitation, a mouse cell, a rat cell, hamster cell, a rodent cell, and a nonhuman primate cell.
  • the target cell is a human cell. It should also be appreciated that the target cell may be of any cell type.
  • the target cell may be a stem cell, which may include embryonic stem cells, induced pluripotent stem cells (iPS cells), fetal stem cells, cord blood stem cells, or adult stem cells (i.e., tissue specific stem cells).
  • the target cell may be any differentiated cell type found in a subject.
  • Cells of interest include both dividing cells and non-dividing cells. Examples of specific target cells of interest include, but are not limited to: hepatocytes, stellate cells, T lymphocytes, B lymphocytes, NK cells, skeletal muscle cells, cardiomyocytes, neurons, astrocytes, oligodendrocytes, dendritic cells, skin cells, etc.
  • Targeted cells may include the cells of a targeted location, such as, e.g., the liver, or cells near or adjacent to hepatocytes, e.g., hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), ductal cells, or combinations thereof.
  • hepatocytes e.g., hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), ductal cells, or combinations thereof.
  • HSCs hepatic stellate cells
  • KCs Kupffer cells
  • LSECs liver sinusoidal endothelial cells
  • ductal cells or combinations thereof.
  • the application of interest is a therapeutic application, for example, in the treatment of a disease.
  • the compositions and methods of the present application may be used to deliver a nucleic acid sequence to a cell to complement a genetic deficiency.
  • compositions of the present application may be used in the treatment of a genetic deficiency that impacts the function of hepatocytes, or in the treatment of a genetic deficiency elsewhere in the body that can be remedied by leveraging hepatocytes as a biofactory to secrete the deficient protein.
  • the terms "individual,” “subject” and “host” are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired.
  • the subject is a mammal.
  • the subject is a human being.
  • the subject is a patient.
  • the subject is a human patient.
  • the subject can have or is suspected of having a disorder or health condition associated with a gene-of-interest (GOI).
  • GOI gene-of-interest
  • the subject is a human who is diagnosed with a risk of disorder or health condition associated with a GOI at the time of diagnosis or later.
  • the diagnosis with a risk of disorder or health condition associated with a GOI can be determined based on the presence of one or more mutations in the endogenous GOI or genomic sequence near the GOI in the genome that may affect the expression of GOI.
  • treatment used referring to a disease or condition means that at least an amelioration of the symptoms associated with the condition afflicting an individual is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom, associated with the condition (e.g., hemophilia A) being treated.
  • a parameter e.g., a symptom
  • treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or eliminated entirely such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition.
  • the terms “may,” “optional,” “optionally,” or “may optionally” mean that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.
  • the phrase “optionally substituted” means that a non-hydrogen substituent may or may not be present on a given atom, and, thus, the description includes structures wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.
  • Acyl refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkenyl-C(
  • alkyl refers to a branched or unbranched saturated hydrocarbon group (i.e., a mono-radical) typically although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like.
  • alkyl groups herein may contain 1 to about 18 carbon atoms, and such groups may contain 1 to about 12 carbon atoms.
  • lower alkyl intends an alkyl group of 1 to 6 carbon atoms.
  • heteroatom-containing alkyl and “heteroalkyl” refer to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom, as described in further detail infra. If not otherwise indicated, the terms “alkyl” and “lower alkyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkyl or lower alkyl, respectively.
  • substituted alkyl is meant to include an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain have been optionally replaced with a heteroatom such as -O-, -N-, -S-, -S(O)n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thio
  • alkenyl refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like.
  • alkenyl groups herein may contain 2 to about 18 carbon atoms, and for example may contain 2 to 12 carbon atoms.
  • lower alkenyl intends an alkenyl group of 2 to 6 carbon atoms.
  • substituted alkenyl refers to alkenyl substituted with one or more substituent groups
  • heteroatom-containing alkenyl and “heteroalkenyl” refer to alkenyl in which at least one carbon atom is replaced with a heteroatom.
  • alkenyl and “lower alkenyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkenyl and lower alkenyl, respectively.
  • alkynyl refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Generally, although again not necessarily, alkynyl groups herein may contain 2 to about 18 carbon atoms, and such groups may further contain 2 to 12 carbon atoms. The term “lower alkynyl” intends an alkynyl group of 2 to 6 carbon atoms.
  • substituted alkynyl refers to alkynyl substituted with one or more substituent groups
  • heteroatomcontaining alkynyl and “heteroalkynyl” refer to alkynyl in which at least one carbon atom is replaced with a heteroatom.
  • alkynyl and “lower alkynyl” include linear, branched, unsubstituted, substituted, and/or heteroatom-containing alkynyl and lower alkynyl, respectively.
  • aryl refers to an aromatic substituent generally, although not necessarily, containing 5 to 30 carbon atoms and containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety).
  • Aryl groups may, for example, contain 5 to 20 carbon atoms, and as a further example, aryl groups may contain 5 to 12 carbon atoms.
  • aryl groups may contain one aromatic ring or two or more fused or linked aromatic rings (i.e., biaryl, aryl -substituted aryl, etc.).
  • substituted aryl refers to an aryl moiety substituted with one or more substituent groups
  • heteroatomcontaining aryl and “heteroaryl” refer to aryl substituent, in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail infra.
  • Aryl is intended to include stable cyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated C3-C14 moieties, exemplified but not limited to phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, and oxazoyl; which may further be substituted with one to five members selected from the group consisting of hydroxy, Ci-Cs alkoxy, Ci-Cs branched or straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (see e.g. Katritzky, Handbook of Heterocyclic Chemistry). If not otherwise indicated, the term "aryl" includes unsubstituted, substituted, and/or heteroatom-containing aromatic substituents.
