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  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
  • C07C275/04—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms
  • C07C275/06—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton
  • C07C275/14—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—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
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  • 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
  • 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
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  • 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
  • C07C271/16—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 singly-bound oxygen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
  • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
  • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D211/40—Oxygen atoms
  • C07D211/44—Oxygen atoms attached in position 4
  • C07D211/46—Oxygen atoms attached in position 4 having a hydrogen atom as the second substituent in position 4
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
  • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
  • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
  • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
  • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain

Definitions

• Lipid formulations have been used in the laboratory for the delivery of nucleic acids into cells.
• Early formulations based on the cationic lipid 1 ,2-dioleoyl-3- trimethylammonium propane (DOTAP) and the ionizable, fusogenic lipid dioleoylphosphatidyl ethanolamine (DOPE) had a large particle size and were problematic when used in vivo, exhibiting too rapid clearance, tropism for the lung, and toxicity.
• DOTAP cationic lipid 1 ,2-dioleoyl-3- trimethylammonium propane
• DOPE fusogenic lipid dioleoylphosphatidyl ethanolamine
• Lipid nanoparticles comprising ionizable cationic lipids have been developed to address these issues to the extent that RNA based products, such as the siRNA ONPATTRO® and two mRNA-based SARS-CoV-2 vaccines have received regulatory approval and entered the market. There is limited ability to control which tissues or cells take up the LNP once administered. LNP administered intravenously are taken up primarily in the liver, lung, or spleen depending to a significant degree on net charge and particle size. It is possible to direct >90% of LNP to the liver by a combination of formulation and intravenous administration. Intramuscular administration can provide a clinically useful level of local delivery and expression.
• LNP can be redirected to other tissues or cell types by conjugating a binding moiety with specificity for the target tissue or cell type, for example, conjugating a polypeptide containing an antigen binding domain from an antibody, to the LNP. Nonetheless, avoiding uptake by the liver remains a challenge. Moreover, with current systems only a minor portion of the encapsulated nucleic acid is successfully delivered to the cells of interest and into the cytoplasm. Current formulations may release only 2-5% of the administered RNA into the cytoplasm (see for example Gilleron et al., Nat. Biotechnol. 31 :638-646, 2013, and Munson et aL, Common. Biol. 4:211 -224, 2021 ).
• this disclosure provides ionizable lipids and lipid nanoparticles to satisfy an urgent need in the field.
• Certain aspects of the disclosure relate to an ionizable cationic lipid having a structure selected from the group consisting of Formula 1 , Formula 2, and Formula 3.
• LNP lipid nanoparticle
• the LNP may further comprise one or more of a phospholipid, a sterol, a co-lipid, a PEG-lipid, or combinations thereof.
• Examples of the phospholipids includes, without limitation, dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), 1 ,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), and combinations thereof.
• Examples of the sterol include, without limitation, cholesterol, campesterol, sitosterol, stigmasterol, and combinations thereof.
• Examples of the co-lipid include, without limitation, cholesterol hemisuccinate (CHEMS), and a quaternary ammonium headgroup containing lipid.
• Examples of the quaternary ammonium headgroup containing lipid include, without limitation, 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1 -(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA), 3£-(N- (N',N'-Dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), and combinations thereof.
• DOTAP 1,2-dioleoyl-3-trimethylammonium propane
• DOTMA N-(1 -(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
• DC-Chol 3£-(N- (N',N'-Dimethylaminoethane)car
• Examples of PEG-lipid may comprise a PEG moiety of 1000-5000 Da molecular weight (MW), and/or fatty acids with a fatty acid chain length of C14-C18.
• Examples of the PEG-lipid include, without limitation, DMG-PEG2000 (1 ,2-dimyristoyl- rglycero-3-methoxypolyethylene glycol-2000), DPG-PEG2000 (1 ,2-dipalmitoyl- glycero-3-methoxypolyethylene glycol-2000), DSG-PEG2000 (1 ,2-distearoyl-glycero- 3-methoxypolyethylene glycol-2000), DGG-PEG2000 (1 ,2-dioleoyl-glycero-3- methoxypolyethylene glycol-2000), DMPE-PEG200 (1 ,2-dimyristoyl-glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPE-PEG2000 (1 ,2- dip
• the PEG-lipid comprises an optically pure glycerol moiety.
• the LNP further comprises a functionalized PEG-lipid.
• the functionalized PEG-lipid comprises fatty acids with a fatty acid chain length of C16-C18.
• the functionalized PEG-lipid comprise a dipalmitoyl lipid or a distearoyl lipid.
• the LNP comprises 40 to 60 mol% ionizable cationic lipid. In certain embodiments, the LNP comprises 7 to 30 mol% phospholipid. In certain embodiments, the LNP comprises 20 to 45 mol% sterol. In certain embodiments, the LNP comprises 1 to 30 mol% co-lipid. In certain embodiments, the LNP comprises 0 to 5 mol% PEG-lipid. In certain embodiments, the LNP comprises 0.1 to 5 mol% functionalized PEG-lipid.
• the LNP further comprises a nucleic acid (e.g., mRNA).
• a nucleic acid e.g., mRNA
• the weight ratio of total lipid to nucleic acid is 10:1 to 50:1.
• aspects of the disclosure relate to a method of delivering a nucleic acid into a cell comprising contacting the cell with one or more LNP’s disclosed herein, wherein at least some of the LNP’s comprise the nucleic acid.
• Figures 1 A-1 F depict a synthetic scheme for compounds having a structure of Formula 1 .
• Figure 1A shows the synthesis starting with readily available reagents through intermediate I -f A.
• Figure 1 D shows the synthesis of intermediates l-c, l-d, l-e, and l-f, which are embodiments of Formulas l-cA, l-dA, l-eA, and l-fA, respectively, wherein p is 1 , n is 1 , and R is C9 alkyl straight chain.
• Figure 1 F shows the synthetic path from
• Figures 2A-2F depict a synthetic scheme for compounds having a structure of Formula 2.
• Figure 2A shows the synthesis starting with readily available reagents through intermediate ll-hA.
• Figure 2D shows the synthesis of intermediates ll-c, ll-d, I l-e, I l-f, I l-g, and I l-h, which are embodiments of Formulas H-cA, ll-dA, H-eA, ll-fA, II- gA, and H-hA, respectively, wherein p is 1 , n is 1 , and R is C9 alkyl straight chain when applicable.
• Figures 3A-3D depict a synthetic scheme for compounds having a structure of Formula 3.
• Figure 3C shows the synthesis starting with readily available reagents to intermediates lll-a, lll-b, lll-c, and Compound A-9, which are embodiments of Formulas IH-aA, IH-bA, HI-cA, and Formula 3, respectively, wherein p is 1 , n is 2, and R c is C9 alkyl straight chain when applicable.
• Figures 4A-B depict synthetic schemes for reagents that may be used to make polyethylene glycol-containing lipid head groups.
• Figure 4A depicts the synthesis of an embodiment of XR125 in which m is 2 and 0 is 3 (see Table 3).
• Figure 4B depicts the synthesis of an embodiment of XR126 in which o is 3 (see Table 3).
• Figures 5A-C depict the viability (5A), frequency of transfection (5B), and level of expression as geometric mean fluorescence intensity (gMFI) of the transfected cells (5C) for HEK293F cells transfected with mCherry mRNA encapsulated in LNP in which the ionizable cationic lipid was one of Compounds A-2, A-11, A-12, A-13, A-14, or A-15.
• gMFI geometric mean fluorescence intensity
• Figure 6 depicts the frequency and level of expression, as determined by flow cytometry, of mCherry mRNA transfected in vitro into mouse splenic T cells by CD5-targeted lipid nanoparticles incorporating the indicated ionizable cationic lipids A- 2, A-1 1 , A-12, A-13, A-14, and A-15, respectively.
• Expression level is presented as the mean fluorescence intensity (MFI; geometric mean) of the positive peak in the flow cytometry histogram and transfection rate is the proportion of CD3 + cells in the positive peak.
• MFI mean fluorescence intensity
• Figure 7 depicts the frequency and level of expression, as determined by flow cytometry, of mCherry mRNA transfected in vivo into mouse splenic T cells by CD5-targeted lipid nanoparticles incorporating the indicated ionizable cationic lipids A- 2, A-1 1 , A-12, A-13, A-14, and A-15, respectively.
• Expression level is presented as the mean fluorescence intensity (MFI; geometric mean) of the positive peak in the flow cytometry histogram and transfection rate is the proportion of CD3 + cells in the positive peak.
• MFI mean fluorescence intensity
• Figure 8 depicts a conceptual biodegradation scheme for Compound A-11 (above the line) and the starting compound and end products of biodegradation (below the line).
• the disclosed ionizable cationic lipids, and particularly Compounds of Formula 1 may undergo biodegradation according to such a conceptual scheme, without being bound to any particular theory.
• the instant disclosure provides ionizable cationic lipids, methods for synthesizing them, as well as intermediates useful in synthesis of these lipids and methods of synthesizing the intermediates.
• the instant disclosure provides ionizable cationic lipids of this disclosure as a component of lipid nanoparticles (LNPs), which LNPs can be used for the delivery of nucleic acids into cells in vivo or ex vivo.
• LNP compositions are also disclosed herein, including LNPs comprising a functionalized PEG-lipid to enable conjugation of a binding moiety to generate targeted LNPs (tLNPs), that is LNPs containing a binding moiety that directs the tLNP to a desired tissue or cell type. Also disclosed herein are methods of delivering a nucleic acid into a cell comprising contacting the cell with a LNP or tLNP of this disclosure.
• any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
• any number range of this disclosure relating to any physical feature, such as polymer subunits, size, or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
• numerical ranges are inclusive of their recited endpoints, unless specifically stated otherwise.
• Derivative refers to a chemically or biologically modified version of a compound that is structurally similar to a parent compound and (actually or theoretically) derivable from that parent compound.
• a “derivative” differs from an "analogue” in that a parent compound may be the starting material to generate a "derivative,” whereas the parent compound may not necessarily be used as the starting material to generate an “analogue.”
• a derivative may have different chemical or physical properties than the parent compound. For example, a derivative may be more hydrophilic or hydrophobic, or it may have altered reactivity as compared to the parent compound.
• Alkyl refers to a saturated hydrocarbon moiety, that is an alkane lacking one hydrogen leaving a bond that connects to another portion of an organic molecule.
• hydrogens are unsubstituted.
• one or more hydrogens of the alkyl group may be substituted with the same or different substituents.
• Alkenyl refers to a hydrocarbon moiety with one or more carbon-carbon double bonds but that is otherwise saturated. In some embodiments, hydrogens are unsubstituted. In other embodiments, one or more hydrogens of the alkenyl group may be substituted with the same or different substituents.
• Alkynoic refers to a carboxylic acid moiety comprising one or more carboncarbon triple bonds. In some embodiments, hydrogens are unsubstituted. In other embodiments, one or more hydrogens of the alkynoic group may be substituted with the same or different substituents.
• Amide refers to a carboxylic acid derivative comprising a carbonyl group of a carboxylic acid bonded to an amine moiety.
• Aryl refers to an aromatic or heteroaromatic ring lacking one hydrogen leaving a bond that connects to another portion of an organic molecule.
• aryl include, without limitation, phenyl, naphthalenyl, pyridine, pyrimidine, pyrazine, pyrrole, furan, thiophene, imidazole, thiazole, oxazole, and the like.
• Aryl-alkyl refers to a moiety comprising one or more aryl rings and one or more alkyl moieties.
• the position of the one or more aryl rings can vary within the alkyl portion of the moiety.
• the one or more aryl rings may be at an end of the one or more alkyl moieties, be fused into the carbon chain of the one or more alkyl moieties, or substitute one or more hydrogens of one or more alkyl moieties; and the one or more alkyl moieties may substitute one or more hydrogens of the one or more aryl rings.
• Branched alkyl is a saturated alkyl moiety wherein the alkyl group is not a straight chain. Alkyl portions such as methyl, ethyl, propyl, butyl, and the like, can be appended to variable positions of the main alkyl chain. In some embodiments, there is a single branch; while in other embodiments, there are multiple branches.
