Ionizable lipids for nucleic acid delivery

Abstract

To provide ionizable lipids capable of transfecting cells with genetic material.SOLUTION: The present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] [Cross - Reference to Related Applications]

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 868,900, filed Jun. 29, 2019, and U.S. Provisional Patent Application No. 63 / 009,042, filed Apr. 13, 2020.

[0002]

[0003] Field

[0004] The disclosed subject matter generally relates to ionizable lipids, particularly ionizable lipids capable of transfecting living cells with genetic material.

Background Art

[0003]

[0006] Related prior art

[0007] The number of nucleic acid treatment strategies for diseases continues to increase, even for diseases without an initial genetic cause. Each nucleic acid therapy has a different form, chemistry, and charge and typically requires a different delivery modality.

[0004]

[0008] Lipofection has been studied since at least 1987 as a means of altering the genetic properties of cells through lipid - mediated gene delivery. Over the years, such lipids have been improved to better fit more situations. Cationic lipids such as DODAC, DOTMA, DDAB, and DOTAP were used in the 1990s but were proven to be too toxic for clinical applications.

[0005]

[0009] Clinically relevant approaches have been the development of ionizable lipids for human use. Examples of ionizable lipids include 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), dilinoleyl methyl-4-dimethylaminobutyrate (DLin-MC3-DMA (see, e.g., U.S. Patent No. 8,158,601), and 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA). DLin-MC3-DMA or "MC3" is used in Onpattro™ (trademark) patisiran, an approved drug, but has issues with shelf-life.

[0006]

[0010] There continues to be a need for more options for clinically relevant transfection lipids.

Summary of the Invention

[0007]

[0012] According to an embodiment of the present invention, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof.

Chem.

[0013] Wherein p is 0 or 1;

[0014] E1 is selected from -O-δ1, -OC(O)O-δ1, -OC(O)-δ1, -OC(O)N(Q)-δ1, -OC(O)S-δ1, -C(O)N(Q)-δ1, -C(O)O-δ1, -N(Q)C(O)-δ1, -N(Q)C(O)O-δ1, -N(Q)C(O)S-δ1, and -N(Q)C(O)N(Q)-δ1; Q is H, or C1-C5 alkyl; δ1 represents a bond linked to the R1 group;

[0015] R1 is

Chem.

[0016] R3 and R4 are each independently selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl; or R3 and R4 together contain oxygen (O) or up to two nitrogens (N) and are each independently optionally substituted with one or two substituents selected from C1-C6 alkyl, cyclopropyl, OH, and C1-C3 alkoxy groups to form a 4- to 6-membered ring;

[0017] R5 is selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, and 2-hydroxyethyl group;

[0018] R6 is selected from H and C1-C6 alkyl group;

[0019] a is 1, 2, 3, 4 or 5;

[0020] b and c are independently 0, 1, or 2;

[0021] c’ is 1, 2, 3, 4, or 5;

[0022] d is 1 or 2;

[0023] e is 0, 1, or 2;

[0024] E2 is selected from -OC(O)-δ2, -OC(O)O-δ2, -OC(O)N(Q)-δ2, -O-δ2, -OCH2CH2O-δ2, and -OC(O)(CH2)6C(O)O-δ2; Q is H or C1-C5 alkyl; δ2 represents a bond linked to the R2 group;

[0025] R2 is

Chemical formula

[0026] L1 and L2 are each independently a direct bond, -O-δ3, -CH2OC(O)-δ3, and -CH2O-δ3; δ3 represents a bond linked to the R7 and R8 groups;

[0027] R7 and R8 are each independently C4-C10 alkyl, C4 - C 10 alkenyl or C4 - C 10 alkynyl;

[0028] f is 0, 1, 2, 3, 4, or 5;

[0029] L3 is

Chemical formula

[0030] R9 is selected from H and C4 - C8 alkyl groups;

[0031] g is an integer in the range of 1 - 18;

[0032] h is 0, 1, 2, or 3;

[0008]

[0033] According to another embodiment of the present invention, a compound of formula (II), or a pharmaceutically acceptable salt thereof, is provided.

Chemical formula

[0034]

[0035] wherein E1 is selected from -OC(O)O - δ1, -OC(O) - δ1, -OC(O)N(Q) - δ1, and -OC(O)S - δ1; Q is H or C1 - C5 alkyl; δ1 represents a bond linked to the R1 group;

[0036] R1 is

Chemical formula

[0037] here,

[0038] each of R3 and R4 is independently selected from C1 - C6 alkyl groups; or R3 and R4 may together form a 5 - to 6 - membered ring optionally substituted with 1 - 2 substituents selected from C1 - C6 alkyl groups and containing 2 or fewer nitrogens (N);

[0039] R5 is selected from C1 - C6 alkyl and C3 - C6 cycloalkyl groups;

[0040] R6 is selected from H and C1-C6 alkyl groups;

[0041] a is 1, 2, 3, or 4;

[0042] b and c are independently 0, 1, or 2;

[0043] c' is 2, 3, or 4;

[0044] d is 2;

[0045] e is 0 or 1;

[0046] E2 is selected from -O-δ2, -OC(O)-δ2, -OCH2CH2O-δ2, and -OC(O)(CH2)6C(O)O-δ2, where δ2 represents the bond linked to the R2 group;

[0047] R2 is

Chemical formula

[0048] L1 and L2 are each independently a direct bond, -O-δ3, -CH2OC(O)-δ3, and -CH2O-δ3; δ3 represents the bond linked to the R7 and R8 groups;

[0049] R7 and R8 are each independently C4-C 10 alkyl, C4-C 10 alkenyl or C4-C 10 alkynyl,

[0050] f is 0, 1, 2, 3, 4, or 5;

[0051] L3 is

Chemical formula

[0052] R9 is selected from H and C4-C8 alkyl groups;

[0053] g is an integer in the range of 1-18;

[0054] h is 0, 1, or 2;

[0009]

[0055] According to another embodiment of the present invention, there is provided a compound of formula (II), or a pharmaceutically acceptable salt thereof.

Chemical formula

[0056] wherein E1 is selected from -OC(O)O-δ1, -OC(O)-δ1, -OC(O)N(Q)-δ1, and -OC(O)S-δ1; Q is H or C1-C5 alkyl; δ1 represents a bond linked to the R1 group,

[0057] R1 is

Chemical formula

[0058] where

[0059] R3 and R4 are each independently selected from C1-C6 alkyl groups; or R3 and R4 together may form a 5- to 6-membered ring containing 2 or fewer nitrogens (N) and optionally substituted with 1 or 2 substituents selected from C1-C6 alkyl groups;

[0060] R5 is selected from C1-C6 alkyl and cyclopropyl groups;

[0061] R6 is selected from H and C1-C6 alkyl groups;

[0062] a is 1, 2, 3, or 4;

[0063] b is 0 or 1;

[0064] c is 0, 1, or 2;

[0065] c' is 2, 3, or 4;

[0066] d is 2;

[0067] e is 1;

[0068] E2 is selected from -O-δ2, -OC(O)-δ2, -OCH2CH2O-δ2, and -OC(O)(CH2)6C(O)O-δ2, where δ2 represents a bond linked to the R2 group,

[0069] R2 is

Chem.