  • alkylene refers to a di-radical alkyl group. Unless otherwise indicated, such groups include saturated hydrocarbon chains containing from 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups, and may be heteroatom-containing. "Lower alkylene” refers to alkylene linkages containing from 1 to 6 carbon atoms. Examples include, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), 2-methylpropylene (-CH2-CH(CH3)-CH2-), hexylene (-(CH2)e-), and the like.
  • alkenylene refers to di-radical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.
  • amino refers to the group -NRR’ wherein R and R’ are independently hydrogen or nonhydrogen substituents, with nonhydrogen substituents including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and/or heteroatom-containing variants thereof.
  • Cycloalkyl refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems.
  • suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like.
  • Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
  • substituted cycloalkyl refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,
  • Heteroaryl refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring.
  • Such heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic, provided that the point of attachment is through an atom of an aromatic ring.
  • the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N— >0), sulfinyl, or sulfonyl moieties.
  • This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.
  • heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thio
  • heterocycle refers to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 15 ring atoms, including 1 to 4 hetero atoms.
  • ring heteroatoms are selected from nitrogen, sulfur and oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring.
  • the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or -SO2- moieties.
  • heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline,
  • heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,
  • substituted as in “substituted alkyl,” “substituted aryl,” and the like, as alluded to in some of the aforementioned definitions, is meant that in the alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents.
  • substituents include, without limitation, functional groups, and the hydrocarbyl moieties C1-C24 alkyl (including Ci-Cis alkyl, further including C1-C12 alkyl, and further including Ci-Ce alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12 alkenyl, and further including C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl, further including C2-C12 alkynyl, and further including C2- Ce alkynyl), C5-C30 aryl (including C5-C20 aryl, and further including C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, and further including C6-C12 aralkyl).
  • C1-C24 alkyl including Ci-Cis alkyl, further including C1-C12 alkyl, and further including Ci-Ce al
  • hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. Unless otherwise indicated, any of the groups described herein are to be interpreted as including substituted and/or heteroatom-containing moieties, in addition to unsubstituted groups.
  • Each M + may independently be, for example, an alkali ion, such as K + , Na + , Li + ; an ammonium ion, such as + N(R 60 )4; or an alkaline earth ion, such as [Ca 2+ ]o.5, [Mg 2+ ]o.5, or [Ba 2+ ]o.5 (“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions).
  • an alkali ion such as K + , Na + , Li +
  • an ammonium ion such as + N(R 60 )4
  • -NR 80 R 80 is meant to include -NH2, -NH-alkyl, A-pyrrolidinyl, 7V-piperazinyl, 47V- methyl-piperazin-l-yl and 7V-morpholinyl.
  • substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R 60 , halo, -O'M + , -OR 70 , -SR 70 , -S“M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO3- M + , -SO3R 70 , -OSO2R 70 , -OSO3-M+ -OSO3R 70 , -PO3' 2 (M + )2, -P(O)(OR 70 )O- M + , -P(O)(OR 70 ) 2 , -C(O)R 70 , -C(S)R 70 , -
  • R 60 , R 70 , R 80 and M + are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -O'M + , -OR 70 , -SR 70 , or -S“M + .
  • substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O M + , -OR 70 , -SR 70 , -S'M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O) 2 R 70 , -S(O)2O'M + , -S(O) 2 OR 70 , -OS(O) 2 R 70 , -OS( O) 2 O'M + , -OS(O) 2 OR 70 , -P(O)(O-) 2 (M + )2, -P(O)(OR 70 )O'M + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70 , - C
  • R 70 , R 80 and M + are as previously defined.
  • a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
  • arylalkyloxycarbonyl refers to the group (aryl)-(alkyl)-O-C(O)-.
  • any of the groups disclosed herein which contain one or more substituents it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
  • the subject compounds include all stereochemical isomers arising from the substitution of these compounds.
  • a substituent may contribute to optical isomerism and/or stereo isomerism of a compound.
  • Salts, solvates, hydrates, and prodrug forms of a compound are also of interest. All such forms are embraced by the present disclosure.
  • the compounds described herein include salts, solvates, hydrates, prodrug and isomer forms thereof, including the pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers thereof.
  • a compound may be a metabolized into a pharmaceutically active derivative.
  • a bond designated as — in a small molecule structure refers to a bond that, in some embodiments, is a single (e.g., saturated) bond, and in some embodiments, is a double (e.g., unsaturated) bond.
  • the following structure is intended to encompass both and
  • reference to an atom is meant to include isotopes of that atom.
  • reference to H is meant to include 1 H, 2 H (i.e., D) and 3 H (i.e., T)
  • reference to C is meant to include 12 C and all isotopes of carbon (such as 13 C).
  • Embodiment 1 An ionizable lipid compound of formula (I): (Z-L-Y)-W n -(X-R) ( n-i)
  • Embodiment 2 The compound of embodiment 1, wherein n is 4 to 6.
  • Embodiment 3 The compound of embodiment 2, wherein n is 4.
  • Embodiment 4 The compound of embodiment 2, wherein n is 5.
  • Embodiment 5 The compound of embodiment 2, wherein n is 6.
  • Embodiment 6 The compound of embodiment 1, wherein n is 3.
  • Embodiment 7 The compound of any one of embodiments 1 to 6, wherein W selected from: wherein:
  • G 1 is H, or a group cyclically linked with Y that together with the carbon atom of W n to which they are attached provide a heterocycle; and G 2 is H or -CH2OH.
  • Embodiment 8 The compound of any one of embodiments 1 to 6, wherein Y is selected from — O— , — C(R 10 ) 2 — , — OC(O)— , — C(O)O— , — OC(O)O— , — OC(O)NR 10 — , — SC(O)NR 10 — , — C(O)NR 10 — , — NR 10 C(O)— , — S— , —NR 10 —, — NR 10 C(O)O — , and — NR 10 C(O)S — , wherein R 10 is selected from H and C1-6 alkyl.