• Branched alkenyl refers to an alkenyl group comprising at least one branch off the main chain which may be formed by substituting one or more hydrogens of the main chain with the same or different alkyl groups, e.g., without limitation, methyl, ethyl, propyl, butyl, and the like.
• a branched alkenyl is a single branch structure, while in other embodiments, a branched alkenyl may have multiple branches.
• Straight chain alkyl is a non-branched, non-cyclic version of the alkyl moiety described above.
• Straight chain alkenyl is a non-branched, non-cyclic version of the alkenyl moiety described above.
• Cycloalkyl refers to a moiety which is a cycloalkyl ring of 3-12 carbons.
• a cycloalkyl is a single ring structure; while in other embodiments, a cycloalkyl may have multiple rings.
• Cycloal kyl-alkyl refers to a moiety which contains one or more cycloalkyl rings of 3-12 carbons, and one or more alkyl moieties.
• the position of the cycloalkyl ring can vary within the alkyl portion of the moiety.
• the one or more cycloalkyl rings may be at an end of the one or more alkyl moieties, be fused into the carbon chain of the one or more alkyl moieties, or substitutes one or more hydrogens of one or more alkyl moieties; and the one or more alkyl moieties may substitute one or more hydrogens of the one or more cycloalkyl rings.
• the cycloalkyl ring is a single ring structure; while in other embodiments, a cycloalkyl-al ky I may have multiple rings.
• Ether refers to an oxygen atom attached to 2 carbon-based moieties that are the same or different.
• Head group refers to the hydrophilic or polar portion of a lipid.
• Imide refers to a moiety comprising a nitrogen bond to two carbonyl groups.
• Sterol refers to a subgroup of steroids that contain at least one hydroxyl (OH) group.
• examples of sterols include, without limitation, cholesterol, ergosterol, p- sitosterol, stigmasterol, stigmastanol, 20-hydroxycholesterol, 22-hydroxycholesterol, and the like.
• Ionizable cationic lipids useful as a component of lipid nanoparticles for the delivery of nucleic acids, including DNA, mRNA, or siRNA into cells are disclosed.
• the ionizable cationic lipids have a c-pKa from 8 to 1 1 and cLogD from 9 to 18 or 1 1 -14. These ranges can lead to a measured pKa in the LNP or tLNP of 6 to 7 facilitating ionization in the endosome. In some embodiments, somewhat greater basicity may be desirable and can be obtained from ionizable cationic lipids with c-pKa and cLogD in the stated ranges.
• the c-pKa is about 8, about 9, about 10, or about 1 1 , or in a range bound by any pair of these values.
• cLogD is about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, or in a range bound by any pair of these values.
• Some embodiments specifically include one or more species or subgenera based on specific choices of R, X, Y, m, n, 0, p, and/or carbon chain length, structure, or saturation. Other embodiments specifically exclude one or more species or subgenera based on specific choices of R, X, Y, m, n, 0, p, and/or carbon chain length, structure, or saturation.
• each R when p is 1 , is independently Cs to C12, C13, or C14 straight-chain alkyl.
• each R from a nearest common branch point is the same. In some embodiments, each R is the same.
• the ionizable cationic lipid has a structure of Formula (Formula 1a) wherein each R is independently Ce to C16 straight-chain alkyl; Ce to C16 straight-chain alkenyl; Ce to C16 branched alkyl; Ce to C16 branched alkenyl; C9 to C16 cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at either end or within the alkyl chain; or Cs to Cis aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain,
• Y is O, NH, N-CH3, or CH 2 , n is an integer from 0 to 4, m is an integer from 1 to 3, and o is an integer from 1 to 4.
• Ce to C12 straight-chain alkenyl Ce to C12 branched alkyl; branched Ce to C12 alkenyl;
• each R is independently Ce to C10 straight-chain alkyl; straight-chain Ce to C10 alkenyl; Ce to C10 branched alkyl; Ce to C10 branched alkenyl; C9 to Cio cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at either end or within the alkyl; or Cs to C12 aryl-alky in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain.
• Some embodiments include one or more species or subgenera based on specific choices of R, X, Y, m, n, o, p, and/or carbon chain length, structure, or saturation. Other embodiments specifically exclude one or more species or subgenera based on specific choices of R, X, Y, m, n, 0, p, and/or carbon chain length, structure, or saturation. In some embodiments, each R from a nearest common branch point is the same. In some embodiments, each R is the same.
• the ionizable cationic lipid has a structure of Formula 2a (Formula 2a) wherein R is Ce to C16 straight-chain alkyl; Ce to C16 straight-chain alkenyl; Ce to C16 branched alkyl; branched Ce to C16 alkenyl; C9 to C16 cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at either end or within the alkyl chain; or Cs to C18 aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain,
• Y is O, NH, N-CH3, or CH2, n is an integer from 0 to 4,
• n is an integer from 1 to 3
• o is an integer from 1 to 4.
• Some embodiments include one or more species or subgenera based on specific choices of Rc, W, X, m, n, 0, p, and/or carbon chain length, structure, or saturation. Other embodiments specifically exclude one or more species or subgenera based on specific choices of R c , W, X, m, n, 0, p, and/or carbon chain length, structure, or saturation. In some embodiments, each R c from a nearest common branch point is the same. In some embodiments, each R c is the same.
• the ionizable cationic lipid has a structure of Formula 3a
• R c is Cs to Cis straight-chain alkyl; Cs to Cis straight-chain alkenyl; Cs to Cis branched alkyl; Cs to G branched alkenyl; Cn to Cis cycloalkyl-alkyl in which the cycloalkyl is C3 to Cs cycloalkyl positioned at either end or within the alkyl chain; or C10 to C20 aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain,
• n is an integer from 0 to 4
• m is an integer from 1 to 3
• o is an integer from 1 to 4.
• all four R groups are identical.
• the two R c groups stemming from a first branchpoint are identical to each other and the two R c groups from a second branchpoint are identical to each other, but the Rc groups stemming from the first branchpoint are different than the R groups stemming from the second branchpoint.
• some embodiments are limited to one, or a subset, of the alternatives for R c , W, X, Y, m, n, 0, and/or p, as applicable.
• Other embodiments specifically exclude one, or a subset, of the alternatives for R c , W, X, Y, m, n, o, p, and/or carbon chain length, structure, or saturation, as applicable.
• Each range of carbon chain length is meant to convey embodiments of all individual lengths and subranges therein.
• R c is straight-chain alkyl and in further instances the chain is unsubstituted. In still further instances, R c is Cs or C9 or C10 to C12.
• cLogD is a calculated measure of lipophilicity that takes into account the state of ionization of the molecule at a particular pH, predicting partitioning of the lipid between water and octanol as a function of pH. More specifically, cLogD is calculated at a specified pH based on cLogP and c-pKa. (LogP is the partition coefficient of a molecule between aqueous and lipophilic phases usually considered as octanol and water.) When higher basicity of the ionizable lipid is desired, it should be balanced by greater lipophilicity as represented by a higher cLogD value.
• Balance of basicity and lipophilicity is used herein to maximize LNP function, including both stability of the LNP and release of the cargo (e.g., a nucleic acid) upon uptake by a cell. Accordingly, as m, n, or p increases, overall lipophilicity of ionizable cationic lipids disclosed herein, as represented by cLogD, can be balanced by shorter chain lengths for R.
• Some embodiments of the ionizable cationic lipid species encompassed by Formulas 1 -3 have a cLogD ranging from about of 9 to about 18 or about 9 to about 22 calculated using ACD Labs Structure Designer v 12.0, cLogP was calculated using ACD Labs Version B; cLogD was calculated at pH 7.4.
• a measured pKa of 6 to 7 for an LNP carrying a nucleic acid load ensures that the ionizable cationic lipid in the LNP will remain essentially neutral in the blood stream and interstitial spaces but ionize after uptake into cells as the endosomes acidify.
• the lipid Upon acidification in the endosomal space, the lipid becomes protonated, and associates more strongly with the phosphate backbone of the nucleic acid, which destabilizes the structure of the LNP and promotes nucleic acid release from the LNP into the cell cytoplasm (also referred to as endosomal escape).
• ionizable cationic lipids constitute means for destabilizing LNP structure (when ionized) or means for promoting nucleic acid release or endosomal escape.
• Ionizable cationic lipids of this disclosure have a branched structure to give the lipid a conical rather than cylindrical shape and such structure helps promote endosomolytic activity. The greater the endosomolytic activity, the more efficient release of the nucleotide cargo.
• the fatty acid tails are designed to comprise esters in a position that minimizes steric hinderance of ester cleavage.
• the presence of two tails leads to the tails extending in opposite directions as this is an energetically favorable conformation. This means one of the tails may extend toward the carbonyl and sterically hinder cleavage of the ester. Accordingly, large branches immediately adjacent to the ester carbonyl were avoided.
• Another consideration potentially contributing to tolerability of the lipid is the extent to which ester cleavage or other catabolism generates fragments or byproducts and whether such fragments or byproducts can be eliminated from the body without involving oxidative degradation in the liver.
• the ionizable cationic lipids of this disclosure are expected to be readily biodegradable-and the fragments easily cleared.
• Figure 8 depicts that esterase cleavage or other hydrolysis of compound A-11 would be predicted to produce tetra-alcohol B and 4 equivalents of nonanoic acid. Cyclization should then result in the production of 2 equivalents of butyrolactone C and 1 equivalent of diol D.
• An advantage of relying, at least in part, on ionizable cationic lipids of this disclosure is that it avoids the toxicity associated with quaternary ammonium cationic lipids.
• Some LNPs based on such lipids, which are effectively permanently cationic, have displayed a fatal hyperacute toxicity in laboratory animals.
• use of ionizable cationic lipids of this disclosure in LNP use of quaternary ammonium cationic lipids can be substantially reduced mitigating or avoiding toxicity.
• use of a LNP or tLNP of this disclosure causes no detectable toxicity to cells or in a subject.
• use of a LNP or tLNP of this disclosure causes no more than mild toxicity to cells or in a subject that is asymptomatic or induces only mild symptoms that do not require intervention. In certain embodiments, use of a LNP or tLNP of this disclosure causes no more than moderate toxicity to cells or in a subject which may impair activities of daily living that requires only minimal, local, or non- invasive interventions.
• Therapeutic window is the dose range from the lowest dose that exhibits a detectable therapeutic effect up to the maximum tolerated dose (MTD); the highest dose that will the desired therapeutic effect without producing unacceptable toxicity.
• Most typically therapeutic index is calculated as the ratio of LD50:ED50 when based on animal studies and TD50:ED50 when based on studies in humans (though this calculation could also be derived from animal studies and is sometime called the protective index), where LD50, TD50, and ED50 are the doses that are lethal, toxic, and effective in 50% of the tested population, respectively.
• toxicity is based on the active agent itself or some other component of the drug product, as for example, the LNP or its components.
• the toxicity is based on the active agent itself or some other component of the drug product, as for example, the LNP or its components.
• an increase in the efficiency of delivering the nucleic acid into the cytoplasm will improve the therapeutic window or index, as an effective amount of the nucleic acid would be deliverable with a smaller dosage of LNP (and its component lipids).
• Toxicities and adverse events are sometimes graded according to a 5-point scale.
• a grade 1 or mild toxicity is asymptomatic or induces only mild symptoms; may be characterized by clinical or diagnostic observations only; and intervention is not indicated.
• a grade 2 or moderate toxicity may impair activities of daily living (such as preparing meals, shopping, managing money, using the telephone, etc.) but only minimal, local, or non-invasive interventions are indicated.
• Grade 3 toxicities are medically significant but not immediately life-threatening; hospitalization or prolongation of hospitalization is indicated; activities of daily living related to self-care (such as bathing, dressing and undressing, feeding oneself, using the toilet, taking medications, and not being bedridden) may be impaired.
• Grade 4 toxicities are lifethreatening and urgent intervention is indicated.
• Grade 5 toxicity produces an adverse event-related death.
• a toxicity is confined to grade 2 or less, grade 1 or less, or produces no observation of the toxicity.
• a LNP and tLNP of this disclosure is used according to a specified regimen, provided at a particular dosage, or administered via a particular route of administration.