[0070] L1 and L2 are each a direct bond;

[0071] R7 and R8 are each independently selected from C4-C 10 alkyl groups;

[0072] f is 0 or 1;

[0073] L3 is

Chem.

[0074] R9 is selected from H and C4-C8 alkyl groups;

[0075] g is an integer in the range of 1 to 18;

[0076] h is 0, 1, or 2.

[0010]

[0077] According to another embodiment of the present invention, there is provided a compound of formula (III), or a pharmaceutically acceptable salt thereof.

Chem.

[0078]

[0079] R1 is

Chem.

[0080] wherein

[0081] R3 and R4 are each independently selected from C1-C6 alkyl groups; or R3 and R4 together may form a 5- or 6-membered ring optionally substituted with 1 or 2 substituents selected from C1-C6 alkyl groups and containing up to 2 nitrogens (N);

[0082] R5 is selected from C1-C6 alkyl and cyclopropyl groups;

[0083] R6 is selected from H and C1-C6 alkyl groups;

[0084] a is 1, 2, 3, or 4;

[0085] b is 0 or 1;

[0086] c is 0, 1, or 2;

[0087] c' is 2, 3, or 4;

[0088] d is 2;

[0089] e is 1;

[0090] E2 is selected from -O-δ2, -OC(O)-δ2, -OCH2CH2O-δ2, and -OC(O)(CH2)6C(O)O-δ2, where δ2 represents the bond linked to the R2 group;

[0091] R2 is

Chemical formula

[0092] L1 and L2 are each a direct bond;

[0093] R7 and R8 are each independently selected from C4-C 10 alkyl groups;

[0094] f is 0 or 1;

[0095] L3 is

Chemical formula

[0096] R9 is selected from H and C4-C8 alkyl groups;

[0097] g is an integer in the range of 1-18;

[0098] h is 0, 1, or 2.

[0011]

[0099] According to an embodiment of the present invention, R1 is one of

[0100]

Chemical formula

[0012]

[0101] In a further embodiment, each R2 is independently

[0102]

Chemical formula

[0013]

[0103] In still another embodiment of the present invention, E1 is selected from

Chemical formula

[0014]

[0104] In still another embodiment of the present invention, E2 is selected from

Chemical formula

[0015]

[0105] According to an embodiment of the present invention, a compound selected from the group consisting of the compounds listed in Table 1 or its pharmaceutically acceptable salt is provided.

Table 1-A

Table 1-B

Table 1-C

Table 1-D

Table 1-E

[0016]

[0106] According to an embodiment of the present invention, compounds listed in Table 2 are also provided.

Table 2-A

Table 2-B

Table 2-C

Table 2-D

Table 2-E

Table 2-F

Table 2-G

Table 2-H

Table 2-I

Table 2-J

Table 2-K

Table 2-L

Table 2-M

Table 2-N

Table 2-O

Table 2-P

Table 2-Q

Table 2-R

Table 2-S

Table 2-T

Table 2-U

Table 2-V

Table 2-W

[0017]

[0107] The ionizable lipid of the present invention has an asymmetric center and exists as a racemate, a racemic mixture, individual enantiomers, an enantiomeric mixture, individual diastereomers, and as a diastereomeric mixture together with all possible isomers such as tautomers and mixtures thereof.

[0018]

[0108] In embodiments of the present invention, the experimental pKa of the formulated lipid nanoparticles containing the lipid of Table 1 is calculated using the 2-(p-toluidinyl)naphthalene-6-sulfonic acid (TNS) assay. The procedure for the TNS assay is described in Example 25.

[0019]

[0109] According to embodiments of the present invention, there is provided a lipid mixed composition comprising any of the above compounds in combination with a structural lipid, a sterol, and a stabilizer and at least one therapeutic agent.

[0020]

[0110] In embodiments, the structural lipid comprises one or more structural lipids selected from the group consisting of DSPC, DSPE, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the structural lipid is DSPC. In other embodiments, the structural lipid is DOPE.

[0021]

[0111] In embodiments, the stabilizer comprises one or more surfactants and polymer-conjugated lipids.

[0022]

[0112] In embodiments, the compound is present at about 10 Mol% - 90 Mol%, the structural lipid is present at about 0 - 50 Mol%, the sterol is present at about 0 - 45 Mol%, the stabilizer is present at 0 - 10 Mol%, and the total mol% of all components is 100 mol%.

[0023]

[0113] In a further embodiment, the compound is present at about 40 Mol% - 60 Mol%, the structural lipid is present at about 11 - 40 Mol%, and the total mol% of all components is 100 mol%.

[0024]

[0114] In embodiments, the molar ratio of the compound to the remaining components is 30 Mol% - 70 Mol%.

[0025]

[0115] In embodiments, the compound is present at 40 Mol%, DSPC is present at 20 Mol%, cholesterol is present at 37.5 Mol%, and polyoxyethylene (10) stearyl ether is present at 2.5 Mol%. In other embodiments, the compound is present at 40 - 47.5 Mol%, DSPC is present at 12.5 Mol%, cholesterol is present at 38.5 - 46 Mol%, and PEG-DMG 2000 is present at 1.5 Mol%. In still other embodiments, the compound is present at 40 - 47.5 Mol%, DOPE is present at 12.5 Mol%, cholesterol is present at 38.5 - 46 Mol%, and PEG-DMG 2000 is present at 1.5 Mol%.

[0026]

[0116] In several embodiments, the lipid mix composition further comprises a targeting moiety. In an embodiment, the sterol is cholesterol. In an embodiment, the therapeutic agent comprises one or more nucleic acids. In an embodiment, the therapeutic agent comprises a polypeptide.

[0027]

[0117] In an embodiment, the stabilizer is selected from the group consisting of PEG-DMG2000, polyoxyethylene (10) stearyl ether, polyoxyethylene (40) stearate, polysorbate 80, polyoxyethylene (4) lauryl ether, polyoxyethylene (20) stearyl ether, polyoxyethylene (23) lauryl ether, and D-α-tocopherol polyethylene glycol 1000 succinate.

[0028]

[0118] According to an embodiment of the present invention, there is provided a lipid mix composition in the form of lipid particles.

[0029]

[0119] Also provided is the use of a compound of the present invention for preparing a therapeutic agent for ex vivo administration of a therapeutic agent to cells. In a further embodiment, the therapeutic agent is a pharmaceutical preparation for use in the treatment of cancer. In some embodiments, the therapeutic agent is a pharmaceutical preparation for use in T cell modification. In yet another embodiment, the therapeutic agent is a vaccine.