  • Embodiment 9 The compound of embodiment 8, wherein Y is selected from — O — , — OC(O)— , and — OC(O)NR 10 — .
  • Embodiment 10 The compound of embodiment 8, wherein Y is — CH2 — .
  • Embodiment 11 The compound of embodiment 7, wherein G 1 is a group that is cyclically linked with Y and together with the carbon atom of W n to which they are attached provides a heterocycle.
  • Embodiment 12 The compound of any one of embodiments 1 to 11, wherein L is (C2- Ce)alkylene or substituted (C2-Ce)alkylene.
  • Embodiment 13 The compound of embodiment 12, wherein L is -(CH 2 ) 2 -.
  • Embodiment 14 The compound of embodiment 12, wherein L is -(CH 2 )3- Embodiment 15. The compound of any one of embodiments 1 to 14, wherein Z comprises a tertiary amino group.
  • Embodiment 16 The compound of embodiment 15, wherein Z is -NR U R 12 , wherein R 11 and R 12 are each independently alkyl or substituted alkyl.
  • Embodiment 17 The compound of embodiment 16, wherein R 11 and R 12 are each C1-6 alkyl.
  • Embodiment 18 The compound of embodiment 17, wherein R 11 and R 12 are each C1-3 alkyl.
  • Embodiment 21 The compound of any one of embodiments 1 to 20, wherein each X is independently selected from — (CH2)sOC(O) — , — (CH2)sC(O)O — , — (CH 2 )sOC(O)O— ,— (CH 2 ) S OC(O)NR 10 — , — (CH 2 )sO— , — (CH 2 ) s SC(O)NR 10 — , — (CH 2 ) S C(O)NR 10 — , — (CH 2 ) s NR 10 C(O)— , — (CH 2 ) S S— , — (CH 2 ) S NR 10 — , — (CH2)SNR 10 C(O)O — , and — (CH2)sNR 10 C(O)S — , wherein R 10 is selected from H and C1-6 alkyl and s is 0-6.
  • Embodiment 22 The compound of any one of embodiments 1 to 21, wherein each X is independently selected from — OC(O) — , — C(O)O — , — OC(O)O — , — O — , — OC(O)NR 10 — , — SC(O)NR 10 — , — C(O)NR 10 — , — NR 10 C(O)— , — S— , —NR 10 —, — NR 10 C(O)O — , and — NR 10 C(O)S — , wherein R 10 is selected from H and C1-6 alkyl.
  • Embodiment 23 The compound of embodiment 22, wherein each X is independently selected from — OC(O)— , — C(O)O— , and — OC(O)O— .
  • Embodiment 24 The compound of embodiment 23, wherein each — X-R is — OC(O)R.
  • Embodiment 31 The compound of any one of embodiments 1 to 25, wherein at least one R is a branched hydrocarbon group optionally comprising a cyclic group.
  • Embodiment 32 The compound of embodiment 31, wherein each R is a branched hydrocarbon group.
  • Embodiment 33 The compound of embodiment 32, wherein the branched hydrocarbon group comprises 8-20 carbon atoms.
  • Embodiment 34 The compound of any one of embodiments 31 to 33, wherein the branched hydrocarbon group is saturated.
  • Embodiment 35 The compound of any one of embodiments 31 to 33, wherein the branched hydrocarbon group is unsaturated.
  • Embodiment 36 The compound of any one of embodiments 31 to 35, wherein R is - CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • Embodiment 37 The compound of embodiment 31, wherein at least one R is a branched hydrocarbon group comprising a cyclic group.
  • Embodiment 38 The compound of embodiment 37, wherein the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, wherein any of the monocyclic or bicyclic groups are optionally substituted.
  • Embodiment 39 The compound of embodiment 1, wherein the compound is of formula (IIA):
  • Embodiment 40 The compound of embodiment 39, wherein Y is selected from — O — , — OC(O) — , and — OC(O)NR 10 — , wherein R 10 is selected from H and Ci-6 alkyl.
  • Embodiment 41 The compound of embodiment 40, wherein Y is — O — .
  • Embodiment 42 The compound of embodiment 40, wherein Y is — OC(O) — ,
  • Embodiment 43 The compound of embodiment 40, wherein Y is — OC(O)NR 10 — .
  • Embodiment 44 The compound of any one of embodiments 39 to 43, wherein L is (C2-
  • Embodiment 45 The compound of embodiment 44, wherein L is -(CH2)2-
  • Embodiment 46 The compound of embodiment 44, wherein L is -(CEb)?-.
  • Embodiment 47 The compound of embodiment 44, wherein L is -(CH2)4-
  • Embodiment 48 The compound of any one of embodiments 39 to 47, wherein Z is -
  • R 11 and R 12 are each independently Ci-6 alkyl or substituted Ci-6 alkyl.
  • Embodiment 49 The compound of embodiment 48, wherein R 11 and R 12 are each Ci -3 alkyl.
  • Embodiment 50 The compound of embodiment 49, wherein R 11 and R 12 are each methyl.
  • Embodiment 51 The compound of embodiment 49, wherein R 11 and R 12 are each ethyl.
  • Embodiment 52 The compound of any one of embodiments 39 to 51, wherein each X is independently selected from — OC(O) — , — C(O)O — , and — OC(O)O — .
  • Embodiment 53 The compound of any one of embodiments 39 to 52, wherein each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl.
  • Embodiment 54 The compound of any one of embodiments 39 to 52, wherein at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms optionally further comprising one or more cyclic group.
  • Embodiment 55 The compound of embodiment 54, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • Embodiment 56 The compound of embodiment 39, wherein the compound is of formula (IIIA): wherein:
  • R 11 and R 12 are each independently C1-3 alkyl; q is 1 to 4;
  • Y is selected from — O — , — OC(O) — , and — OC(O)NR 10 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R 7 )2, and -(CH 2 )tJ(CH 2 )u, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and t and u are each independently 1-10.