• Structural symmetries and convergent rather than linear synthesis pathways can be used to simplify the synthesis of the ionizable lipids.
• the instant disclosure provides a method of synthesizing an ionizable cationic lipid of Formula 1.
• the method comprises converting an intermediate having a structure of l-f A to the ionizable cationic lipid of Formula 1.
• the method further comprises synthesizing the intermediate having a structure of l-fA (e.g., Figure 1 A).
• Y O, NH, or N-CH3
• the method further comprises reacting I- fA with carbonyl diimazole to provide l-gA.
• the method further comprises coupling l-gA and X-(CH2)n+2-YH.
• the coupling reaction of l-gA and X-(CH2)n+2-YH is performed in the presence of an alkylating agent.
• the alkylating agent is MeOTf, as shown in Figure 1 B.
• the coupling reaction of l-hA with X-(CH 2 ) n+ 2-YH is carried out in the presence of a base, e.g., without limitation, NaH, or EtsN.
• the method comprises coupling an intermediate having a structure of l-fA with X-(CH2)n+3-COOH to provide the ionizable cationic lipid of Formula 1.
• the coupling method is carried out in the presence of DMAP and EtsN, e.g., as shown in Figure 1 C.
• the method comprises coupling an intermediate of l-dA with (HO-CH2-(CH2)p)2-N-PG to provide an amine-protected derivative of l-fA, wherein PG is a protecting group of amine.
• PG is -CCtef-Bu as shown in Figure 1 A.
• the amine-protected derivative of l-f A is deprotected to provide l-f A.
• the deprotecting reagent can be TFA in dimethyl chloride as shown in Figure 1 A.
• the method further comprises synthesis of l-dA.
• the synthesis method of l-dA comprises preparing a carboxylic acid derivative of l-dA wherein the carboxylic acid moiety of l-dA is protected with a protecting group that can be deprotected selectively over the hydrolysis of the R-COO- moiety.
• the carboxylic acid derivative of l-dA is l-cA which is a t-Butyl ester of l-d A, e.g., see Figure 1 A.
• the carboxylic acid derivative of l-dA is prepared by reacting the desired diol carboxylic acid derivative (e.g., l-b, wherein the carboxylic acid derivative is a t- Butyl ester, in other embodiments, the derivative can be other forms) and R-COOH.
• the diol carboxylic acid derivative is prepared by hydrogenation of an alkenyldiol carboxylic acid derivative (e.g., l-a, wherein the carboxylic acid derivative is a t-Butyl ester, in other embodiments, the derivative can be other forms).
• the alkenyldiol carboxylic acid derivative is prepared by reacting dihydroxyacetone and an alkyloxycarbonyl methylene triphenyl phosphorane (e.g., the alkyl can be t-butyl as shown in Figure 1 A).
• the method of synthesizing an ionizable cationic lipid of Formula 1 proceeds according to the synthetic scheme of Figures 1 D-F. In some embodiments, the method of synthesizing an ionizable cationic lipid of Formula 1 proceeds according to Examples 5-16 and 24 (for example, Compounds A-1 1 thru A- 15), 17-23 (for example, Compound A-2), or 25-33 (for example, Compound A-16); analogs of these Compounds with different m, n, o, p, R, X, and/or Y can be made by substituting reactants as described herein.
• the method is a method of synthesizing an ionizable cationic lipid of Formula 1 a. In some instances, the method is a method of synthesizing Compound A-1 , Compound A-2, Compound A-3, Compound A-4, Compound A-11 , Compound A-12, Compound A-13, Compound A-14, Compound A-15 or Compound A-16. In some embodiments, the method specifies only a single step, or subset of steps, depicted in Figures 1 D-F or Examples 5-16 and 24, 17-23, or 25-33, resulting in the final product.
• a further aspect is a method of synthesizing an intermediate of the synthetic scheme of Figures 1 D-F, wherein the intermediate is l-d, l-e, l-f, l-g, or l-h.
• the method specifies only a final step to generate the intermediate as depicted in Figures 1 D-F.
• the method specifies all or a subset of the steps as depicted in Figures 1 D-F to reach the intermediate.
• FIG. 1 For example l-d2, 1 -e2, I- f2, l-g2, or l-h2 as shown in Examples 8-12 and Examples 18-22.
• n 1 resulting from the reaction of intermediate l-h with 3-dimethylamino-1 -propanol, N,N- dimethyl-1 ,3-propanediamine, N,N,N’-trimethyl-1 ,3-propanediamine, in the presence of base to generate Compounds A-1 to A-3, respectively, in which Y is O, NH, or N- CH3, respectively.
• Compounds in which n is 0 or 2 to 4 can be synthesized by substituting the propanediamine moiety with an analogous C2, C4, C5 or Ce moiety.
• Compound A-4 in which Y is CH2, is obtained by reacting a salt of intermediate l-f with 5-dimethylamino-pentanoic acid.
• Compounds in which n is 0 or 2 to 4 can be synthesized by substituting the pentanoic acid moiety with an analogous C4, Ce, C7, or Cs moiety.
• R is C9, resulting from the use of decanoic acid in the conversion of intermediate l-b to intermediate l-c. Substitution of -oic acids of the corresponding chain length and structure can be used to obtain R of Ce-Cs or C10-C18, as appropriate.
• X is N(CHs)2.
• Compounds according to Formula 1 having alternative definitions of X can be synthesized by reacting alternative head group pieces from Tables 1 -3 with l-h to obtain analogues of Compounds A-1 to A-3, respectively, or reacting alternative head group pieces from Table 4 with l-f to obtain analogues of Compound A-4, as disclosed in Example 1 (below).
• Synthesis of head group pieces not previously disclosed in the art can be made analogously to their shorter congeners or, for polyethylene glycol-containing head group pieces, made according to the synthetic schemes shown in Figures 4A-B and disclosed in Example 4, or as described in Examples 25-32, (below).
• That product is then converted into acyl-imidazolide l-g ( Figure 1 E) or an acyl-imidazolide with a structure of l-gA ( Figure 1 B) upon reaction with carbonyl diimidazole and triethylamine in dichloromethane.
• the acyl- imidazolium intermediate is then reacted with: 3-dimethylamino-1 -propanol in the presence of triethylamine, to provide Compound A-1 ( Figure 1 E) or analogues with different p (e.g., Figure 1 B); with N,N-dimethyl-1 ,3-propanediamine and triethylamine to provide Compound A-2 ( Figure 1 E; see also Example 23) or analogues with different p (e.g., Figure 1 B); or with N,N,N’-trimethyl-1 ,3-propanediamine and triethylamine to provide Compound A-3 ( Figure 1 E), in each case in dichloromethane, or analogues with different p ( Figure 1 B).
• Compound A-16 a species of Formula 1 a in which Y is N-CH3, X is , n is 1 , m is 2, and 0 is 1 , the head group piece terminates in a small polyethylene glycol moiety.
• Compound A-16 can be made according to the synthetic scheme presented in Example 4 and has also been synthesized as shown in Examples 25-33. In these latter examples, ultimately l-d2 is reacted with V-15 in the presence of ⁇ MAP and EDC-HCI in dichloromethane. Analogues of l-d2 with different hydrocarbon tails (e.g., l-dA in Figure 1A) can be used to generate analogues of Compound A-16 with different R.
• V-6a V-5a in dioxane is exposed slowly added acid, for example, HCI, stirred for several hours, and solvent removed.
• acid for example, HCI
• the crude V-6a is dissolved in dichloromethane and tert-butylmethyl(3-oxopropyl)carbamate is added.
• NaBH(OAc)s is added in several portions over a time interval and incubated further.
• Water is then added, and pH adjusted to 8 with Na2COs.
• the mixture is extracted with dichloromethane, the organic phases combined and dried over Na2SC>4, and solids removed by filtration. Silica gel is added to the filtrate and concentrated to dryness.
• V-7a eluted with a gradient of dichloromethane:methanol and dried to a yellow oil.
• V-7a is dissolved in dioxane and exposed to slowly added acid, for example, HCL After incubation the solvent was removed to afford crude V-8a as a while solid.
• GDI and EtsN are added in order to a solution of V-12 in dichloromethane, the resulting solution incubated with stirring, and then cast into water. The organic phase was separated, and the aqueous phase extracted with dichloromethane. Combined organic phases are washed successively with saturated NH4CI and 5% aqueous NaHCOs, and dried. Filtration and concentration affords V-13 as a pale yellow oil.
• V-15 BFs-OEt2 is slowly added to a solution of V-14 in THF. The mixture is incubated with stirring for several hours and poured onto water. The pH is adjusted to 8.0 with saturated aqueous NaHCOs and the solvent removed to about a fifth of its original volume. The remaining solution is purified by flash chromatography using a water:acetonitrile gradient. Fractions containing V-14 are pooled and concentrated to provide V-14 as an off-white oil.
• the present disclosure provides a method of synthesizing an ionizable cationic lipid of Formula 2.
• the method comprises converting an intermediate having a structure of I l-gA to the ionizable cationic lipid of Formula 2
• the method further comprises synthesizing the intermediate having a structure of ll-gA ( Figure 2A).
• Y O, NH, or N-CH3
• the method further comprises reacting II- gA with carbonyl diimazole to provide ll-hA.
• the method further comprises coupling ll-hA and X-(CH2)n+2-YH.
• the coupling reaction of ll-hA and X-(CH2)n+2-YH is performed in the presence of an alkylating agent.
• the alkylating agent is MeOTf, as shown in Figure 2B.
• the coupling reaction of H-iA with X-(CH2)n+2-YH is carried out in the presence of a base, e.g., without limitation, NaH, or EtsN. See Figure 2B.
• the method comprises coupling an intermediate having a structure of H-gA with X-(CH2)n+3-COOH to provide the ionizable cationic lipid of Formula 2.
• the coupling method is carried out in the presence of DMAP and EtsN, e.g., as shown in Figure 2C.
• the method comprises coupling an intermediate of ll-eA with R-COOH to provide an amine- protected derivative of H-gA, also referred to as H-fA.
• the amine protecting group is -CO2FBU as shown in Figure 2A.
• the amine-protected derivative of H-gA is deprotected to provide H-gA.
• the deprotecting reagent can be TFA in dimethyl chloride as shown in Figure 2A.
• the method further comprises synthesis of H-eA.
• the synthesis method of H-eA comprises preparing a derivative of H-eA wherein the hydroxyl groups of H-eA are protected (i.e., the OH-protected ll-eA).
• the OH-protected H-eA can be ll-cA which can be prepared by reacting the sodium salt of BOC- N((CH2)p+iCH2-OH)2 with I l-a.
• the OH- protected H-eA can be H-dA which can be prepared by reacting the sodium salt of BOC-N((CH 2 )p + iCH2-OH)2 with ll-b.
• the method of synthesizing an ionizable cationic lipid of Formula 2 proceeds according to the synthetic scheme of Figures 2A-F.
• the method is a method of synthesizing an ionizable cationic lipid of Formula 2a.
• the method is a method of synthesizing Compound A- 5, Compound A-6, Compound A-7, or Compound A-8.
• the method specifies only a single step, or subset of steps, depicted in Figures 2A-F.
• the present disclosure provides methods of synthesizing an intermediate of the synthetic scheme of Figures 2D-F, wherein the intermediate is I l-e, I l-f, ll-g, I l-h, or ll-i.
• the method comprises only a final step to generate the intermediate as depicted in Figures 2D-F.
• the method comprises all or a subset of the steps as depicted in Figures 2D-F to reach the intermediate.
• n 1 resulting from the reaction of intermediate ll-i with the sodium salt of 3-dimethylamino- 1 -propanol, N,N-dimethyl-1 ,3-propanediamine, N,N,N’-trimethyl-1 ,3-propanediamine to generate Compounds A-5 to A-7, respectively, in which Y is O, NH, or N-CH3, respectively.
• Compounds in which n is 0 or 2 to 4 can be synthesized by substituting the propanediamine moiety with an analogous C2, C4, C5, or Ce moiety.
• Compound A- 8 in which Y is CH2, is obtained by reacting a salt of intermediate ll-g with 5- dimethylamino-pentanoic acid.