[0030]

[0120] In some embodiments, the therapeutic agent comprises a nucleic acid therapeutic. In these embodiments, the nucleic acid therapeutic is mRNA, siRNA, miRNA, guide RNA, synthetic guide RNA, artificial chromosome, circular or linearized DNA, DNA minicircle, or msDNA. In some of these embodiments, the mRNA is a self-replicating RNA molecule.

[0031]

[0121] According to an embodiment of the present invention, there is provided the use of a lipid mix composition for the preparation of a vaccine. In an embodiment, the vaccine relates to the prevention of viral diseases. In an embodiment, the vaccine relates to coronavirus infection.

[0032]

[0122] According to an embodiment of the present invention, there is provided the use of the lipid mix composition for use in a therapeutic or cancer vaccine. According to an embodiment of the present invention, there is provided the use of the lipid mix composition for use in protein regulation in vivo or ex vivo.

[0033]

[0123] In a plurality of embodiments, the mRNA is a self-replicating RNA molecule.

[0034]

[0124] According to an embodiment of the present invention, there is provided the use of the lipid mix composition in the preparation of an agent for regulating human T cells, CAR-T, TCR, gene editing, or allogeneic T cells.

[0035]

[0125] Further provided are embodiments of the use of the lipid mix composition of the present invention in the preparation of an agent for regulating T cells, where the T cells are isolated from a patient or are T cells that have been specifically genetically engineered against T cells or allogeneic T cells.

[0036]

[0126] In an embodiment, the lipid mix composition further comprises a polypeptide. In an embodiment, the lipid mix composition further comprises both a polypeptide and a nucleic acid.

[0037]

[0127] In an embodiment, the lipid mix composition further comprises a ribonucleoprotein.

[0038]

[0128] According to the present invention, there is provided a compound in which one of the hydrogens is substituted with a halogen. In an embodiment, the halogen is iodine or fluorine.

[0039]

[0129] According to an embodiment of the present invention, there is provided the above compound, or a pharmaceutically acceptable salt thereof, wherein the experimental pKa of the nanoparticle is in the range of 5.6 to 7.1.

[0040]

[0130] According to an embodiment of the present invention, there is provided a pharmaceutical composition comprising the above compound and at least one pharmaceutically acceptable carrier or excipient.

[0041]

[0131] Further features and advantages of the present disclosure will become apparent from the following detailed description in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0042]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Mode for Carrying Out the Invention

[0043]

[0146] Throughout the accompanying drawings, similar features are denoted by similar reference numerals.

[0044]

[0147] In the present disclosure, the term "comprising" is used in a non-limiting sense, meaning that the item preceding that term is included, but items not specifically recited are not excluded. In embodiments that include or may include a defined feature or variable or parameter, alternative embodiments are understood to consist of or consist essentially of such feature or variable or parameter. Reference to an element by the indefinite article "a" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the element.

[0045]

[0148] In the present disclosure, a description based on the endpoints of a numerical range includes all numbers, including all whole numbers, all integers, and all intermediate fractional numbers within that range (for example, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.). In the present disclosure, the singular forms “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to a composition containing “a compound” includes a mixture of substances.

[0046]

[0149] In the present disclosure, the term “or” is generally used in the sense of including “and / or” unless the context clearly indicates otherwise.

[0047]

[0150] “Lipid” can be a fatty acid derivative or a sterol, or a lipid such as a lipidoid (e.g., C12 - 200) or a substance within a polymer conjugate lipid, and means a structurally diverse group of organic compounds characterized by being insoluble in water but soluble in many organic solvents.

[0048]

[0151] “Lipid mix composition”. The lipid mix composition refers to the type of components, the ratio of components, and the ratio of all components to the nucleic acid payload. For example, a lipid mix composition of 40 Mol% ionizable lipid, 20 Mol% structural lipid, 17.5 Mol% sterol, and 2.5 Mol% stabilizer would be a lipid mix composition.

[0049]

[0152] As used herein, “N / P” is the ratio of the number of moles of the amine groups of the ionizable lipid to the number of moles of the phosphate groups of the nucleic acid. In embodiments of the present invention, the N / P ratio is from 4 to 10, and the most preferred ratio is N / P from 4 to 12. In one embodiment, the N / P ratio is 10. The nucleic acid component binds to this lipid mix composition at a pre - considered ratio, for example, the ratio of the amine (N) of the ionizable lipid to the phosphate (P) of the nucleic acid being N / P 4, N / P 6, N / P 8, N / P 10, N / P 12, or another relevant specific N / P ratio to form lipid - nucleic acid particles, or LNPs.

[0050]

[0153] "Lipid particles". The present invention provides lipid particles produced from the lipid mix composition described above. Lipid particles exhibit the physical organization between the lipid mix composition and the therapeutic agent and between the components. Lipid nanoparticles are lipid particles. Lipid particles are generally spherical aggregates of lipids, nucleic acids, cholesterol, and stabilizers. Positive and negative charges, ratios, and hydrophilicity and hydrophobicity determine the physical structure of lipid particles with respect to the size and orientation of the components. The structural organization of these lipid particles can result in an aqueous interior having a minimal bilayer, such as a liposome, or can have a solid interior, such as a solid nucleic acid lipid nanoparticle. There can be single or multiple forms of phospholipid monolayers or bilayers. Lipid particles are between 1 and 1000 μm in size.

[0051]

[0154] When referring to cells in vitro, "viability" means the ability to continue to grow, divide, and continue to grow and divide as normal for the cell type or tissue culture strain. The survival of cells is affected by harsh conditions or treatments. The viability of cells is very important in ex vivo therapy or parenteral administration.

[0052]

[0155] "Ionizable lipid". The compounds of the present invention contain ionizable lipids. As used herein, the term "ionizable lipid" means a lipid that is cationic or becomes ionizable (protonated) when the pH drops below the pKa of the ionizable group of the lipid, but is more neutral at higher pH values. At pH values lower than the pKa, the lipid can bind to negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term "ionizable lipid" includes either a lipid that bears a positive charge upon a decrease in pH and many lipid species that bear a net positive charge at selected pHs, such as physiological pH.

[0053]

[0156] Ionizable lipids or compounds are present in the lipid compositions according to other embodiments of the present invention, preferably in some embodiments in an amount of about 30 to about 70 Mol%, in other embodiments about 30 Mol%, in other embodiments about 40 Mol%, in yet other embodiments about 50 Mol%, about 60 Mol% (where "Mol%" means the percentage of the total number of moles of a specific component). The term "about" in this paragraph means a range of plus or minus 5 Mol%. DODMA or 1,2-dioleoyl-oxy-3-dimethylaminopropane is an ionizable lipid such as DLin-MC3-DMA or (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate.

[0054]

[0157] Lipid particles can be generated from lipid formulations containing the ionizable lipids of the present invention.

[0055]

[0158] Structural lipids, also known as "helper lipids" or "neutral lipids", are incorporated into the lipid formulations and lipid particles of the present invention in some embodiments. The lipid formulations and lipid particles of the present invention contain one or more structural lipids in an amount of about 10 to 40 Mol% of the composition. Suitable structural lipids support the formation of particles during manufacture. Structural lipids refer to any one of many lipid species that exist in either an anionic, uncharged or neutral zwitterionic form at physiological pH. Representative structural lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramide, sphingomyelin, dihydrosphingomyelin, kephalin, and cerebroside.