  • Embodiment 57 The compound of embodiment 1, wherein the compound is of formula (IIB):
  • Embodiment 58 The compound of embodiment 57, wherein Y is selected from — O — , — OC(O)— , — OC(O)NR 10 — , — NR 10 C(O)— , — NR 10 C(O)O— , and — NR 10 C(O)S — , wherein R 10 is selected from H and C1-6 alkyl.
  • Embodiment 59 The compound of embodiment 58, wherein Y is selected from — NHC(O)— , — NHC(O)O— , and — NHC(O)S— .
  • Embodiment 60 The compound of any one of embodiments 57 to 59, wherein L is (C2- Ce)alkylene or substituted (C2-Ce)alkylene.
  • Embodiment 61 The compound of embodiment 60, wherein L is -(CH2)2- Embodiment 62.
  • Embodiment 63 The compound of embodiment 60, wherein L is -(CH2)4-
  • Embodiment 64 The compound of any one of embodiments 57 to 59, wherein Z is -
  • Embodiment 66 The compound of embodiment 64, wherein R 11 and R 12 are each methyl.
  • Embodiment 67 The compound of any one of embodiments 57 to 66, wherein each X is independently selected from — OC(O) — , — C(O)O — , and — OC(O)O — .
  • Embodiment 68 The compound of any one of embodiments 57 to 67, wherein each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl.
  • Embodiment 69 The compound of any one of embodiments 57 to 67, wherein at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms optionally further comprising one or more cyclic groups.
  • Embodiment 70 The compound of embodiment 69, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • Embodiment 71 The compound of embodiment 57, wherein the compound is of formula (IIIB): wherein:
  • R 11 and R 12 are each independently C1-3 alkyl; q is 1 to 4; Y is selected from — NHC(O) — , — NHC(O)O — , and — NHC(O)S — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R 7 )2, and -(CH 2 )tJ(CH 2 )u, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and t and u are each independently 1-10.
  • Embodiment 72 The compound of embodiment 1, wherein the compound is of formula (IIC):
  • Embodiment 73 The compound of embodiment 72, wherein Y is selected from — O — , — OC(O) — , — OC(O)NR 10 — , and — C(R 10 )2 — , wherein R 10 is selected from H and Ci -6 alkyl.
  • Embodiment 74 The compound of embodiment 73, wherein Y is — O — .
  • Embodiment 75 The compound of embodiment 73, wherein Y is — C(R 10 )2 — . — .
  • Embodiment 76 The compound of any one of embodiments 72 to 75, wherein L is (C2-
  • Embodiment 77 The compound of any one of embodiments 76, wherein L is -(CH2)2-
  • Embodiment 78 The compound of any one of embodiments 76, wherein L is -(CTb)?-.
  • Embodiment 79 The compound of any one of embodiments 76, wherein L is -(CEb ⁇ -.
  • Embodiment 80 The compound of any one of embodiments 72 to 79, wherein Z is -
  • R 11 and R 12 are each independently Ci-6 alkyl or substituted Ci-6 alkyl.
  • Embodiment 81 The compound of embodiment 80, wherein R 11 and R 12 are each C1-3 alkyl.
  • Embodiment 82 The compound of embodiment 81, wherein R 11 and R 12 are each methyl.
  • Embodiment 83 The compound of any one of embodiments 72 to 82, wherein each X is independently selected from — (CH2)sOC(O) — , — (CH2)sC(O)O — , — (CH2)sOC(O)O — , wherein s is 0-6.
  • Embodiment 84 The compound of any one of embodiments 72 to 82, wherein each s is Embodiment 85.
  • Embodiment 86 The compound of any one of embodiments 72 to 82, wherein each s is 3.
  • Embodiment 88 The compound of any one of embodiments 72 to 86, wherein at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms optionally further comprising one or more cyclic group.
  • Embodiment 89 The compound of embodiment 88, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • Embodiment 90 The compound of embodiment 72, wherein the compound is of formula (IIIC): wherein:
  • R 11 and R 12 are each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
  • Y is selected from — O — , and — C(R 10 )2 — ; each s is independently 0, 1 or 2;
  • W is — O — , and — C(R 10 )2 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R 7 )2, and -(CH 2 )tJ(CH 2 )u, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and each of t and u are 1-10.
  • Embodiment 91 The compound of any one of embodiments 1 to 90, wherein each R is independently
  • Cy A and Cy B is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
  • R x and R y is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C20 aliphatic group; and r, p, and q is each independently an integer from 0 to 20.
  • Embodiment 92 The compound of embodiment 91, wherein at least one R comprises a
  • each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
  • Embodiment 93 A lipid nanoparticle comprising an ionizable lipid compound according to any one of embodiments 1 to 92.
  • Embodiment 94 The lipid nanoparticle of embodiment 93, further comprising a neutral lipid and a lipid capable of reducing aggregation.
  • Embodiment 95 The lipid nanoparticle of embodiment 94, wherein the neutral lipid comprises a phospholipid.
  • Embodiment 96 The lipid nanoparticle of embodiment 94 or 95, wherein the neutral lipid comprises cholesterol.
  • Embodiment 97 The lipid nanoparticle of embodiment 96, comprising: a. a nucleic acid, b. an ionizable lipid, c. a phospholipid, d. cholesterol, and e. a lipid capable of reducing aggregation.
  • Embodiment 98 The lipid nanoparticle of embodiment 97, wherein the nucleic acid comprises DNA.
  • Embodiment 99 The lipid nanoparticle of embodiment 98, wherein the nucleic acid comprises RNA.
  • Embodiment 100 The lipid nanoparticle of embodiment 98, wherein the nucleic acid comprises DNA and RNA.
  • Embodiment 101 The lipid nanoparticle of embodiment 100, wherein the RNA is selected from mRNA, gRNA, and siRNA.