• Compounds in which n is 0 or 2 to 4 can be synthesized by substituting the pentanoic acid moiety with an analogous C4, Ce, C7, or Cs moiety.
• R is C9, resulting from the use of decanoic acid in the conversion of intermediate ll-e to intermediate ll-f.
• Substitution of -oic acids of the corresponding chain length and structure can be used to obtain R of Ce-Cs or C10-C18, as appropriate.
• X is N(CHs)2.
• Compounds according to Formula 2 having alternative definitions of X can be synthesized by reacting alternative head group pieces from Tables 1 -3 with ll-i to obtain analogues of Compounds A-5 to A-7, respectively, or reacting alternative head group pieces from Table 4 with ll-g to obtain analogues of Compound A-8, as disclosed in Example 2 (below).
• Synthesis of head group pieces not previously disclosed in the art can be made analogously to their shorter congeners or, for polyethylene glycol-containing head group pieces, made according to the synthetic schemes shown in Figures 4A-B and disclosed in Example 4, or as described in Examples 25-32, (below).
• an intermediate with a structure of ll-f A is treated with TFA in dichloromethane to remove the BOC blocking group to afford the amine salt I l-g ( Figure 2D) or its analogues with different R and/or p ( Figure 2A).
• the amine salt I l-g or its analogues is reacted with carbonyl diimidazole and triethylamine in dichloromethane to yield the acylimidazole ll-h or its analogues H-hA ( Figure 2A).
• the acyl-imidazolium intermediate is then reacted with 3-dimethylamino-1 - propanol in the presence triethylamine, to provide Compound A-5 ( Figure 2E) or analogues with different R and/or p ( Figure 2B); with N,N-dimethyl-1 ,3- propanediamine and triethylamine to provide Compound A-6 ( Figure 2E) or analogues with different R and/or p ( Figure 2B); or with N,N,N’-trimethyl-1 ,3-propanediamine and triethylamine to provide Compound A-7 ( Figure 2E), in each case in dichloromethane, or analogues with different R and/or p ( Figure 2B).
• the present disclosure provides a method of synthesizing an ionizable cationic lipid of Formula 3.
• the method comprises converting an intermediate having a structure of IH-cA to the ionizable cationic lipid of Formula 3.
• the method further comprises synthesizing the intermediate having a structure of IH-cA.
• the method further comprises converting IH-cA to III- fA, and IH-fA reacting with R-COOH to provide the ionizable cationic lipid of Formula 3. See, e.g., Figure 3B.
• the method further comprises preparing a derivative of HI-fA wherein the hydroxyl groups of HI-fA are protected (i.e., the OH-protected IH-fA).
• the OH-protected HI-fA can be IH-dA which can be prepared by reacting the sodium salt of IH-cA with ll-a.
• the OH-protected IH-fA can be HI-eA which can be prepared by reacting the sodium salt of HI-cA with H-b.
• HI-cA is prepared by reduction of carbonyl groups of IH-bA, e.g., by I AIH4 as shown in Figure 3A.
• HI-bA is prepared by reacting HI-aA with HN((CH2)p-CH2OH)2.
• reaction of IH-aA and HN((CH2)p-CH2OH)2 is in the presence of 4-(4,6-dimethoxy-1 ,3,5-triazin- 2-yl)-4-methylmorpholinium chloride, e.g., as shown in Figure 3A.
• the method of synthesizing an ionizable cationic lipid of Formula 3 proceeds according to the synthetic scheme of Figures 3A-D.
• the method is a method of synthesizing an ionizable cationic lipid of Formula 3a.
• the method is a method of synthesizing Compound A- 9 or Compound A-10.
• the method comprises only a single step, or subset of steps, depicted in Figures 3A-D.
• a method of synthesizing an intermediate of the synthetic scheme of Figures 3C-D wherein the intermediate is lll-d, lll-e, or lll-f.
• the method specifies only a final step to generate the intermediate as depicted in Figures 3C-D.
• the method specifies all or a subset of the steps as depicted in Figures 3C-D to reach the intermediate.
• n 2 resulting from the coupling of intermediate lll-a with diethanolamine (4-(4,6-dimethoxy- 1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride), and subsequent reduction.
• Compounds in which n is 0 to 1 or 3 to 4 can be synthesized by substituting malonic acid, maleic anhydride, 1 ,6-hexanedioic acid, 1 ,7-heptanedioic acid in the coupling reaction with dimethyl amine and subsequent addition of the amide-acid with the amino alcohol.
• W is CH2 in the synthesis of Compound A-10 depicted in Figure 3D.
• Rc is C9, resulting from the use of decanoic acid in the conversion of intermediate lll-c or lll-f to Compound 9 or 10, respectively.
• Substitution of -oic acids of the corresponding chain length and structure can be used to obtain R c of Ce-Cs or C10-C20, as appropriate.
• X is N(CHs)2.
• Compounds according to Formula 3 having alternative definitions of X can be synthesized by reacting alternative head group pieces from Table 4 (instead of lll-a) with diethanolamine to obtain analogues of Compounds A-9 and A-10, as disclosed in Example 3 (below).
• Synthesis of head group pieces not previously disclosed in the art can be made analogously to their shorter congeners or, for polyethylene glycol- containing head group pieces, made according to the synthetic schemes shown in Figures 4A-B and disclosed in Example 4, or as described in Examples 25-32, (below).
• THF can be substituted, for example, without limitation, with DMF, diethyl ether, methyl t-butyl ether, dioxane, or 2-methyl THF.
• Ethyl acetate can be substituted with, for example, without limitation, isopropyl acetate, THF, 2-methyl THF, dioxane, or methyl t-butyl ether.
• Dichloromethane can be substituted with, for example, without limitation, ethyl acetate, isopropyl acetate, THF, methyl t-butyl ether, 2-methly THF, dioxane, or heptane.
• Methanol can be substituted with, for example, without limitation, ethanol, or aqueous THF.
• LNPs Lipid Nanoparticles
• tLNPs Targeted LNPs
• lipid nanoparticle means a solid particle, as distinct from a liposome having an aqueous lumen.
• the core of a LNP like the lumen of a liposome, is surrounded by a layer of lipid that may be, but is not necessarily, a continuous lipid monolayer, a bilayer as found in a liposome, or multi-layer having three or more lipid layers.
• the present disclosure provides a lipid nanoparticle (LNP) comprising an ionizable cationic lipid of Formula 1 , Formula 2, or Formula 3, or a combination thereof.
• an LNP comprises an ionizable cationic lipid of Formula 1 , Formula 2, or Formula 3, or a combination thereof, and a phospholipid, a sterol, a co-lipid, or a PEGylated lipid, or a combination thereof.
• the PEG-lipids are not functionalized PEG-lipids.
• the LNP comprises at least one PEG-lipid that is functionalized and at least one that is not.
• the present disclosure provides a targeted lipid nanoparticle (tLNP) comprising an ionizable cationic lipid of Formula 1 , Formula 2, or Formula 3, or a combination thereof.
• the aforementioned tLNP may further comprise one or more of a phospholipid, a sterol, a co-lipid, and a PEG- lipid, or a combination thereof, and a functionalized PEG-lipid.
• “functionalized PEG-lipid” refers to a PEG-lipid in which the PEG moiety has been derivatized with a chemically reactive group that can be used for conjugating a targeting moiety to the PEG-lipid.
• the functionalized PEG-lipid can be reacted with a binding moiety after the LNP is formed, so that the binding moiety is conjugated to the PEG portion of the lipid.
• the conjugated binding moiety can thus serve as a targeting moiety for the tLNP.
• a phospholipid comprises dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), or 1 ,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), or a combination thereof.
• DOPE dioleoylphosphatidyl ethanolamine
• DMPC dimyristoylphosphatidyl choline
• DSPC distearoylphosphatidylcholine
• DMPG dimyristoylphosphatidyl glycerol
• DPPC dipalmitoyl phosphatidylcholine
• DAPC 1 ,2-diarachidoyl-sn-glycero-3-phosphocholine
• Phospholipids can contribute to formation of a membrane, whether monolayer, bilayer, or multi-layer, surrounding the core of the LNP or tLNP. Additionally, phospholipids such as DSPC, DMPC, DPPC, DAPC impart stability and rigidity to membrane structure. Phospholipids, such as DOPE, impart fusogenicity. Further phospholipids, such as DMPG, which attains negative charge at physiologic pH, facilitates charge modulation. Thus, phospholipids constitute means for facilitating membrane formation, means for imparting membrane stability and rigidity, means for imparting fusogenicity, and means for charge modulation.
• a sterol is cholesterol, 20-hydroxycholesterol, 22-hydroxycholesterol, or a phytosterol.
• the phytosterol comprises campesterol, sitosterol, or stigmasterol, or combinations thereof.
• the cholesterol is not animal-sourced but is obtained by synthesis using a plant sterol as a starting point.
• LNPs incorporating C-24 alkyl (such as methyl or ethyl) phytosterols have been reported to provide enhanced gene transfection. The length of the alkyl tail, the flexibility of the sterol ring, and polarity related to a retain C-3 -OH group are important to obtaining high transfection efficiency.
• Sterols serve to fill space between other lipids in the LNP or tLNP and influence LNP or tLNP shape. Sterols also control fluidity of lipid compositions, reducing temperature dependence.
• sterols such as cholesterol, 20-hydroxycholesterol, 22- hydroxycholesterol, campesterol, fucosterol, p-sitosterol, and stigmasterol constitute means for controlling LNP shape and fluidity or sterol means for increasing transfection efficiency.
• a co-lipid is absent or comprises an ionizable lipid, anionic or cationic.
• a co-lipid can be used to adjust various properties of an LNP or tLNP, such as surface charge, fluidity, rigidity, size, stability, etc.
• a co-lipid is an ionizable lipid, such as cholesterol hemisuccinate (CHEMS) or an ionizable lipid of this disclosure.
• CHEMS cholesterol hemisuccinate
• a co-lipid is a charged lipid, such as a quaternary ammonium headgroup containing lipid.
• a quaternary ammonium headgroup containing lipid comprises 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1 - (2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA), or 3P-(N-(N',N'- Dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or combinations thereof.
• these compounds a chloride, bromide, mesylate, or tosylate salt.
• the disclosed ionizable lipids of Formulas 1 , 2, and 3 have a measured pKa between 6 and 7, they can contribute substantial endosomal release activity to an LNP or tLNP containing the ionizable lipid. More acidic or basic ionizable lipids of Formulas 1 , 2, and 3 can contribute surface charge and thus serve as a co- lipid as described immediately above. In such cases, it can be advantageous to incorporate another lipid with fusogenic activity into a LNP or tLNP of this disclosure. Surface charge is known to influence the tissue tropism of LNPs or tLNPs; for example, positively charged LNPs or tLNPs have shown a tropism for spleen and lung.
• a PEG-lipid that is, a lipid conjugated to a polyethylene glycol (PEG)
• PEG polyethylene glycol
• a PEG-lipid is a C14-C20 lipid conjugated with a PEG.
• PEG-lipids with fatty acid chain lengths less than C14 are too rapidly lost from the (t)LNP while those with chain lengths greater than C20 are prone to difficulties with formulation.
• a PEG is of 500-5000 or 10DO- 5000 Da molecular weight (MW).
• the PEG unit has a MW of 2000 Da.
• the MW2000 PEG-lipid comprises DMG-PEG2000 (1 ,2- dimyristoyl-glycero-3-methoxypolyethylene glycol-2000), DPG-PEG2000 (1 ,2- dipalmitoyl-glycero-3-methoxypolyethylene glycol-2000), DSG-PEG2000 (1 ,2- distearoyl-glycero-3-methoxypolyethylene glycol-2000), DOG-PEG2000 (1 ,2-dioleoyl- glycero-3-methoxypolyethylene glycol-2000), DMPE-PEG200 (1 ,2-dimyristoyl- glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPE- PEG2000 (1 ,2-dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DSPE-PEG2000 (1 ,2-distearoyl-glycero-3-phospho
• the PEG unit has a MW of 2000 Da.