[0056]

[0159] Exemplary structural lipids include zwitterionic lipids such as distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (trans DOPE). In one preferred embodiment, the structural lipid is distearoyl phosphatidylcholine (DSPC).

[0057]

[0160] In another embodiment, the structural lipid is any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol such as dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacyl phosphatidylserine, diacyl phosphatidic acid, and other anionic modifying groups attached to neutral lipids. Other suitable structural lipids include glycolipids (e.g., monosialoganglioside GM1).

[0058]

[0161] Stabilizing agents are included in embodiments of lipid formulations to ensure the integrity of the mixture. Stabilizing agents are molecules that disrupt or assist in the formation of intermolecular hydrophobic-hydrophilic interactions. Suitable stabilizing agents include, but are not limited to, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate), Myrj 52 (polyoxyethylene (40) stearate), and Brij(™) S10 (polyoxyethylene (10) stearyl ether). Polyethylene glycol-conjugated lipids may also be used. Stabilizing agents may be used alone or in combination with each other.

[0059]

[0162] In some embodiments, the stabilizing agent occupies about 0.1 to 3 Mol% of the total lipid mixture. In some embodiments, the stabilizing agent occupies about 0.5 to 2.5 Mol% of the total lipid mixture. In some embodiments, the stabilizing agent is present in more than 2.5 Mol%. In some embodiments, the stabilizing agent is present at 5 Mol%. In some embodiments, the stabilizing agent is present at 10 Mol%. In some embodiments, the stabilizing agent is about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, etc. In other embodiments, the stabilizing agent is 2.6 to 10 Mol% of the lipid mixture. In other embodiments, the stabilizing agent is present in more than 10 Mol% of the lipid mixture.

[0060]

[0163] Steroids are included in a preferred lipid mix composition for several applications, and the lipid particles created therefrom contain sterols such as cholesterol and plant sterols. In some embodiments, in the lipid mix of the present invention, cholesterol is present at about 30-50 Mol% of the final lipid mix. Alternatively, cholesterol is present at about 35-41 Mol% of the final lipid mix. In other embodiments, no sterol is present.

[0061]

[0164] Nucleic acids. The lipid mix compositions and lipid particles of the present invention are useful for systemic or local delivery of nucleic acids. As used herein, the term "nucleic acid therapeutic" (NAT) means any oligonucleotide or polynucleotide whose delivery to cells causes a desired effect. This definition includes diagnostic agents and research reagents that follow the same physical principles provided by the present invention. Fragments containing up to 50 nucleotides are generally referred to as oligonucleotides, and longer fragments are called polynucleotides. In certain embodiments, the oligonucleotides of the present invention are 20-50 nucleotides in length. In embodiments of the present invention, the oligonucleotide is 996-4500 nucleotides in length, such as in the case of messenger RNA.

[0062]

[0165] The term "nucleic acid" also means ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar-backbone oligonucleotides, other nucleotides, nucleotide analogs, and combinations thereof, and can be single-stranded, double-stranded, or can contain portions of both double-stranded and single-stranded sequences as appropriate. mRNA can be modified or unmodified, can be base-modified, and can contain different types of capping structures, such as Cap1.

[0063]

[0166] As used herein, the terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to single-stranded and double-stranded polymers of nucleotide monomers, such as internucleotide phosphodiester linkages, such as 3'-5' and 2'-5', inverted linkages, such as 3'-3' and 5'-5', branched structures, or 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by nucleotide analogs. Polynucleotides have associated counterions such as H+, NH4+, trialkylammonium, Mg2+, Na+, etc. Polynucleotides can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or a chimeric mixture thereof. Polynucleotides can consist of internucleotides, nucleobases, and / or sugar analogs.

[0064]

[0167] As used herein, the term "polypeptide" encompasses "oligopeptides" and "proteins" which are therapeutic agents in some embodiments, as well as their tertiary and quaternary structures. Oligopeptides generally consist of 2 to 20 amino acids. A polypeptide is a single straight chain of any length of many amino acids held together by amide bonds. Proteins can consist of one or more and can include structural proteins, energy catalysts, albumin, hemoglobin, immunoglobulins, and enzymes.

[0065]

[0168] "Ribonucleoprotein" is in some embodiments a complex of Cas9 protein and guide RNA. In some embodiments, ribonucleoprotein is a therapeutic agent referred to in several aspects of the present invention.

[0066]

[0169] Currently, nucleic acid therapeutic substances include deoxyribonucleic acid, complementary deoxyribonucleic acid, complete genes, ribonucleic acid, oligonucleotides, and ribozymes for gene therapy targeting various diseases such as cancer, infectious diseases, genetic diseases, and neurodegenerative diseases. As described herein, nucleic acid therapeutic substances (NATs) are incorporated therein during the formation of lipid particles together with the compounds of the present invention. More than one nucleic acid therapeutic substance can be incorporated in this way. They may be of natural origin or, more generally, may be synthetic or cultured and propagated. Examples of nucleic acid therapeutic substances include, but are not limited to, antisense oligonucleotides, ribozymes, microRNAs, mRNAs, ribozymes, tRNAs, tracrRNAs, sgRNAs, snRNAs, siRNAs, shRNAs, ncRNAs, miRNAs, mRNAs, pre-concentrated DNA, pDNA, or aptamers. Nucleic acid reagents are used to silence genes (e.g., with siRNA), express genes (e.g., with mRNA), edit the genome (e.g., with CRISPR / Cas9), and reprogram cells to return to the originating organism (e.g., reprogramming immune cells for cancer treatment in ex vivo cell therapy; autologous or allogeneic transport).

[0067]

[0170] The nucleic acids present in the lipid particles according to the present invention include nucleic acids in any known form. The nucleic acids used in the present invention can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA include structural genes, genes containing regulatory and terminal regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA include siRNA and other RNA interference reagents. Single-stranded nucleic acids include antisense oligonucleotides, guide RNAs such as CRISPR-Cas9 gRNA, ribozymes, microRNAs, mRNAs, and oligonucleotides that form triplexes. More than one nucleic acid can be incorporated into the lipid particles, for example, mRNA and guide RNA together, or each of different types, or in combination with proteins.

[0068]

[0171] Plasmid DNA is a preferred nucleic acid incorporated in embodiments of the present invention. A plasmid is a separate DNA molecule that is separated from chromosomal DNA within a cell and can replicate independently. Plasmids range in size from less than 1000 nucleotides to tens of thousands of nucleotides. The most common form is a small circular double-stranded DNA. Plasmids can be synthesized and delivered to mammalian cells for therapeutic purposes. Synthetic plasmids are used as vectors in molecular cloning and serve to drive the replication of recombinant DNA sequences within a host organism. Plasmids can be introduced into cells by transformation using physical methods such as electroporation or by transfection enhanced by lipid particles using chemical means as in the present invention. These lipid mix compositions of the present invention have several advantages over physical techniques, for example, i) high biocompatibility and low toxicity in cell and tissue systems, ii) relative ease of manufacture, iii) the lipophilic matrix is less susceptible to the erosion phenomena observed in polymer systems, iv) increased in vivo circulation half-life due to being invisible to the immune system.