  • Embodiment 102 The lipid nanoparticle of any one of embodiments 97 to 101, wherein the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG), and derivatives thereof.
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PG phosphatidylglycerol
  • Embodiment 103 The lipid nanoparticle of embodiment 102, wherein the phospholipid is a phosphatidylethanolamine (PE).
  • PE phosphatidylethanolamine
  • Embodiment 104 The lipid nanoparticle of embodiment 103, wherein the phospholipid is a phosphatidylcholine (PC).
  • PC phosphatidylcholine
  • Embodiment 105 The lipid nanoparticle of any one of embodiments 97 to 104, wherein the phospholipid comprises hydrocarbon chains each independently having 12-24 carbons.
  • Embodiment 106 The lipid nanoparticle of embodiment 105, wherein the phospholipid comprises hydrocarbon chains each independently having 16-20 carbons.
  • Embodiment 107 The lipid nanoparticle of embodiment 105 or 106, wherein the hydrocarbon chains are saturated.
  • Embodiment 108 The lipid nanoparticle of embodiment 105 or 106, wherein the hydrocarbon chains are unsaturated and/or further comprise a carbocyclyl.
  • Embodiment 109. The lipid nanoparticle of embodiment 108, wherein the hydrocarbon chains each independently comprise 1-4 double bonds.
  • Embodiment 110 The lipid nanoparticle of any one of embodiments 94 to 109, wherein the phospholipid comprises two different hydrocarbon chains.
  • Embodiment 111 The lipid nanoparticle of embodiment 103, wherein the phospholipid comprises l,2-dioleyl-sn-glycero-3 -phosphoethanolamine (DOPE).
  • DOPE l,2-dioleyl-sn-glycero-3 -phosphoethanolamine
  • Embodiment 112. The lipid nanoparticle of embodiment 103, wherein the phospholipid comprises l-stearoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (SOPE).
  • SOPE l-stearoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine
  • Embodiment 113 The lipid nanoparticle of embodiment 104, wherein the phospholipid comprises l,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine (A9A9-Cis PC).
  • Embodiment 114 The lipid nanoparticle of embodiment 106, wherein the lipid nanoparticle comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • DSPC l,2-distearoyl-sn-glycero-3-phosphocholine
  • Embodiment 115 The lipid nanoparticle of embodiment 104, wherein the lipid nanoparticle comprises l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC).
  • DOPC l,2-dioleoyl-sn-glycero-3 -phosphocholine
  • Embodiment 116 The lipid nanoparticle of any one of embodiments 103 to 115 wherein the lipid capable of reducing aggregation is a PEG-lipid.
  • Embodiment 117 The lipid nanoparticle of embodiment 116, wherein the PEG- lipid is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG- DMG[2K]) or PEG-1, 2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG- DSG[2K]).
  • Embodiment 118 The lipid nanoparticle of any one of embodiments 94 to 117, further comprising a targeting ligand.
  • Embodiment 119 The lipid nanoparticle of embodiment 118, wherein the targeting ligand comprises GalNAc.
  • Embodiment 120 The lipid nanoparticle of embodiment 118 or 119, wherein the targeting ligand is linked to the lipid capable of reducing aggregation.
  • Embodiment 121 The lipid nanoparticle of embodiment 120, wherein the lipid capable of reducing aggregation is PEG-l,2-distearoyl-rac-glycero-3- methylpolyoxyethylene 2000 (PEG-DSG[2K]).
  • Embodiment 122 The lipid nanoparticle of any one of embodiments 94 to 121, wherein the N/P ratio (ratio of moles of the amine groups of cationic lipids to those of the phosphate ones of DNA) is from 5 to 30.
  • Embodiment 123 The lipid nanoparticle of embodiment 122, wherein the N/P ratio is 7.
  • Embodiment 124 The lipid nanoparticle of embodiment 122, wherein the N/P ratio is 14.
  • Embodiment 125 The lipid nanoparticle of embodiment 122, wherein the N/P ratio is 28.
  • Embodiment 126 The lipid nanoparticle of any one of embodiments 94 to 125, comprising: a. an ionizable lipid at 40 to 60 mol % of the total lipid present; b. a phospholipid at 6 to 20 mol % of the total lipid present; c. cholesterol at 35 to 45 mol % of the total lipid present; and d. a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
  • Embodiment 127 The lipid nanoparticle of any one of embodiments 94 to 125, comprising: a. an ionizable lipid at 40 to 60 mol % of the total lipid present; b. a phospholipid at 10 to 20 mol % of the total lipid present; c. cholesterol at 35 to 45 mol % of the total lipid present; and d. a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
  • Embodiment 128 The lipid nanoparticle of any one of embodiments 94 to 125, comprising: a. e) an ionizable lipid at 40 to 49 mol % of the total lipid present; b. f) a phospholipid at 10 to 20 mol % of the total lipid present; c. g) cholesterol at 35 to 45 mol % of the total lipid present; and d. h) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
  • Embodiment 129 A pharmaceutical composition comprising a lipid nanoparticle of any one of embodiments 94 to 128 and a pharmaceutically acceptable excipient, carrier, or diluent.
  • Embodiment 130 A method for delivering a nucleic acid into a cell, the method comprising contacting the cell with a lipid nanoparticle of any one of embodiments 94 to 128.
  • Embodiment 132 The method according to embodiment 130, wherein the cell is in vivo.
  • Embodiment 133 A method for delivering a nucleic acid for in vivo production of target protein, the method comprising: administering systemically to a subject in need thereof a pharmaceutical composition of embodiment 129, wherein the nucleic acid encodes a target protein and is encapsulated within the lipid nanoparticles, and the administering of the pharmaceutical composition results in the prolonged stable expression of the target protein.