• the MW2000 PEG-lipid comprises DMrG-PEG2000 (1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000), DPrG-PEG2000 (1 ,2-dipalmitoyl-rac-glycero-3- methoxypolyethylene glycol-2000), DSrG-PEG2000 (1 ,2-distearoyl-rac-glycero-3- methoxypolyethylene glycol-2000), DOrG-PEG2000 (1 ,2-dioleoyl-glycero-3- methoxypolyethylene-rac-glycol-2000), DMPEr-PEG200 (1 ,2-dimyristoyl-rac-glycero- 3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPEr-PEG2000 (1 ,2- dipalmitoyl-rac-glycero-3-
• optically pure antipodes of the glycerol portion can be employed, that is, the glycerol portion is homochiral.
• optically pure means >95% of a single enantiomer (D or L).
• the enantiomeric excess is >98%.
• the enantiomeric excess is >99%. Additional PEG-lipids, including achiral PEG-lipids built on a symmetric dihydroxyacetone scaffold, a symmetric 2-(hydroxymethyl)butane-1 ,4-diol, or a symmetric glycerol scaffold, are disclosed in U.S. Provisional Application No.
• a PEG-moiety provides a hydrophilic surface on the LNP, inhibiting aggregation or merging of LNP, thus contributing to their stability and reducing polydispersity. Additionally, a PEG moiety may impede binding by the LNP, including binding to plasma proteins. These plasma proteins include apoE which is understood to mediate uptake of LNP by the liver so that inhibition of binding can lead to an increase in the proportion of LNP reaching other tissues. These plasma proteins also include opsonins so that inhibition of binding reduces recognition by the reticuloendothelial system. The PEG-moiety can also be functionalized to serve as an attachment point for a targeting moiety.
• PEG-lipid can thus serve as means for inhibiting LNP binding
• PEG- lipid conjugated to a binding moiety can serve as means for LNP-targeting.
• a “binding moiety” or “targeting moiety” refers to a protein, polypeptide, oligopeptide, peptide, carbohydrate, nucleic acid, or combination thereof that is capable of specifically binding to a target or multiple targets.
• a binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest.
• Exemplary binding moieties of this disclosure include an antibody, a Fab', F(ab')2, Fab, Fv, rlgG, scFv, hcAbs (heavy chain antibodies), a single domain antibody, VHH, VNAR, sdAbs, nanobody, receptor ectodomains or ligand-binding portions thereof, or ligands (e.g., cytokines, chemokines).
• a “Fab” fragment antigen binding
• a binding moiety such as a binding moiety comprising immunoglobulin light and heavy chain variable domains (e.g., scFv), can be incorporated into a variety of protein scaffolds or structures as described herein, such as an antibody or an antigen binding fragment thereof, a scFv-Fc fusion protein, or a fusion protein comprising two or more of such immunoglobulin binding domains.
• scFv immunoglobulin light and heavy chain variable domains
• An antibody or other binding moiety “specifically binds” a target if it binds the target with an affinity or Ka (i.e. , an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 5 M’ 1 , while not significantly binding other components present in a test sample.
• Binding domains (or fusion proteins thereof) may be classified as “high affinity” binding domains (or fusion proteins thereof) and “low affinity” binding domains (or fusion proteins thereof).
• “High affinity” binding domains refer to those binding domains with a Ka of at least 10 8 M’ 1 , at least 10 9 M’ 1 , at least 1 O 10 M’ 1 , at least 10 11 M’ 1 , at least 10 12 M’ 1 , or at least 10 13 M’ 1 , preferably at least 10 8 M -1 or at least 10 9 M’ 1 .
• “Low affinity” binding domains refer to those binding domains with a Ka of up to 10 8 M’ 1 , up to 10 7 M' 1 , up to 10 6 M' 1 , up to 10 5 M' 1 .
• affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10' 5 M to 10' 13 M).
• binding domain polypeptides and fusion proteins can be readily determined using conventional techniques (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51 :660, 1949; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).
• Some embodiments of the disclosed ionizable cationic lipids have head groups with small ( ⁇ 250 Da) PEG moieties. These lipids are not what is meant by the term PEG-lipid as used herein. These small PEG moieties are generally too small to impede binding to a similar extent as the larger PEG moieties of the PEG-lipids disclosed above, though they will impact the lipophilicity of ionizable cationic lipid. Moreover, the PEG-lipids are understood to be primarily located in an exterior facing lamella whereas much of the ionizable cationic lipid is in the interior of the LNP.
• a binding moiety of a tLNP comprises an antigen binding domain of an antibody, an antigen, a ligand-binding domain of a receptor, or a receptor ligand.
• the binding moiety comprising an antigen binding domain of an antibody comprises a complete antibody, an F(ab)2, an Fab, a minibody, a single-chain Fv (scFv), a diabody, a VH domain, or a nanobody, such as a VHH or single domain antibody.
• the receptor ligand is a carbohydrate, for example, a carbohydrate comprising terminal galactose or N- acetylgalactosamine units, which are bound by the asialoglycoprotein receptor.
• These binding moieties constitute means for LNP targeting. Some embodiments specifically include one or more of these binding moieties. Other embodiments specifically exclude one or more of these binding moieties.
• antibody refers to a protein comprising an immunoglobulin domain having hypervariable regions determining the specificity with which the antibody binds antigen; so-called complementarity determining regions (CDRs).
• CDRs complementarity determining regions
• the term antibody can thus refer to intact or whole antibodies as well as antibody fragments and constructs comprising an antigen binding portion of a whole antibody. While the canonical natural antibody has a pair of heavy and light chains, camelids (camels, alpacas, llamas, etc.) produce antibodies with both the canonical structure and antibodies comprising only heavy chains.
• the variable region of the camelid heavy chain only antibody has a distinct structure with a lengthened CDR3 referred to as VHH or, when produced as a fragment, a nanobody.
• Antigen binding fragments and constructs of antibodies include F(ab)2, F(ab), minibodies, Fv, single-chain Fv (scFv), diabodies, and VH. Such elements may be combined to produce bi- and multi-specific reagents, such as BiTEs.
• Antibodies can be obtained through immunization, selection from a naive or immunized library (for example, by phage display), alteration of an isolated antibody-encoding sequence, or any combination thereof. Numerous antibodies that could be used as binding moieties are known in the art.
• a functionalized PEG-lipid of a tLNP comprises one or more fatty acid tails, each that is no shorter than Ci6 nor longer than C20 for straight-chain fatty acids. For branched chain fatty acids, tails no shorter than C14 fatty acids nor longer than C20 are acceptable. In some embodiments, fatty acid tails are C16. In some embodiments, the fatty acid tails are C18. In some embodiments, the functionalized PEG-lipid comprises a dipalmitoyl lipid. In some embodiments, the functionalized PEG-lipid comprises a distearoyl lipid.
• the fatty acid tails serve as means to anchor the PEG-lipid in the tLNP to reduce or eliminate shedding of the PEG-lipid from the tLNP. This is a useful property for the PEG-lipid whether or not it is functionalized but has greater significance for the functionalized PEG-lipid as it will have a targeting moiety attached to it and the targeting function could be impaired if the PEG-lipid (with the conjugated binding moiety) were shed from the tLNP.
• Any suitable chemistry may be used to conjugate the binding moiety to the PEG of the PEG-lipid, including maleimide (see Parhiz et aL, Journal of Controlled Release 291 :106-1 15, 2018) and click (see Kolb et aL, Angewandte Chemie International Edition 40(1 1 ):2004-2021 , 2001 ; and Evans, Australian Journal of Chemistry 60(6):384-395, 2007) chemistries.
• Reagents for such reactions include lipid-PEG-maleimide, lipid-PEG-cysteine, lipid-PEG-alkyne, lipid, PEG- dibenzocyclooctyne (DBCO), and lipid-PEG-azide.
• an existing cysteine sulfhydryl or derivatize the protein by adding a sulfur containing carboxylic acid, for example, to the epsilon amino of a lysine to react with a maleimide, bromomaleimide, alkylnoic amide, or alkynoic imide.
• a sulfur containing carboxylic acid for example, to the epsilon amino of a lysine to react with a maleimide, bromomaleimide, alkylnoic amide, or alkynoic imide.
• an alkyne to a sulfhydryl or an epsilon amino of a lysine to participate in a click chemistry reaction.
• the molar ratio of the lipids is about 40 to about 60 mol% ionizable cationic lipid. In some embodiments of the LNP or the tLNP, the molar ratio of the lipids is about 7 to about 30 mol% phospholipid. In some embodiments of the LNP or the tLNP, the molar ratio of the lipids is about 20 to about 45 mol% sterol. In some embodiments of the LNP or the tLNP, the molar ratio of the lipids is 1 to 30 mol% co-lipid.
• the molar ratio of the lipids is 0 to 5 mol% PEG-lipid. In some embodiments of the LNP or the tLNP, the molar ratio of the lipids is 0.1 to 5 mol% functionalized PEG-lipid. In some embodiments, the functionalized PEG-lipid is conjugated to a binding moiety.
• the LNP or tLNP Due to physiologic and manufacturing constraints LNP or tLNP for in vivo use, particles with a hydrodynamic diameter of about 50 to about 150 nm are desirable. Accordingly, in some embodiments, the LNP or tLNP has a hydrodynamic diameter of 50 to 150 nm and in some instances the hydrodynamic diameter is ⁇ 120, ⁇ 1 10, ⁇ 100, or ⁇ 90 nm. Uniformity of particle size is also desirable with a polydispersity index (PDI) of ⁇ 0.2 (on a scale of 0 to 1 ) being acceptable. Both hydrodynamic diameter and polydispersity index are determined by dynamic light scattering (DLS). Particle diameter as assessed from cryo-transmission electron microscopy (Cryo-TEM) can be smaller than the DLS-determined value.
• DLS dynamic light scattering
• LNPs or tLNPs of this disclosure further comprise a nucleic acid.
• a nucleic acid is an mRNA, a self-replicating RNA, a siRNA, a miRNA, DNA, a gene editing component (for example, a guide RNA, a tracr RNA, a sgRNA), a gene writing component, an mRNA encoding a gene or base editing protein, a zinc- finger nuclease, a Talen, a CRISPR nuclease, such as Cas9, a DNA molecule to be inserted or serve as a template for repair), and the like, or a combination thereof.
• a gene editing component for example, a guide RNA, a tracr RNA, a sgRNA
• a gene writing component for example, a guide RNA, a tracr RNA, a sgRNA
• a gene writing component for example, a guide RNA, a tracr RNA,
• an mRNA encodes a chimeric antigen receptor (CAR).
• an mRNA encodes a gene-editing or base-editing or gene writing protein.
• a nucleic acid is a guide RNA.
• an LNP or tLNP comprises both a gene- or base-editing or gene writing proteinencoding mRNA and one or more guide RNAs.
• CRISPR nucleases may have altered activity, for example, modifying the nuclease so that it is a nickase instead of making double-strand cuts or so that it binds the sequence specified by the guide RNA but has no enzymatic activity.
• Base-editing proteins are often fusion proteins comprising a deaminase domain and a sequence-specific DNA binding domain (such as an inactive CRISPR nuclease).
• the ratio of total lipid to nucleic acid is about 10:1 to about 50:1 on a weight basis. In some embodiments, the ratio of total lipid to nucleic acid is about 10:1 , about 20:1 , about 30:1 , or about 40:1 to about 50:1 , or 10:1 to 20:1 , 30:1 , 40:1 or 50:1 , or any range bound by a pair of these ratios.
• the present disclosure provides a method of making a LNP or tLNP comprising mixing of an aqueous solution of a nucleic acid and an alcoholic solution of the lipids.
• the mixing is rapid.
• the aqueous solution is buffered at pH of about 3 to about 5, for example, without limitation, with citrate or acetate.
• an alcohol can be ethanol, isopropanol, t- butanol, or a combination thereof.
• the rapid mixing is accomplished by pumping the two solutions through a T-junction or with an impinging jet mixer.
• Microfluidic mixing through a staggered herringbone mixer (SHM) or a hydrodynamic mixer (microfluidic hydrodynamic focusing), microfluidic bifurcating mixers, and microfluidic baffle mixers can also be used.