[0069]

[0172] In some cases, the nucleic acid encodes a genetically engineered receptor that specifically binds to a ligand, such as a recombinant receptor, and a molecule involved in a metabolic pathway or a functional portion thereof. Alternatively, the molecule involved in the metabolic pathway is a recombinant molecule that includes an exogenous entity. The genetically engineered receptor and the molecule involved in the metabolic pathway can be encoded by one nucleic acid or two or more different nucleic acids. In some examples, the first nucleic acid may encode a genetically engineered receptor that specifically binds to a ligand, or the second nucleic acid may encode a molecule involved in a metabolic pathway.

[0070]

[0173] As used herein, "therapeutic agent" includes the nucleic acid therapeutic substances described herein, the polypeptides described herein, as well as polysaccharides, salts, small molecules, inorganic ions, and radionuclides.

[0071]

[0174] Lipid particles according to some embodiments of the present invention can be characterized by electron microscopy. Particles of the present invention having a substantially solid core have an electron-dense core as seen by electron microscopy. One such structure is disclosed by Cullis et al. in US Patent No. 9,758,795. Electron-dense is defined as the area-average electron density (as seen in 2-D cryo-EM images) of the inner 50% of the projected area of ​​the solid core particle is equal to or greater than x% (x=20%, 40%, 60%) of the maximum electron density at the periphery of the particle. Electron density is calculated as the absolute value of the difference of the image intensity of the region of interest from the background intensity of regions not containing nanoparticles.

[0072]

[0175] The lipid particles of the present invention can be sized using a device such as a Malvern™ Zetasizer™, which measures the size of particles in solution. The particles have an average particle diameter of about 15 to about 300 nm. Another term for lipid particles is "LNP," which stands for "lipid nanoparticle." In some embodiments, the average particle diameter is greater than 300 nm. In some embodiments, the lipid particles have a diameter of about 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, or 50 nm or less. In one embodiment, the lipid particles have a diameter of about 50 to about 150 nm. Smaller particles generally exhibit increased in vivo circulation lifetimes compared to larger particles. Smaller particles have increased ability to reach tumor sites than larger nanoparticles. In one embodiment, the lipid particles have a diameter of about 15 to about 50 nm.

[0073]

[0176] Mixing. Lipid particles according to embodiments of the present invention can be prepared by standard T-tube mixing techniques, turbulent mixing, trituration mixing, agitation-promoted sequential self-assembly, or passive mixing of all elements involving self-assembly of elements into nanoparticles. A variety of methods have been developed for formulating lipid nanoparticles (LNPs) containing gene drugs. Suitable methods are disclosed, for example, in U.S. Patent No. 5,753,613 by Ansell, Mui and Hope, and in U.S. Patent No. 6,734,171 by Saravolac et al. These methods involve mixing pre-formed lipid particles with a nucleic acid therapeutic substance (NAT) in the presence of ethanol or mixing lipids dissolved in ethanol with an aqueous medium containing NAT, to obtain lipid particles with an NAT encapsulation efficiency of 65-99%. Both of these methods rely on the presence of ionizable lipids to achieve encapsulation of NAT and stabilizers to inhibit aggregation and formation of large structures. The properties of the resulting lipid particle systems, such as size and NAT encapsulation efficiency, are sensitive to various lipid mix composition parameters, such as ionic strength, lipid and ethanol concentrations, pH, NAT concentration, and mixing rate. 1

[0177] Microfluidic two-phase droplet technology has been applied to produce monodisperse polymer microparticles for drug delivery or large vesicles for encapsulation of cells, proteins, or other biomolecules. The use of hydrodynamic flow focusing, a common microfluidic technique that provides rapid mixing of reagents, has been demonstrated for creating monodisperse liposomes of controlled size.

[0074]

[0178] Generally, parameters such as the relative lipid and NAT concentrations during mixing, as well as the mixing speed, are difficult to control using current formulation techniques, resulting in variability in the properties of NAT generated both within and between preparations. Automated microfluidic devices, such as the NanoAssemblr® instrument (Precision Nanosystems Inc, Vancouver, Canada), enable the rapid and controlled manufacture of nanomedicines (liposomes, lipid nanoparticles, and polymeric nanoparticles). The NanoAssemblr® instrument achieves controlled molecular self-assembly of nanoparticles through a microfluidic mixing cartridge that enables millisecond mixing of nanoparticle components at the nanoliter, microliter, or larger scale, with custom production or parallelization. Rapid mixing at small scales enables reproducible control of particle synthesis and quality that is not possible with larger devices.

[0075]

[0179] Preferred methods include devices such as microfluidic mixing devices like the NanoAssemblr® Spark™, Ignite™, Benchtop™, and NanoAssemblr® Blaze™ instruments to achieve encapsulation of nearly 100% of the nucleic acid used in the formation process into particles in one step. In one embodiment, lipid particles are prepared by a process in which about 90% to about 100% of the nucleic acid used in the formation process is encapsulated into the particles.

[0076]

[0180] U.S. Patent Nos. 9,758,795 and 9,943,846 by Cullis et al. describe methods using small-volume mixing techniques and novel formulations derived therefrom. U.S. Patent Application Publication No. 20160022580 by Ramsay et al. describes a more advanced method of formulating different materials using small-volume mixing techniques and products. U.S. Patent No. 9,943,846 by Walsh et al. discloses a microfluidic mixer having different channels and wells for elements to be mixed. International Publication No. 2017117647 by Wild, Leaver, and Taylor discloses a microfluidic mixer having a disposable sterile passageway. U.S. Patent No. 10,076,730 by Wild, Leaver, and Taylor discloses a branched donut-shaped micro mixing geometry and its application to micro mixing. International Publication No. 2018006166 by Chang, Klaassen, Leaver, et al. discloses a programmable automated micromixer and a mixing chip therefor. U.S. Design Nos. D771834, D771833, D772427, D803416 by Wild and Leaver, and U.S. Design Nos. D800335, D800336, and D812242 by Chang et al. disclose mixing cartridges having microfluidic channels and mixing geometries for mixer devices sold by Precision Nanosystems Inc.

[0077]

[0181] In embodiments of the present invention, a device for biological microfluidic mixing is used to prepare lipid particles according to embodiments of the present invention. The device includes a flow of a first and a second reagent, which are supplied to a microfluidic mixer, and the lipid particles are collected at an outlet or appear in a sterile environment.

[0078]

[0182] The first flow includes a therapeutic agent in a first solvent. Suitable first solvents include solvents in which the therapeutic agent is soluble and miscible with a second solvent. Suitable first solvents include aqueous buffers. Representative first solvents include citrate and acetate buffers or other low pH buffers.