  • LNP formulation LNPs encapsulating nucleic acid payloads are prepared by mixing an organic solution of lipids with an aqueous solution of nucleic acid (e.g., DNA only, mRNA only, or DNA/mRNA mixtures) as described in Prud’Neill et al. (J Pharm Sci 2018). Briefly, the lipidic excipients mixture (ionizable lipid, helper lipid, cholesterol, PEG- lipid and potentially other targeting moieties) is dissolved in an organic solvent. An aqueous solution of the nucleic acid is prepared in a low pH buffer of range pH 3.0 - 4.0.
  • nucleic acid e.g., DNA only, mRNA only, or DNA/mRNA mixtures
  • the lipid mixture is then mixed with the aqueous nucleic acid solution at a flow ratio of 1 :3 (V/V) using a commercially available mixer device.
  • the resulting solution is immediately diluted with a buffer pH range of 5.0-6.5.
  • the diluted LNP is subjected to dialysis purification against a secondary buffer with the pH range of 7.0-8.0.
  • the LNP solution is concentrated by using 100,000 MWCO Amicon Ultra centrifuge tubes (Millipore Sigma) followed by filtration through 0.2 pm PES sterilizing-grade filter. Particle size is determined by dynamic light scattering (Horiba nanoPartica SZ-100). Encapsulation efficiency is calculated by using Quant-it RiboGreen assay kit.
  • EPO and cytokine detection in serum Blood is collected via a retro-orbital bleed into serum separator tubes and processed to serum. The serum samples may be stored at -80C from collection until analysis.
  • the serum levels of human EPO protein driven by expression from the DNA payload are quantified using the U-PLEX Human EPO Assay from MSD according to the manufacturer’s instructions.
  • the serum levels of mouse cytokines resulting from exposure to DNA-LNPs were quantified using the Mouse Prolnflammatory 7-Plex Tissue Culture Kit from MSD according to the manufacturer’s instructions.
  • the plasma samples may be stored at -80C from collection until analysis.
  • the plasma levels of human FIX following administration of LNPs were quantified using a U-Plex assay on the MSD platform. Briefly, a monoclonal mouse anti-human FIX antibody (Prolytix, clone AHIX-5041) was conjugated to biotin and used as the capture reagent on streptavidin-coated plates.
  • a polyclonal goat anti-human FIX antibody (Cedarlane, clone CL20040AP) was conjugated to Sulfo-TAG and used as the detection reagent with the standard setup for quantification of electrochemiluminescence (ECL) signal using the QuickPlex SQ 120MM instrument from MSD.
  • ECL electrochemiluminescence
  • Pooled normal human plasma (Affinity Biologicals, FRNCP0125), which is a pool of normal citrated human plasma collected from a minimum of 20 donors, was used to generate a standard curve and calculate % of normal human FIX levels. The assay was confirmed to be specific for human FIX and not to cross-react with mouse FIX, demonstrating very low levels of background in untreated mouse plasma samples.
  • silica gel type: ZCX-2, 100-200 mesh, 20 w/w.
  • silica gel type: ZCX-2, 100-200 mesh, 10 w/w.
  • the product was purified using CombiFlash, then eluted with CH2CI2 / MeOH (9:1) (gradient from 100:0 to 90: 10, collected every 200 ⁇ 10 mL).
  • the reaction solution was decompressed and vacuum concentrated, 200 mL DCM was added, and washed with 5% citric acid solution (3*100), then washed with saturated salt water three times, dried with anhydrous sodium sulfate and concentrated.
  • the organic solvent was removed under reduced pressure and basified to pH 8 with saturated Na2CCh (aq.).
  • the resulting mixture was extracted with heptane (3 xl50 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase HP-flash chromatography with the following conditions: column, XSelect CSH Prep C18 5 pm; mobile phase, B: C LCN, A: Water (0.1% TFA), 50% to 95% gradient in 15 min; Flow: 50 mL/min: detector, ELSD. The organic solvent was removed under reduced pressure and basified to pH 8 with saturated Na2CCh (aq.). The aqueous layer was extracted with heptane (3x100 mL). The resulting mixture was concentrated under reduced pressure.
  • Trideca-l,12-dien-7-ol (10 g, 50.934 mmol, 1 equiv) and Toluene (200 mL) at room temperature.
  • NaH 4.00 g, 166.554 mmol, 3.27 equiv
  • the resulting mixture was stirred for an additional overnight at 85°C.
  • (3- chloropropyl) dimethylamine hydrochloride (15.28 g, 101.868 mmol, 2 equiv) in portions at 80°C. The resulting mixture was stirred for an additional 8 h at 80°C.
  • the resulting mixture was stirred for an additional 16 h at room temperature.
  • the resulting mixture was diluted with water (50 mL).
  • the resulting mixture was extracted with heptane (3x20 mL).
  • the combined organic layers were washed with sat. Na2CCh (2x20 mL), Me0H/H20 (4: 1, 5x20 mL), H2O (3x20 mL) and brine (20 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.
  • the residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeCN in Water (0.1% TFA), 35% to 70% gradient in 10 min; detector, Ms.
  • the resulting mixture was stirred for 18 h at 45°C under N2 atmosphere.
  • the resulting mixture was diluted with DCM (120 mL).
  • the combined organic layers were washed with 0.05M HC1 (1x100 mL) and Brine (1x200 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.
  • the residue was dissolved in DCM (50 mL) and 30 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w./w.) was added. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C.
  • silica gel type: ZCX-2, 100-200 mesh, 20.00 w/w.
  • the last step prepared dry silica gel which absorbed the reaction mixture.
  • Eluted with PE / EA (5: 1) grade from 20: 1 to 4: 1, collected every 200 ⁇ 10 mL).
  • silica gel type: ZCX-2, 100-200 mesh, 20.00 w/w.
  • the last step prepared dry silica gel which absorbed the reaction mixture.