• buffer for example phosphate, HEPES, or Tris
• the diluted LNPs are purified either by dialysis or ultrafiltration or diafiltration using tangential flow filtration (TFF) against a buffer in a pH range of 6 to 8.5 (for example, phosphate, HEPES, or Tris) to remove the alcohol.
• THF tangential flow filtration
• the buffer is exchanged with like buffer containing a cryoprotectant (for example, glycerol or a sugar such as sucrose, trehalose, or mannose).
• a cryoprotectant for example, glycerol or a sugar such as sucrose, trehalose, or mannose.
• the LNPs are concentrated to a desired concentrated, followed by 0.2 pm filtration through, for example, a polyethersulfone (PES) or modified PES filter and filled into glass vials, stoppered, capped, and stored frozen.
• PES polyethersulfone
• a lyoprotectant is used and the LNP lyophilized for storage instead of as a frozen liquid.
• One aspect is a method of making a tLNP comprising rapid mixing of an aqueous solution of a nucleic acid and an alcoholic solution of the lipids as disclosed for LNP.
• the lipid mixture includes functionalized PEG-lipid, for later conjugation to a targeting moiety.
• functionalized PEG-lipid refers to a PEG-lipid in which the PEG moiety has been derivatized with a chemically reactive group (such as, maleimide, NHO ester, Cys, azide, alkyne, and the like) that can be used for conjugating a targeting moiety to the PEG-lipid, and thus, to the LNP comprising the PEG-lipid.
• the functionalized PEG-lipid is inserted into and LNP subsequent to initial formation of an LNP from other components.
• the targeting moiety is conjugated to functionalized PEG-lipid after the functionalized PEG-lipid containing LNP is formed. Protocols for conjugation can be found, for example, in Parhiz et al. J. Controlled Release 291 :106-1 15, 2018, and Tombacz et aL, Molecular Therapy 29(11 ):3293- 3304, 2021 , each of which is incorporated by reference for all that it teaches about conjugation of PEG-lipids to binding moieties.
• the targeting moiety can be conjugated to the PEG-lipid prior to insertion into pre-formed LNP.
• the method comprises: i). forming an initial LNP by mixing all components of the tLNP except for the one or more functionalized PEG-lipids and the one or more targeting moieties; ii). forming a pre-conjugation tLNP by mixing the initial LNP with the one or more functionalized PEG-lipids; and iii). forming the tLNP by conjugating the pre-conjugation tLNP with the one or more targeting moieties.
• the method comprises: i). forming a pre-conjugation tLNP by mixing all components of the tLNP, including the one or more functionalized PEG-lipids, except for the one or more targeting moieties; and ii). forming the tLNP by conjugating the pre-conjugation tLNP with the one or more targeting moieties.
• the method comprises: i). forming one or more conjugated functionalized PEG-lipids by conjugating the one or more functionalized PEG-lipids with the one or more targeting moieties; and ii) forming the tLNP by mixing all components of the tLNP, including the one or more conjugated functionalized PEG-lipids.
• the method comprises: i). forming one or more conjugated functionalized PEG-lipids by conjugating the one or more functionalized PEG-lipids with the one or more targeting moieties; ii) forming an LNP by mixing all components of the tLNP, except the one or more conjugated functionalized PEG-lipids; and iii) forming the tLNP by mixing the initial LNP with the one or more conjugated functionalized PEG-lipids.
• tLNPs are purified by dialysis, tangential flow filtration, or size exclusion chromatography, and stored, as disclosed above for LNPs.
• the encapsulation efficiency of the nucleic acid by the LNP or tLNP is typically determined with a nucleic acid binding fluorescent dye added to intact and lysed aliquots of the final LNP or tLNP preparation to determine the amounts of unencapsulated and total nucleic acid, respectively.
• Encapsulation efficiency is typically expressed as a percentage and calculated as 100 x (T-U)/T where T is the total amount of nucleic acid and U is the amount of unencapsulated nucleic acid. In various embodiments, the encapsulation efficiency is >80%, >85%, >90%, or >95%.
• contacting takes place ex vivo.
• the contacting takes place in vivo.
• the in vivo contacting comprises intravenous, intramuscular, subcutaneous, intranodal or intralymphatic administration.
• toxicity is confined (or largely confined) to grades of 0 or 1 or two, as discussed above.
• Dihydroxy acetone can react with tert- butoxycarbonylmethylene)triphenylphosphorane to provide alkene l-a.
• Hydrogenation of l-a provides l-b and the coupling (EDC-HCI, DMAP) of l-b with decanoic acid results in tri-ester l-c.
• Hydrolysis of the t-butyl ester (CF3CO2H, CH2CI2) results in a mono- acid l-d.
• Coupling of l-d with BOC-blocked di-ethanolamine affords l-e.
• BOC-removal (CF3CO2H, CH2CI2) provides salt l-f (see Figure 1 D) which is converted to acyl- imidazolide l-g upon reaction with carbonyl diimidazole Reaction of l-g with methyl triflate produced acyl-imidazolium l-h, which is an intermediate to be converted to Compounds A-1 to A-3.
• Compound A-4 can be obtained from the reaction of salt l-f with 5- dimethylamino-pentanoic acid (EDC-HCI, DMAP, EtsN) (see Figure 1 F).
• the headgroup in Compound A-1 that is, X in Formula 1 is derived from 3- dimethylamino-1 -propanol.
• the compounds of Table 1 may be used to substitute for 3-dimethylamino-1 -propanol in the conversion of l-h.
• Reagents XR1 -XR9, XR12-XR18, XR21-XR27, XR30-XR38, and XR41 -49 are known in the art, as reported by the Chemical Abstract Society’s SciFinder® with XR1 -XR5, XR7, XR12-XR15, XR21 -XR25, XR30-XR31 , XR33, and XR41 being commercially available.
• the polyethylene glycol-containing reagents can be synthesized as described in Example 4, as shown below.
• the headgroup in Compound A-2 that is, X in Formula 1 is derived from N,N-dimethyl-1 ,3-propanediamine.
• the compounds of Table 2 may be used to substitute N,N-dimethyl-1 ,3-propanediamine in the conversion of I- h.
• Reagents XR52-XR60, XR63-XR70, XR73-XR81 , XR84-XR92, and XR95- XR103 are known in the art, as reported by the Chemical Abstract Society’s SciFinder® with XR52-XR57, XR63-XR66, XR73-XR77, XR84, XR86-XR87, and XR95 being commercially available.
• the polyethylene glycol-containing reagents can be synthesized as described in Example 4, as shown below.
• the headgroup in Compound A-3 that is, X in Formula 1 is derived from N,N,N’-trimethyl-1 ,3-propanediamine.
• the compounds of Table 3 may be used to substitute N,N,N’-trimethyl-1 ,3-propanediamine in the conversion of l-h.
• Reagents XR106-XR114, XR1 17-XR124, XR127-XR131 , XR134, XR138- XR142, XR149-XR153, and XR156 are known in the art, as reported by the Chemical Abstract Society’s SciFinder® with XR106-XR1 10, XR117-XR120, and XR127 being commercially available.
• XR132-XR133, XR135, XR143-XR146, XR154-XR154, and XR156 are prepared analogously to their shorter congeners.
• the polyethylene glycol- containing reagents are synthesized as disclosed in Example 4, as shown below.
• the headgroup in Compound A-4 that is, X in Formula 1 is derived from 4- dimethylamino-butanoic acid.
• the compounds of Table 4 may be used to substitute4-dimethylamino-butanoic acid in the conversion of l-f.
• Reagents XR160-XR168, XR171 -XR178, XR181 -XR189, XR192-XR196, And XR203-XR206 are known in the art, as reported by the Chemical Abstract Society’s SciFinder® with XR160, XR162-XR164, and XR181 being commercially available.
• XR196-XR199 and XR207-XR211 can be prepared analogously to their shorter congeners.
• the polyethylene glycol-containing reagents can be synthesized
• Benzylidene acetal ll-d can be deprotected with hydrogen and Pd/C to provide ll-e.
• decanoic acid EDC-HCI, DMAP
• ll-f decanoic acid
• CF3CO2H CF3CO2H
• amine salt ll-g The reaction of amine salt ll-g with carbonyl diimidazole leads to ll-h, followed by the reaction of the acylimidazole I l-h with methyl triflate to provide the intermediate that can be used for the synthesis of Compounds A-5 to A-7, acyl-imidazolium ll-i ( Figure 2D).
• Amide Compound A-8 is obtained from the reaction of salt ll-g with 4- dimethylamino-butanoic acid (EDC-HCI, DMAP, EtsN) ( Figure 2F).
• the headgroup in Compound A-5 that is, X in Formula 2 is derived from 3- dimethylamino-1 -propanol.
• X in Formula 2 is derived from 3- dimethylamino-1 -propanol.
• the compounds of Table 1 can be used to substitute 3-dimethylamino-1 -propanol in the conversion of ll-i.
• the headgroup in Compound A-6 that is, X in Formula 2 is derived from N,N-dimethyl-1 ,3-propanediamine.
• X in Formula 2 is derived from N,N-dimethyl-1 ,3-propanediamine.
• the headgroup in Compound A-7 that is, X in Formula 2 is derived from N,N,N’-trimethyl-1 ,3-propanediamine.
• X in Formula 2 is derived from N,N,N’-trimethyl-1 ,3-propanediamine.
• the headgroup in Compound A-8 that is, X in Formula 2 is derived from 4- dimethylamino-butanoic acid.
• X in Formula 2 is derived from 4- dimethylamino-butanoic acid.
• the compounds of Table 4 (above) can be used to substitute 4-dimethylamino-butanoic acid in the conversion of ll-g.
• the headgroup in Compounds A-9 and A-10 that is, X in Formula 3 is derived from 5-(dimethylamino)-5-oxopentanoic acid (lll-a), reacted with diethanolamine.
• X in Formula 3 is derived from 5-(dimethylamino)-5-oxopentanoic acid (lll-a), reacted with diethanolamine.
• the carboxylic acids of Table 4 can be used to substitute 5-(dimethylamino)-5-oxopentanoic acid (lll-a) according to the scheme: and the reactions are completed according to Figure 3C-D, as appropriate.
• polyethylene glycol-containing reagents are synthesized as disclosed in Example 4, below.
• Shorter chain PEG-containing head group entities can be obtained by substituting the known / commercially available shorter chain mesylates V-9 (known) and V-10 (commercially available) for the 2-(2-(2-methoxyethoxy)ethoxy)ethyl methanesulfonate utilized in the schemes above.
• V-8 For variants of V-8 one uses appropriate analogues of the 1 ,1 -dimethylethyl N- (3-hydroxypropyl)-N-methylcarbamate to bring in different values of m and analogues of the 1 ,1 -dimethylethyl N-methyl-N-(3-oxopropyl)carbamate to bring in different values of n or definitions of Y to create the desired PEG-containing head group piece.
• the dry silica gel was placed onto a gravity column of silica gel (3700g, type: ZCX-2, 100-200 mesh, packed with petroleum ether), and the resulting column was eluted with a gradient of petroleum ether: ethyl acetate (100:0 to 50:50).
• Compound l-a eluted with petroleum ether: ethyl acetate 50:50 and the fractions of l-a were concentrated in vacuo to provide 1 (235.0g) containing PhsPO (purity 73.8% by HNMR, 55% yield of l-a).
• the dry silica gel was placed onto a gravity column of silica gel (4000g, type: ZCX-2, 100-200 mesh, packed with petroleum ether), and the resulting column was eluted with a gradient of petroleum ether: THF (100:0 to 95:5).
• l-c2 leads to a lipid in which R is straight-chain Cs
• l-c leads to a lipid in which R is straight-chain C9.
• l-d2 leads to a lipid in which R is straightchain Cs
• l-d leads to a lipid in which R is straight-chain C9.
• the solution was concentrated in vacuo to ca. 1 .5L volume and silica gel (350g, type: ZCX-2, 100-200 mesh) was added and the mixture was concentrated in vacuo to dryness.
• silica gel was placed onto a gravity column of silica gel (2100g, type: ZCX-2, 100- 200 mesh, packed with heptane), and the resulting column was eluted with a gradient of heptane: THF (100:0 to 90:10).
• l-e2 leads to a lipid in which R is straight-chain Cs
• l-e leads to a lipid in which R is straight-chain C9.