[0079]

[0183] The second stream contains the lipid mixture substance in a second solvent. Suitable second solvents include solvents in which the ionizable lipid according to the embodiments of the present invention is soluble and which are miscible with the first solvent. Suitable second solvents include 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, and alcohols. Representative second solvents include 90% aqueous ethanol or absolute ethanol.

[0080]

[0184] In one embodiment of the present invention, a suitable device includes one or more microchannels (i.e., channels having a maximum dimension of less than 1 millimeter). In one example, the microchannels have a diameter of about 20 to about 300 μm. In a plurality of examples, at least one region of the microchannel has one or more surfaces having a main flow direction and at least one groove or protrusion defined therein, and the groove or protrusion forms an angle with the main direction as described in U.S. Patent No. 9,943,846 (e.g., a zigzag herringbone mixer), or has a branched donut-shaped flow as described in U.S. Patent No. 10,076,730. To achieve the maximum mixing rate, it is advantageous to avoid excessive fluid resistance in front of the mixing region. Thus, one example of the device has a non-microfluidic channel having a dimension greater than 1000 μm for delivering fluid to a single mixing channel.

[0081]

[0185] Less automated micro mixing methods and devices, such as those disclosed in Zhang, S-h et al. 2, and U.S. Patent Application Publication No. 20040262223 to Stroock A et al., and Jeffs, LB et al. 3, are also useful for creating the lipid particle compositions of the present invention.

[0082]

[0186] The ionizable lipids of the present invention can be used to deliver therapeutic agents to cells in vitro or in vivo. In certain embodiments, the therapeutic agent is a nucleic acid that is delivered to cells using the nucleic acid-lipid particles of the present invention. The nucleic acid can be siRNA, miRNA, LNA, plasmid, replicon, mRNA, guide RNA, transposon, or a single gene. In other embodiments, the therapeutic agent delivered to one or more cells is a gene editing technology. Gene editing technology is a group of scientific and technological methods that can change the DNA of an organism and enable the addition, removal, or modification of genetic material at specific positions within the genome. There are several methods for genome editing, such as CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 9), TALEN, and ZFN4.

[0083]

[0187] In other embodiments, the therapeutic agent is an oligopeptide, polypeptide, or protein that is delivered to cells using the peptide-lipid particles of the present invention. In other embodiments, the therapeutic agent is a mixture of a nucleic acid and a protein component, such as Cas9. The methods and lipid mix compositions can be readily adapted for the delivery of any suitable therapeutic agent for the treatment of any disease or disorder that would benefit from such treatment.

[0084]

[0188] In certain embodiments, the present invention provides a method for introducing nucleic acids into cells (i.e., transfection). Transfection is a technique commonly used in molecular biology for the introduction of nucleic acid therapeutic substances (or NATs) from the extracellular to the intracellular space for the purpose of transcription, translation, and expression of the gene(s) to be delivered. Transfection efficiency is generally measured by i) the percentage of live or fixed cells imaged (for detection of fluorescent proteins) and by flow cytometry among the total treated population of cells showing positive expression of the delivered gene, or ii) the intensity or amount of protein expressed by the treated cell(s) analyzed by live or fixed cell imaging or flow cytometry, or iii) defined using protein quantification techniques such as ELISA or Western blot. These methods can be carried out by contacting the particles or lipid mix compositions of the present invention with the cells for a time sufficient for intracellular delivery to occur.

[0085]

[0189] Typical applications include providing intracellular delivery to knockdown or silence specific cell targets in vitro and in vivo using well-known procedures. Alternatively, the application can include delivery of DNA or mRNA sequences encoding therapeutically useful polypeptides. In this way, treatment for genetic diseases is provided by supplying the missing or non-existent gene product. The methods of the present invention can be carried out in vitro, ex vivo, or in vivo. For example, the lipid mix compositions of the present invention can also be used for in vivo delivery of nucleic acids to cells using methods known to those skilled in the art. In another example, the lipid mix compositions of the present invention can be used to deliver nucleic acids ex vivo to a sample of patient cells and then returned to the patient.

[0086]

[0190] The delivery of nucleic acid therapeutic substances by the lipid particles of the present invention is described below.

[0087]

[0191] In the case of in vivo administration, the pharmaceutical composition is preferably administered parenterally (e.g., intra-articularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseously, intramuscularly, or intratumorally). In certain embodiments, the pharmaceutical composition is administered intravenously, intrathecally, or intraperitoneally by bolus injection. Other routes of administration include topical (skin, eye, mucosa), oral, pulmonary, intranasal, sublingual, rectal, and intravaginal.

[0088]

[0192] In the case of ex vivo use, the pharmaceutical composition is preferably administered to a biological sample taken from a living being, after which the cells are washed and reimplanted into the living being. The living being can be a mammal, particularly a human. This process is used, for example, in cell reprogramming, gene repair, and immunotherapy.

[0089]

[0193] In one embodiment, the present invention provides a method for regulating the expression of a target polynucleotide or polypeptide. These methods generally involve contacting a cell with a lipid particle of the present invention that is bound to a nucleic acid capable of regulating the expression of the target polynucleotide or polypeptide. As used herein, the term "regulating" means altering the expression of a target polynucleotide or polypeptide. Regulating can mean increasing or enhancing, or it can mean decreasing or reducing.

[0090]

[0194] In related embodiments, the present invention provides a method for treating a disease or disorder characterized by overexpression of a polypeptide in a subject, the method comprising supplying to the subject a pharmaceutical composition of the present invention, wherein the therapeutic agent is selected from siRNA, microRNA, antisense oligonucleotides, and plasmids capable of expressing siRNA, microRNA, or antisense oligonucleotides, and the siRNA, microRNA, or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide encoding the polypeptide, or its complement.

[0091]

[0195] In related embodiments, the present invention provides a method of treating a disease or disorder characterized by underexpression of a polypeptide in a subject, comprising administering to the subject a pharmaceutical composition of the present invention, wherein the therapeutic agent is selected from an mRNA, a self-amplifying RNA (SAM), a self-replicating DNA, or a plasmid that specifically encodes or expresses the underexpressed polypeptide or its complement, a nucleic acid therapeutic substance.

[0092]

[0196] In embodiments, the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology or developed in the future. Generally, such preparation methods include the step of combining the active ingredient with an excipient and / or one or more other accessory components.

[0093]

[0197] Methods of delivering biologically active agents for the treatment of diseases

[0198] In one embodiment, the compounds, compositions, methods and uses of the present invention are for delivering a biologically active agent to liver cells (e.g., hepatocytes). In one embodiment, the compounds, compositions, methods and uses of the present invention are for delivering a biologically active agent to a tumor or tumor cells (e.g., primary tumor or metastatic cancer cells). In another embodiment, the compounds, compositions, methods and uses are for delivering a biologically active agent to skin fat, muscle and lymph nodes (subcutaneous administration).