  • Eluted with CH2CI2 / MeOH (20: 1) gradient from 50: 1 to 10: 1, collected every 200 ⁇ 10 mL).
  • the resulting mixture was filtered, the filter cake was washed with EA (2x100 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in Heptane (150 mL). The resulting mixture was washed with 2x100 mL of ACN/H2O (5: 1, 50 mL), 1x100 mL of ACN (50 mL). The heptane phase was concentrated under reduced pressure.
  • the reaction was quenched by the addition of sat. NH4Q (aq.) (300 mL) at 0°C.
  • the resulting mixture was extracted with Heptane (3 x 200 mL).
  • the combined organic layers were washed with aqueous sat. Na2CCh (2x50 mL), water (2x50 mL), MeOH / H2O (4: 1, 4x50 mL), water (2x50 mL) and brine (1x50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.
  • the resulting mixture was concentrated under reduced pressure.
  • the resulting mixture was diluted with heptane (500 mL).
  • the solid was filtered out; the filter cake was washed with heptane (2x50 mL).
  • the filtrate was washed with 2x200 mL of water, 3x200 mL of water/MeOH (1 :4), 2x200 mL of aqueous citric acid (5% w/w), 2x200 mL of sat. NaHCCh and brine (200 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.
  • the resulting mixture was stirred for an additional 2 h at 40°C.
  • the resulting mixture was diluted with water (20 mL).
  • the resulting mixture was extracted with CH2CI2 (3 x 20 mL).
  • the combined organic layers were washed with brine (1 x 20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.
  • the resulting solution was diluted with 250 mL of DCM.
  • the resulting solution was washed with 1 x 200 mL of citric acid (5%, aq.), 2 x 200 mL of water, and 1 x 200 mL of brine, dried with anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.
  • Example 21 Synthesis of 3-[3-(dimethylamino)propoxy]-l,4,5-tris( ⁇ [3-(octahydro-lH- inden-2-yl)propanoyl]oxy ⁇ )pentan-2-yl 3-(octahydro-lH-inden-2-yl)propanoate oxylic acid
  • reaction mixture was stirred at 20 °C for 10 min, NalCb (3.09 g, 14.430 mmol, 5.0 equiv) and 2,6-lutidine (1.55 g, 14.430 mmol, 5.0 equiv) was added at 20 °C.
  • the reaction mixture was stirred at 20 °C for 18 h.
  • the resulting mixture was diluted with EA (200 mL).
  • the resulting mixture was washed with 3 x 60 mL of water, brine (60 mL).
  • the resulting solution was dried over anhydrous Na2SO4.
  • the resulting mixture was concentrated under reduced pressure.
  • the resulting mixture was washed with 3x28 mL of water, brine (28 mL), dried over anhydrous Na2SO4. After filtration. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeCN in water (0.05% CF3COOH), 30% to 90% gradient in 26 min; detector, ELSD. The collect liquid was added 28 mL NaHCOs (2M, aq). The resulting mixture was extracted with n-Heptane (3 x 70 mL). The combined organic layers were washed with 3 x 28 mL of MeOH/H2O(4/l), water (28 mL).
  • L-24 is synthesized in a manner similar to L-18, substituting 4-oxoheptanedioic acid instead of 5-oxononanedioic acid.
  • Example 25 Synthesis of 3-(3-(dimethylamino)propoxy)-l,5-bis((3-(octahydro-lH- inden-2-yl)propanoyl)oxy)pentane-2,4-diyl bis(decanoate) (L-25)
  • L-25 is synthesized in a manner similar to L-21, but instead the xylitol core is acylated first with 3-(octahydro-lH-inden-2-yl)propanoic acid at primary then the secondary alcohols are acylated with decanoyl chloride to provide the final product.
  • L-26 is synthesized in a manner similar to L-5 and L-23.
  • Example 27 Preparation and analysis of lipid nanoparticle formulations with varying ionizable lipids
  • DNA payloads were formulated into lipid nanoparticles (LNPs) comprising an ionizable lipid, as described above and detailed in Fig. 1 A.
  • LNPs lipid nanoparticles
  • Wild type female BALB/c mice (approximately 7-8 weeks old) were dosed once by a single i.v. bolus injection into the tail vein at 5 mL/kg body weight.
  • the DNA-LNPs were administered at 1 mg/kg based on the weight of the DNA payload.
  • blood was collected via a retro-orbital bleed and EPO levels in serum determined as presented in FIG. IB.
  • the DNA-LNP formulation with ionizable lipid L-3 produced robust EPO expression levels, comparable to the exemplary ionizable lipid ALC-0315 (FIG. IB).
  • the DNA-LNP formulation with ionizable lipid L-2 produced measurable but somewhat lower EPO expression levels, as did the exemplary ionizable lipid MC3 (FIG. IB).
  • blood was collected via a retro-orbital bleed and IL-6 levels in serum determined as presented in FIG. 1C.
  • the DNA-LNP formulations with ionizable lipids L-2 and L-3 generated similar serum levels of IL-6 compared to the exemplary ionizable lipid MC3, which were higher than the IL-6 levels generated by formulations with the exemplary ionizable lipid ALC-0315 (FIG. 1C).
  • Example 28 Preparation and analysis of further LNP formulations with varying additional ionizable lipids
  • DNA payloads were formulated into lipid nanoparticles (LNPs) comprising an ionizable lipid, as described above.
  • LNPs lipid nanoparticles
  • the LNP formulations using DSPC as the phospholipid are detailed in Fig. 2A.
  • the LNP formulations using DOPE as the phospholipid are detailed in Fig. 2B.
  • Wild type female BALB/c mice (approximately 8 weeks old) were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight.
  • the DNA-LNPs were administered at 1 or 0.3 mg/kg based on the weight of the DNA payload. Seven days after dosing, blood was collected via a retro-orbital bleed and EPO levels in serum determined as presented in FIG. 2C.