• Compound A-11 eluted with CH2Cl2:MeOH 95:5 and the fractions of Compound A-11 were concentrated in vacuo to provide Compound A-11 as a yellow oil (12.0g, HPLC purity 88%).
• Compound A-11 was further purified by reverse phase flash chromatography (WelFlash XSelect CSH Prep C18, 5mm OBD, Regular 30x150mm column; Solvents: A: 0.1 % formic acid in water, B: acetonitrile, gradient 50-80%, 20 minutes, flow 55mL/min). Fractions containing Compound A-11 were pooled, and concentrated in vacuo and the residue was dissolved in heptane (150mL).
• Compound A-12 eluted with CH2Cl2:MeOH 95:5 and the fractions of Compound A-12 were concentrated in vacuo to provide Compound A-12 as a yellow oil (1 1 .2g, HPLC purity 85%).
• Compound A-12 was further purified by reverse phase flash chromatography (WelFlash XSelect CSH Prep C18, 5mm OBD, Regular 30x150mm column; Solvents: A: 0.1 % formic acid in water, B: acetonitrile, gradient 50-80%, 20 minutes, flow 55mL/min). Fractions containing Compound A-12 were pooled and concentrated in vacuo and the residue was dissolved in heptane (150mL).
• Compound A-13 eluted with CH2Cl2:MeOH 97:3 and the fractions of Compound A-13 were concentrated in vacuo to provide Compound A-13 as a yellow oil (18.0g, HPLC purity 83%).
• Compound A-13 was further purified by reverse phase flash chromatography (WelFlash XSelect CSH Prep C18, 5mm OBD, Regular 30x150mm column; Solvents: A: 0.1 % formic acid in water, B: acetonitrile, gradient 50-80%, 20 minutes, flow 55mL/min). Fractions containing Compound A- 13 were pooled and concentrated in vacuo and the residue was dissolved in heptane (150ml_).
• silica gel 40g, type: ZCX-2, 100-200mesh was added to the solution, and the mixture was concentrated in vacuo to dryness.
• the dry silica gel was placed onto a gravity column of silica gel (250g, type: ZCX-2, 100-200 mesh, packed with CH2CI2), and the resulting column was eluted with a gradient of CH2Cl2:MeOH (100:0 to 90:10).
• Compound A-14 eluted with CH2Cl2:MeOH 97:3 and the fractions of Compound A-14 were concentrated in vacuo to provide Compound A-14 as a yellow oil (18.0g, HPLC purity 88%).
• Compound A-14 was further purified by reverse phase flash chromatography (WelFlash XSelect CSH Prep C18, 5mm OBD, Regular 30x150mm column; Solvents: A: 0.1 % formic acid in water, B: acetonitrile, gradient 50-80%, 20 minutes, flow 55mL/min). Fractions containing Compound A-14 were pooled and concentrated in vacuo and the residue was dissolved in heptane (500mL).
• the dry silica gel was placed onto a gravity column of silica gel (900g, type: ZCX-2, 100-200 mesh, packed with petroleum ether), and the resulting column was eluted with a gradient of petroleum ether: ethyl acetate (100:0 to 95:5).
• silica gel (187.5g, type: ZCX- 2, 100-200 mesh) was added to the filtrate and the mixture was concentrated in vacuo to dryness.
• the dry silica gel was placed onto a gravity column of silica gel (1 125g, type: ZCX-2, 100-200 mesh, packed with heptane), and the resulting column was eluted with a gradient of heptane: THF (100:0 to 90:10).
• the solids were removed by filtration, the filtrate was concentrated in vacuo to provide crude Compound A-2 as a viscous yellow oil.
• the crude product was dissolved in ethyl acetate (300mL) and was washed with 5% aq. Na2CC>3 (2x300mL), brine (300mL) and dried (MgSC ).
• the solids were removed by filtration and silica gel (40g, type: ZCX-2, 100-200mesh) was added to the solution, and the mixture was concentrated in vacuo to dryness.
• the dry silica gel was placed onto a gravity column of silica gel (200g, type: ZCX-2, 100-200 mesh, packed with CH2CI2), and the resulting column was eluted with a gradient of CH2Cl2:MeOH (100:0 to 90:10).
• Compound A-2 eluted with CH2Cl2:MeOH 95:5 and the fractions of Compound A-2 were concentrated in vacuo to provide Compound A-2 as a yellow oil (12.5g).
• Compound A-2 was dissolved in heptane (150mL), washed with MeOH/H2O (80:20, 2x150mL), brine (150mL) and dried (MgSO4). The solids were removed by filtration, the filtrate was concentrated in vacuo to provide Compound A-2 as a pale, yellow oil (1 1 .96g, purity 94% by HPLC, 57% yield.
• the aqueous phase was extracted with CH2CI2 (2x300mL), and the combined organic phases were washed with brine (300mL) and dried (Na2SC>4).
• the solids were removed by filtration and silica gel (30g, type: ZCX- 2, 100-200mesh) was added to the filtrate, and the mixture was concentrated in vacuo to dryness.
• the dry silica gel was placed onto a gravity column of silica gel (150g, type: ZCX-2, 100-200 mesh, packed with heptane), and the resulting column was eluted with a gradient of heptane:ethyl acetate (100:0 to 0:100).
• Compound A- 15 eluted with heptane:ethyl acetate 70:30 and the fractions of Compound A-15 were concentrated in vacuo to provide Compound A-15 as a yellow oil (12.0g, HPLC purity 88%).
• Compound A-15 was further purified by reverse phase flash chromatography (WelFlash XSelect CSH Prep C18, 5mm OBD, Regular 30x150mm column; Solvents: A: 0.05% formic acid in water, B: acetonitrile, gradient 50-80%, 20 minutes, flow 55mL/min). Fractions containing Compound A-15 were pooled and concentrated in vacuo and the residue was dissolved in heptane (150mL).
• the mixture was extracted with CH2CI2 (3x200mL) and the combined organic phases were dried (Na2SC ).
• the solids were removed by filtration and silica gel (40g, type: ZCX-2, 100-200mesh) was added to the filtrate, and the mixture was concentrated in vacuo to dryness.
• the dry silica gel was placed onto a gravity column of silica gel (500g, type: ZCX-2, 100-200 mesh, packed with CH2CI2, eluted with a gradient of CH2Cl2:MeOH 100:0 to 90:10).
• Fractions containing V-7a (CH2Cl2:MeOH 95:5) were concentrated in vacuo to provide V-7a as a yellow oil (8.0g, 25.1 mmol, 31%).
• V-13 Filtration and concentration in vacuo afforded crude V-13 as a yellow oil which was dissolved in heptane (250mL). The solution was washed with MeOH / H2O (80:20, 1.15L) and dried (MgSC ). Filtration and concentration in vacuo gave V-13 (270.0g, 0.631 mol, 91 %) as a pale, yellow oil.
• the aqueous layer was extracted with dichloromethane (2x200mL) and the combined organic phases were concentrated in vacuo.
• the resulting crude V-14 was dissolved in heptane (300mL) and the solution was extracted with MeOH / H2O (75:25, 2x100ml_).
• the combined aqueous phases were extracted with heptane (6x200mL), and the combined organic phases were washed with brine (400mL).
• the organic phase was dried (MgSC ). After filtration, silica gel (60g, type: ZCX-2, 100-200mesh) was added to the filtrate, and the mixture was concentrated in vacuo to dryness.
• the dry silica gel was placed onto a gravity column of silica gel (330g, type: ZCX-2, 100- 200 mesh, packed with CH2CI2, eluted with a gradient of CH2CI2 1 MeOH 100:0 to 90:10). Fractions containing V-14 (CH2CI2 / MeOH 93:7) were concentrated in vacuo to provide V-14 as a yellow oil (1 1 .15g, 22. Ommol, 46%).
• the resulting crude Compound A-16 was dissolved in heptane (150mL) and the resulting solution was washed with MeOH I water (80:20,1 OOmL), brine (1 OOmL), and dried (Na2SO4).
• the solids were removed by filtration and silica gel (25g, type: ZCX-2, 100-200mesh) was added to the filtrate, and the mixture was concentrated in vacuo to dryness.
• the dry silica gel was placed onto a gravity column of silica gel (175g, type: ZCX-2, 100-200 mesh, packed with CH2CI2, eluted with a gradient of CH2CI2 / MeOH 100:0 to 90:10).
• cLogD and c-pKa were calculated as noted above.
• the measured pKa of a lipid was determined as formulated in a lipid nanoparticle using the TNS assay as described in the following Example.
• Past experience leads to the expectation that the difference between c- pKa and the measured pKa in an LNP (ApKa) will be between 2 and 3 units; however, all of Compounds A-2 and A-1 1 thru A-15 surprisingly had a ApKa of less than 2.
• the activity of ionizable amino lipids for promoting endosomal escape of the nucleic acid cargo is typically greatest for lipids with a pKa of between 6 and 7.
• lipid stock solution was prepared by dissolution of the lipid in isopropanol at the concentration of 5 mg/mL. A requisite volume of the lipid-isopropanol solution was then diluted to 100 pM concentration at a total volume of 1 .0 mL with 50:50 (v/v) ethanol/water. Ten microliters of this 100 pM solution was spiked into 1 .0 mL of mouse plasma (BiolVT, Lot MSE394920, CD-1 mouse, anticoagulant: sodium heparin, not filtered) that was prewarmed to 37 °C and stirred at 50 rpm with a magnetic stir bar.
• the starting concentration of lipids in plasma was thus 1 pM. Aliquots (50 pL) were taken after 0, 15, 30, 45, 60, and 120 minutes, transferred to microcentrifuge tubes and quenched with three volumes (150 pL) of ice cold acetonitrile/methanol (4:1 ). Positive control incubations utilized the same plasma, with Benfluorex (1 pM) as the substrate with Labetalol (1 .0 pgl) as the in situ disappearance. The quenched solutions were vortexed, centrifuged for 5 minutes at 13,000 rpm, and supernatant (100 pL) transferred to a 96-well plate and diluted with water (200 pL, 0.1 % FA).
• Elution gradient was as follows: time, 0.5min: 20% B; 0.5-2 min: 20-100% B; 2-4.8 min: 100% B; 4.8-5.45 min: 100-20% B.
• Mass spectrometry was in positive scanning mode from 600-1100 m/z.
• the peak of the molecular ion of the lipids was integrated in extracted ion chromatography (XIC) using Xcalibur software (Thermo Fisher).
• the relative peak area compared to T 0, after normalization by the peak area of the internal standard, was used as the percentage of the lipid remaining at each time point.
• T 1/2 values were calculated using the first-order decay model.
• Example 35 LNP Encapsulation of mRNA
• mCherry mRNA was synthesized by T7 RNA polymerase mediated in vitro transcription (IVT) of a linearized DNA template, using full substitution of uridine with N1 -Methylpseudouridine. A Cap1 structure was added to the 5’ end of the mRNA co- transcriptionally and a 3’ polyadenosine tail was encoded by the DNA template. Post IVT, mRNA was purified using a two-step chromatography process using OligoDT affinity chemistry for bulk capture and ion-pair reverse phase chemistry to remove residual impurities.
• TNF Tris buffer dilution and tangential flow filtration
• N nitrogen
• P negatively-charged nucleic acid phosphate
• the diameter of the nanoparticles was measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) instrument. Size measurement was carried out in pH 7.4 Tris buffer at 25°C in relevant disposable capillary cells. A non-invasive back scatter system (NIBS) with a scattering angle of 173° was used for size measurements.
• NIBS non-invasive back scatter system
• Frozen LNP were thawed and diluted to 100 pg mRNA/mL with sterile water for injection. An appropriate volume of LNP was added to provide 0, 0.3, 0.6, or 2 pg RNA per well in duplicate and mixed by re-pipetting. The cells were then incubated for 1 hour at 37°C in a CO2 incubator, washed three times with phosphate buffered saline, resuspended in 400 pL of medium in a deep-well 96-well plate, and incubated at 37°C in a CO2 incubator on an orbital shaker at 900 RPM.
• N nitrogen
• P nucleic acid phosphate
• an anti-CD5 mAb was conjugated to the above LNP to generate tLNP.