[0094]

[0199] In the case of delivering a biologically active agent to the liver or liver cells, in one embodiment, the composition of the present invention is contacted with the liver or liver cells by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, portal vein injection, catheterization, stent implantation) to facilitate delivery. In the case of delivering a biologically active agent to the kidney or kidney cells, in one embodiment, the composition of the present invention is contacted with the patient's kidney or kidney cells by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, catheterization, stent implantation) to facilitate delivery. In the case of delivering a biologically active agent to a tumor or tumor cells, in one embodiment, to facilitate delivery, the composition of the present invention is contacted with the patient's tumor or tumor cells by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, catheterization, stent implantation).

[0095]

[0200] In the case of delivering a biologically active agent to the CNS or CNS cells, in one embodiment, the composition of the present invention is contacted with the patient's CNS or CNS cells (e.g., brain cells and / or spinal cord cells) by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, catheterization, stent implantation, osmotic pump administration (e.g., intramedullary or ventricular)) to facilitate delivery. In the case of delivering a biologically active agent to the Peripheral Nervous System (PNS) or PNS cells, in one embodiment, the composition of the present invention is contacted with the patient's PNS or PNS cells by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection) to facilitate delivery. In the case of delivering a biologically active agent to the lung or lung cells, in one embodiment, the composition of the present invention is contacted with the patient's lung or lung cells by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct intralung administration to lung tissue and cells) to facilitate delivery.

[0096]

[0201] In the case of delivery of a biologically active agent to the vasculature or vascular cells, in one embodiment, the composition of the present invention is administered parenterally (e.g., intravenously, intramuscularly, subcutaneously) or topically (e.g., clamping, catheterization, stent implantation) to contact the vasculature or vascular cells of a patient in order to facilitate delivery.

[0097]

[0202] In the case of delivering a biologically active agent to the skin or skin cells (e.g., dermal cells and / or follicular cells), in one embodiment, the composition of the present invention is administered parenterally (e.g., intravenously, intramuscularly, subcutaneously) or topically (e.g., direct skin application, iontophoresis) to contact the skin or skin cells (e.g., dermal cells and / or follicular cells) of a patient to facilitate delivery. In the case of delivering a biologically active agent to the eye or eye cells (e.g., macula, fovea, cornea, retina), in one embodiment, the composition of the present invention is administered parenterally (e.g., intravenously, intramuscularly, subcutaneously) or topically (e.g., direct injection, intravitreal injection, periocular injection, subretinal, iontophoresis, use of eye drops, transplantation) to contact the eye or eye cells (e.g., macula, fovea, cornea, retina) of a patient to facilitate delivery. In the case of delivering a biologically active agent to the ear or ear cells (e.g., cells of the inner ear, middle ear and / or outer ear), in one embodiment, the composition of the present invention is contacted with the ear or ear cells (e.g., cells of the inner ear, middle ear and / or outer ear) of a patient by parenteral administration (e.g., intravenously, intramuscularly, subcutaneously) or topical administration (e.g., direct injection) as widely known in the art to facilitate delivery. In the case of delivering a biologically active agent (e.g., RNA encoding an immunogen) to cells of the immune system (e.g., antigen-presenting cells including specialized antigen-presenting cells), in one embodiment, the composition of the present invention is delivered intramuscularly, and then immune cells invade the delivery site and can process the delivered RNA and / or the encoded antigen produced by non-immune cells such as muscle cells. Such immune cells can include macrophages (e.g., bone marrow-derived macrophages), dendritic cells (e.g., bone marrow-derived plasmacytoid dendritic cells and / or bone marrow-derived myeloid dendritic cells), monocytes (e.g., human peripheral blood monocytes), etc. (see, for example, International Publication No. WO 2012 / 006372 by Geall, Andy et al.).

[0098]

[0203] Immunization. For the purpose of immunization, the compositions of the present invention are generally prepared as injectable substances, pulmonary or nasal aerosols, or incorporated into delivery devices (such as syringes, nebulizers, atomizers, inhalers, skin patches, etc.). This delivery device can be used to administer the pharmaceutical composition to a subject, such as a human, for immunization.

[0099]

[0204] According to the present invention, for the purpose of immunization, in some embodiments, the present invention includes delivering RNA encoding an immunogen. This immunogen elicits an immune response that recognizes the immunogen and provides immunity against a pathogen, an allergen, or a tumor antigen. Immunization against diseases and / or infections caused by pathogens is preferred.

[0100]

[0205] The RNA is delivered together with the lipid composition of the present invention (formulated, for example, as liposomes or LNPs). In some embodiments, the present invention utilizes LNPs in which the RNA encoding the immunogen is encapsulated. Encapsulation within the LNP can protect the RNA from digestion by RNase. The encapsulation efficiency does not need to be 100%. The presence of outer RNA molecules (e.g., on the outer surface of liposomes or LNPs) or "naked" RNA molecules (RNA molecules not bound to liposomes or LNPs) is acceptable. Preferably, for a composition containing lipid and RNA molecules, at least half of the RNA molecules (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the RNA molecules) are encapsulated in the LNP or complexed LNPs.

[0101]

[0206] Some lipid nanoparticles may contain a lipid core (e.g., the composition may include a mixture of LNPs and nanoparticles having a lipid core). In such cases, the RNA molecules are encapsulated by LNPs having an aqueous core and can be complexed with LNPs having a lipid core by non-covalent interactions (e.g., ionic interactions between negatively charged RNA and cationic lipids). Encapsulation and complexation with LNPs (whether lipid or aqueous core) can protect the RNA from RNase digestion. The encapsulation / complexation efficiency need not be 100%. The presence of "naked" RNA molecules (RNA molecules not bound to liposomes) is acceptable. Preferably, in a composition comprising a population of LNPs and a population of RNA molecules, at least half of the population of RNA molecules (e.g., at least, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the RNA molecules) are encapsulated within the LNPs or complexed with the LNPs.

[0102]

[0207] In the case of delivery of RNA encoding an immunogen, the preferred range of LNP diameter is in the range of 60 to 180 nm, and in a more specific embodiment, in the range of 80 to 160 nm. The LNP can be part of a composition comprising a population of LNPs, and the LNPs within the population can have a range of diameters. For a composition comprising a population of LNPs of different diameters, (I) at least 80% by number of the LNPs have a diameter in the range of 60 to 180 nm, for example, in the range of 80 to 160 nm, (ii) the average diameter of the population (by intensity, for example, Z-average) is ideally in the range of 60 to 180 nm, for example, in the range of 80 to 160 nm; and / or preferably the diameter within a plurality of them has a polydispersity index of less than 0.2. To obtain LNPs of the desired diameter(s), mixing can be carried out using a process in which two feed streams of an aqueous RNA solution are combined with one stream of an ethanolic lipid solution within a single mixing zone, all at the same flow rate, for example, within a microfluidic flow channel. See also other descriptions regarding the NanoAssemblr® microfluidic mixer sold by Precision Nanosystems Inc., Vancouver, Canada.