  • the DNA-LNP with ionizable lipid L-15 When formulated with DSPC, the DNA-LNP with ionizable lipid L-15 produced robust EPO expression levels, higher than the exemplary ionizable lipids ALC- 0315, MC3, and LP01, and comparable to the exemplary ionizable lipids SM102 and ARCT (FIG. 2C).
  • the DNA-LNP with ionizable lipid L-15 produced robust EPO expression levels, higher than the exemplary ionizable lipids MC3, LP01, and SM102, and comparable to the exemplary ionizable lipids ALC-0315 and ARCT (FIG. 2C).
  • the DNA-LNPs with ionizable lipid L-9 When formulated with DSPC or DOPE, the DNA-LNPs with ionizable lipid L-9 produced somewhat lower EPO expression levels than L-15, comparable to several of the exemplary ionizable lipids (FIG. 2C). When formulated with DSPC or DOPE, the DNA-LNPs with ionizable lipid L-5 produced substantially lower EPO expression levels, below the levels of the exemplary ionizable lipids (FIG. 2C). Four hours after dosing, blood was collected via a retro-orbital bleed and IL-6 levels in serum determined as presented in FIG. 2D.
  • the DNA-LNPs with ionizable lipids L-5, L-15, and L-9 generated similar serum levels of IL-6 compared to the exemplary ionizable lipid ALC-0315, which were lower than the IL-6 levels generated by formulations with the exemplary ionizable lipids MC3, LP01, SMI 02, and ARCT (FIG. 2D).
  • the DNA- LNPs with ionizable lipids L-5, L-15, and L-9 generated serum levels of IL-6 that were generally similar compared to all the exemplary ionizable lipids (FIG. 2D).
  • the DNA- LNPs with ionizable lipids L-5, L-15, and L-9 generated similar, or lower, serum levels of cytokines compared to the exemplary ionizable lipid ALC-0315, which were typically lower than the cytokine levels generated by formulations with the exemplary ionizable lipids MC3, LP01, SMI 02, and ARCT (FIG. 2E).
  • the DNA-LNPs with ionizable lipids L-5, L-15, and L-9 generated serum levels of cytokines that were generally similar compared to all the exemplary ionizable lipids (FIG. 2F).
  • Example 29 Preparation and analysis of further LNP formulations with varying additional ionizable lipids
  • DNA payloads were formulated into lipid nanoparticles (LNPs) comprising an ionizable lipid, as described above.
  • LNPs lipid nanoparticles
  • the LNP formulations using DSPC as the phospholipid are detailed in Fig. 3 A.
  • the LNP formulations using DOPE as the phospholipid are detailed in Fig. 3B.
  • Wild type female BALB/c mice (approximately 8 weeks old) were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight.
  • the DNA-LNPs were administered at 1 or 0.3 mg/kg based on the weight of the DNA payload.
  • blood was collected via a retro-orbital bleed and EPO levels in serum determined as presented in FIG. 3C.
  • the DNA-LNP with ionizable lipid L-12 produced robust EPO expression levels, higher than the exemplary ionizable lipids ALC- 0315 and ssOP, and comparable to the exemplary ionizable lipid A9 (FIG. 3C).
  • the DNA-LNPs with ionizable lipids L-13 and L-14 produced robust EPO expression levels, higher than the exemplary ionizable lipid ssOP, and comparable to the exemplary ionizable lipid ALC-0315 (FIG. 3C).
  • the DNA- LNPs with ionizable lipids L-12, L-13, and L-14 produced robust EPO expression levels, higher than the exemplary ionizable lipids ALC-0315 and ssOP, and comparable to the exemplary ionizable lipid A9 (FIG. 3C).
  • IL-6 levels in serum determined as presented in FIG. 3D.
  • the DNA-LNP with ionizable lipid L-14 resulted in substantially lower IL-6 serum levels compared to the other formulations tested (FIG. 3D).
  • the DNA-LNPs with ionizable lipids L-12 and L-13 generated similar serum levels of IL-6 compared to the exemplary ionizable lipid A9, which were lower than the IL-6 levels generated by formulations with the exemplary ionizable lipids ALC-0315 and ssOP (FIG. 3D).
  • the DNA-LNPs with ionizable lipids L- 12 and L-14 resulted in substantially lower IL-6 serum levels compared to the other formulations tested (FIG. 3D).
  • the DNA-LNP with ionizable lipid L-13 generated similar serum levels of IL-6 compared to the exemplary ionizable lipids ALC-0315, A9, and ssOP (FIG. 3D).
  • blood was collected via a retro-orbital bleed and serum levels of multiple cytokines determined as presented in FIG. 3E and FIG. 3F.
  • the DNA-LNPs with ionizable lipids L-12, L-13, and L-14 produced generally similar, or lower, serum levels of cytokines compared to the exemplary ionizable lipids ALC-0315, A9, and ssOP, although for several cytokines L-14 produced the lowest levels (FIG. 3F).
  • the DNA-LNPs with ionizable lipids L-17, L-19, and L-21 produced generally similar serum levels of IL-6 compared to the exemplary ionizable lipids ALC-0315, ARCT, and CL1 (FIG. 5C).

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Abstract

L'invention concerne de nouveaux lipides ionisables. L'invention concerne également de nouvelles compositions de nanoparticules lipidiques pour l'administration de matériau d'acide nucléique à des cellules in vitro et in vivo avec des profils pharmacocinétiques différents et améliorés par rapport à ce qui est typiquement observé dans l'état de la technique. L'invention concerne en outre des méthodes d'utilisation des compositions en recherche et en tant qu'agents thérapeutiques.
EP23892517.6A 2022-11-16 2023-11-15 Lipides ionisables et compositions de nanoparticules lipidiques pour l'administration d'acides nucléiques Pending EP4619378A2 (fr)

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