• Purified rat anti-mouse CD5 antibody, clone 53-7.3 (BioLegend) was coupled to LNP via N-succinimidyl S-acetylthioacetate (SATA)-maleimide conjugation chemistry.
• SATA N-succinimidyl S-acetylthioacetate
• LNPs with DSPE-PEG(2000)-maleimide incorporated were formulated and stored at 4°C on the day of conjugation.
• the antibody was modified with SATA (Sigma-Aldrich) to introduce sulfhydryl groups at accessible lysine residues allowing conjugation to maleimide.
• the particle size (hydrodynamic diameter) and polydispersity index of the targeted lipid nanoparticles were determined using dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Size measurement was carried out in pH 7.4 Tris buffer at 25°C in relevant disposable capillary cells. A non-invasive back scatter system (NIBS) with a scattering angle of 173° was used for size measurements.
• NIBS non-invasive back scatter system
• the apparent pKa of ionizable lipid in the lipid nanoparticle was determined using 6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt (TNS salt, Toronto Research Chemicals, Toronto, ON, Canada). Lipid nanoparticles were diluted in I xDulbecco's PBS to a concentration of 1 mM total lipids. TNS salt was prepared as a 1 mg/mL stock solution in DMSO and then further diluted using distilled water to a working solution of 60 pg/mL (179 mM).
• Diluted lipid nanoparticle samples were further diluted to 90 pM total lipids in 165 pL of buffered solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCI, and final TNS concentration of 1 .33 pg/mL (4 pM) with the pH ranging from 3.5 to 12.2.
• fluorescence intensity was measured at room temperature in a BioTek Synergy H1 plate reader using excitation and emission wavelengths of 321 and 445 nm, respectively.
• the fluorescence signal was blank subtracted and plotted as a function of the pH, then analyzed using a nonlinear (Boltzmann) regression analysis with the apparent pKa determined as the pH giving rise to half maximal fluorescence intensity as calculated by the Henderson-Hasselbalch equation.
• the tLNPs made in this Example are based on a reasonably conventional lipid composition, plus a functionalized PEG-lipid for conjugation of the targeting moiety and the herein disclosed ionizable cationic lipids.
• the conventional composition provides a good platform for assessing the contribution of the ionizable lipid to the tLNP’s properties and a baseline from which to assess further optimization of the overall compositions.
• all of the tLNP incorporating Compounds A-2 or A-1 1 thru A-15 had hydrodynamic diameters and polydispersity indices within the acceptable ranges of 50-150 nm and ⁇ 0.2 for PDI. Encapsulation efficiency is acceptable at >80% although >85% and >90% are preferred. All of the tested Compounds exceeded the >90% threshold (although one of the benchmark lipids, 10a, did not).
• mice splenic T cells were isolated from mechanically dissociated mouse spleens using a standard T cell isolation kit (Stem Cell Technologies #19851 ). Isolated T cells were cultured in complete RPMI medium supplemented with murine interleukin-2 in the presence of CD3/CD28 T cell activation beads (Gibco #1 14530) for 3 days. Following activation, T cells were magnetically separated from the activation beads and transferred to a 96-well plate at a concentration of 2x10 5 cells per well in 100 pL of complete RPMI medium.
• tLNP formulations as described in Example 35 were diluted to 100 pg/mL and 6 pL (0.6 pg) of tLNP was added to each well of cells to be tested.
• Cells were incubated with tLNPs at 37°C for 1 hour before tLNPs were washed away by centrifuging the plate, removing the supernatant, and replacing with fresh medium. Transfected cells were then returned to the incubator overnight. The next day, cells were washed and resuspended in stain buffer containing fluorescently tagged antibodies against T cells markers for 30 minutes before a final wash. After washing, cells were resuspended in stain buffer and run on the Novocyte Quanteon flow cytometer to detect mCherry expression as well as murine T cell markers. Results for CD3 + T cells are depicted in Figure 6.
• tLNP incorporating Compound A-1 1 and benchmark lipid 10p gave robust and comparable results with transfections rates about or over 80% and a high level of expression. Transfection with tLNP incorporating Compounds A-12 thru A-15 or the benchmark lipids 10a and 10f all resulted in similar levels of expression, less than A-11 and 10p but still substantial. Transfections rates varied from about 20% to about 60%. By comparison, the results for tLNP incorporating Compound A-2 were poor, but still positive. The superior performance of Compound A-1 1 among the disclosed compounds tested here correlates with it being the only one of those Compounds with a measured pKa between 6 and 7. However, performance of the other Compounds did not correlate with the size of their deviation from the preferred range for measured pKa showing that outside this range other factors dominate.
• Example 39 Targeted Transfection of T cells in vivo
• tLNP test articles were thawed at room temperature for 30 minutes and then diluted 1 :2 with sterile water for injection to achieve a final dose concentration of 100 pg/mL. 100 pL (10 pg) of each test article was then injected via the tail vein into 8-week-old female C57BI/6 mice. All treated mice were then sacrificed at 24 hours post-treatment and their spleens collected.
• tLNP incorporating Compound A-11 performed markedly better than any of the others with a transfection rate of around 7% and MFI distinctly greater than that achieved with the other tLNP.
• tLNP incorporating the three benchmark lipids performed comparably to each other with a transfection rate of around 2% while tLNP incorporating the other tested Compounds were not clearly distinguishable from background. It was surprising that of the Compounds tested here only Compound A-1 1 had a measured pKa between 6 and 7, but in light of that, the poorer performance of Compounds A-2 and A-12 thru A-15 was not unexpected. That tLNP incorporating Compound A-11 performed substantially better than tLNP incorporating the benchmark lipids, which do have measured pKa’s between 6 and 7 confirms that pKa is not the only determinant of performance.
• Example 40 Further embodiments
• Embodiment 1 An ionizable cationic lipid having a structure of
• Embodiment 3 An ionizable cationic lipid having a structure of
• Embodiment 4 The ionizable cationic lipid of Embodiment 1 or 2, wherein Y is O.
• Embodiment 5 The ionizable cationic lipid of Embodiment 1 or 2, wherein Y is NH.
• Embodiment 6 The ionizable cationic lipid of Embodiment 1 or 2, wherein Y is N-CH3.
• Embodiment 7 The ionizable cationic lipid of Embodiment 1 or 2, wherein Y is CH2.
• Embodiment 8 The ionizable cationic lipid of Embodiment 1 or 2,
• Embodiment 10 The ionizable cationic lipid of any one of
• Embodiments 1-9 comprising an R or Rcthat is straight-chain alkyl.
• Embodiment 11 The ionizable cationic lipid of any one of Embodiments 1-9, comprising an R or Rcthat is straight-chain alkenyl.
• Embodiment 12 The ionizable cationic lipid of any one of Embodiments 1-9, comprising an R or Rcthat is branched alkyl.
• Embodiment 13 The ionizable cationic lipid of any one of Embodiments 1-9, comprising an R that is branched alkenyl
• Embodiment 14 The ionizable cationic lipid of any one of Embodiments 1-9, comprising an R or Rcthat is cycloalkyl-alkyl.
• Embodiment 15 The ionizable cationic lipid of any one of Embodiments 1-9, comprising an R or Rcthat is aryl-alkyl.
• Embodiment 16 The ionizable cationic lipid of any one of Embodiments 1-15, wherein each R or Regroup is the same.
• Embodiment 17 The ionizable cationic lipid of any one of Embodiments 1-15, wherein both R or Regroups stemming from a first branchpoint are the same and both R or Regroups stemming from a second branchpoint are the same, but the R or R c groups stemming the first branchpoint are different than the R or R c groups stemming from the second branchpoint.
• Embodiment 18 A lipid nanoparticle (LNP), comprising the ionizable cationic lipid of any one of Embodiments 1-17.
• Embodiment 19 The LNP of Embodiment 18, further comprising one or more of a phospholipid, a sterol, a co-lipid, and a PEG-lipid, or combinations thereof.
• Embodiment 20 The LNP of Embodiment 18, wherein the phospholipid comprises dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), or 1 ,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), or a combination thereof.
• DOPE dioleoylphosphatidyl ethanolamine
• DMPC dimyristoylphosphatidyl choline
• DSPC distearoylphosphatidylcholine
• DMPG dimyristoylphosphatidyl glycerol
• DPPC dipalmitoyl phosphatidylcholine
• DAPC 1,2-diarachidoyl-s
• Embodiment 22 The LNP of any one of Embodiments 18-21 , wherein the co-lipid comprises cholesterol hemisuccinate (CHEMS) or a quaternary ammonium headgroup containing lipid.
• CHEMS cholesterol hemisuccinate
• Embodiment 23 The LNP of Embodiment 22, wherein the quaternary ammonium headgroup containing lipid comprises 1 ,2-dioleoyl-3- trimethylammonium propane (DOTAP), N-(1 -(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium (DOTMA), or 3p-(N-(N',N'- Dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or combinations thereof.
• DOTAP 1,2-dioleoyl-3- trimethylammonium propane
• DOTMA N-(1 -(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium
• DC-Chol 3p-(N-(N',N'- Dimethylaminoethane)carbamoyl)cholesterol
• Embodiment 24 The LNP of any one of Embodiments 18-23, wherein the PEG-lipid comprises a PEG moiety of 1000-5000 Da molecular weight (MW).
• Embodiment 25 The LNP of any one of Embodiments 18-24, wherein the PEG-lipid comprises fatty acids with a fatty acid chain length of C14-C18.
• Embodiment 26 The LNP of any one of Embodiments 18-25, wherein the PEG-lipid comprises DMG-PEG2000 (1 ,2-dimyristoyl-rglycero-3- methoxypolyethylene glycol-2000), DPG-PEG2000 (1 ,2-dipalmitoyl-glycero-3- methoxypolyethylene glycol-2000), DSG-PEG2000 (1 ,2-distearoyl-glycero-3- methoxypolyethylene glycol-2000), DGG-PEG2000 (1 ,2-dioleoyl-glycero-3- methoxypolyethylene glycol-2000), DMPE-PEG200 (1 ,2-dimyristoyl-glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPE-PEG2000 (1 ,2- dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DSPE
• Embodiment 27 The LNP of any one of Embodiments 18-26, wherein the PEG-lipid comprises an optically pure glycerol moiety.
• Embodiment 28 The LNP of any one of Embodiments 18-27, further comprising a functionalized PEG-lipid.
• Embodiment 29 The LNP of Embodiment 28, wherein the functionalized PEG-lipid has been conjugated with a binding moiety.
• Embodiment 30 The LNP of Embodiment 29, wherein the binding moiety comprises an antigen-binding domain of an antibody.
• Embodiment 31 The LNP of any one of Embodiments 28-30, wherein the functionalized PEG-lipid comprises fatty acids with a fatty acid chain length of C16-C18.
• Embodiment 32 The LNP of Embodiment 31 , wherein the functionalized PEG-lipid comprises a dipalmitoyl lipid or a distearoyl lipid.
• Embodiment 33 The LNP of any one of Embodiments 18-32, comprising 40 to 60 mol% ionizable cationic lipid.
• Embodiment 34 The LNP of any one of Embodiments 19-33, comprising 7 to 30 mol% phospholipid.
• Embodiment 35 The LNP of any one of Embodiments 19-34, comprising 20 to 45 mol% sterol.
• Embodiment 36 The LNP of any one of Embodiments 19-35, comprising 1 to 30 mol% co-lipid.
• Embodiment 37 The LNP of any one of Embodiments 19-36, comprising 0 to 5 mol% PEG-lipid.
• Embodiment 38 The LNP of any one of Embodiments 19-37, comprising 0.1 to 5 mol% functionalized PEG-lipid.
• Embodiment 39 The LNP of any one of Embodiments 18-38, further comprising a nucleic acid.
• Embodiment 40 The LNP of Embodiment 39, wherein the weight ratio of total lipid to nucleic acid is 10:1 to 50:1 .
• Embodiment 41 The LNP of Embodiment 39 or 40, comprising mRNA.
• Embodiment 42 A method of delivering a nucleic acid into a cell comprising contacting the cell with the LNP of any one of Embodiments 39-41.

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