[0103]

[0208] A mixture of lipids useful for forming a lipid composition (e.g., LNPs) for immunization comprises a lipid of formula (I); cholesterol; and a stabilizer, such as PEG-DMG. This mixture can also include a neutral zwitterionic lipid, such as DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) or DSPE. In certain embodiments, the lipid composition (e.g., LNPs) provided by the present invention has adjuvant activity, i.e., in the absence of an immunogen, such as a protein antigen or a nucleic acid (DNA or RNA), such as a nucleic acid encoding such an antigen.

[0104]

[0209] RNA molecules. After in vivo administration of the immunization composition, the delivered RNA is released and translated intracellularly to provide the immunogen in situ. In certain embodiments, the RNA is a plus (“+”) strand and can be translated by the cell without the need for intervening replication steps such as reverse transcription. In certain embodiments, the RNA is self-replicating RNA. A self-replicating RNA molecule (replicon) can produce multiple daughter RNAs by transcription from itself (through the antisense copies it generates from itself) even without protein when delivered to a vertebrate cell. Thus, a self-replicating RNA molecule is in certain embodiments: a (+) strand molecule that can be translated directly after delivery to the cell, and this translation provides an RNA-dependent RNA polymerase, which then produces both antisense and sense transcripts from the delivered RNA. The RNA thus delivered produces multiple daughter RNAs. These daughter RNAs, as well as the collinear subgenomic transcripts, can be translated to themselves to provide in situ expression of the encoded immunogen or transcribed to provide additional transcripts of the same sense as the delivered RNA, which are translated to provide in situ expression of the immunogen. The overall result of this series of transcriptions of this sequence is an amplification of the number of introduced replicon RNAs, such that the immunogen encoded thus becomes the major polypeptide product of the host cell.

[0105]

[0210] One system suitable for achieving self-replication is to use an alphavirus-based RNA replicon. These (+) strand replicons are translated after delivery into cells to produce a replicase (or replicase-transcriptase). The replicase is translated as a polyprotein, which self-cleaves to provide a replication complex that generates a genomic (-) strand copy of the delivered (+) strand RNA. These (-) strand transcripts can themselves be transcribed to give further copies of the parental (+) strand RNA and also subgenomic transcripts encoding an immunogen. Thus, translation of the subgenomic transcripts causes in situ expression of the immunogen by the infected cells. Suitable alphavirus replicons can use replicases from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc.

[0106]

[0211] A mutant or wild-type viral sequence, such as the attenuated TC83 mutant of VEEV, can be used in the replicon. Preferred self-replicating RNA molecules thus (I) have an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule and (ii) encode an immunogen. The polymerase can be, for example, an alphavirus replicase comprising one or more alphavirus proteins nsP1, nsP2, nsP3, and nsP4. The native alphavirus genome encodes structural virion proteins in addition to the non-structural replicase polyprotein in certain embodiments, but the self-replicating RNA molecules of the present invention do not encode alphavirus structural proteins. Thus, certain self-replicating RNAs can cause the production of their genomic RNA copies intracellularly, but do not produce virions containing the RNA. The alphavirus structural proteins required for perpetuation within the wild-type virus are not present in the self-replicating RNAs of the present invention, and their place is occupied by the gene(s) encoding the immunogen of interest, and the subgenomic transcript encodes an immunogen rather than a structural alphavirus virion protein. Thus, self-replicating RNA molecules useful in the present invention can have two open reading frames: one, for example, the first (5') open reading frame encodes a replicase; the other open reading frame, for example, the second (3') open reading frame encodes an immunogen. In some embodiments, the RNA may have additional (e.g., downstream) open reading frames, for example, to encode additional immunogens or to encode accessory polypeptides. The self-replicating RNA molecule can have a 5' sequence compatible with the encoded replicase. The self-replicating RNA molecule can have various lengths, but is typically about 5000 to 25000 nucleotides in length, such as 8000 to 15000 nucleotides, or 9000 to 12000 nucleotides. Thus, the RNA is longer than that seen in conventional mRNA delivery.In some embodiments, the self-replicating RNA is greater than about 2000 nucleotides in length, such as about: 9000, 12000, 15000, 18000, 21000, 24000, or more nucleotides.

[0107]

[0212] The RNA molecule can have a 5' cap (e.g., 7-methylguanosine). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of the RNA molecule useful in the present invention can have a 5' triphosphate group. In capped RNA, this can be linked to 7-methylguanosine via a 5'-5' bridge. The 5' triphosphate can enhance RIG-I binding and thus promote the adjuvant effect. The RNA molecule can have a 3' polyA tail and can also contain a polyA polymerase recognition sequence (e.g., AAUAAA) near its 3' end. The RNA molecule useful for immunization purposes in the present invention is typically single-stranded. Single-stranded RNA can generally initiate an adjuvant effect by binding to TLR7, TLR8, RNA helicase, and / or PKR. RNA delivered in double-stranded form (dsRNA) can bind to TLR3, and this receptor can also be activated by dsRNA formed during replication of single-stranded RNA or within the secondary structure of single-stranded RNA.

[0108]

[0213] RNA molecules for immunization purposes can be conveniently prepared by in vitro transcription (IVT). IVT can use a (cDNA) template that is created and propagated in plasmid form in bacteria or is synthesized (e.g., by gene synthesis and / or polymerase chain reaction (PCR) engineering methods). As described by Hekele, Armin et al. in International Publication No. 2011 / 005799, self-replicating RNA can contain one or more nucleotides with modified nucleobases (in addition to the 5' cap structure). For example, self-replicating RNA can contain one or more modified pyrimidine nucleobases, such as pseudouridine and / or 5-methylcytosine residues. However, in some embodiments, the RNA may contain no modified nucleobases and may not contain modified nucleotides, i.e., all nucleotides of the RNA are standard A, C, G, and U ribonucleotides (except for the 5' cap structure that may contain 7'-methylguanosine). In other embodiments, the RNA may contain a 5' cap containing 7'-methylguanosine, and the first I, 2, or 3 5' ribonucleotides may be methylated at the 2' position of the ribose. The RNA used for immunization purposes in the present invention ideally contains only phosphodiester bonds between nucleosides, but in some embodiments contains phosphoramidate, phosphorothioate, and / or methylphosphonate bonds. The present invention includes embodiments in which a number of RNA species, such as 2, 3, 4, or more RNA species, different types of RNA (e.g., mRNA, siRNA, self-replicating RNA, and combinations thereof) are formulated with the lipid compositions provided by the present invention.

[0109]

[0214] In some embodiments, the immunogenic RNA molecules used for immunization purposes in the present invention encode polypeptide immunogens. In these embodiments, after administration, the RNA is translated in vivo and the immunogen can elicit an immune response in the recipient. The immunogen can elicit an immune response against a pathogen (e.g., bacteria, virus, fungus or parasite), but in some embodiments it can elicit an immune response against an allergen or tumor antigen. The immune response can include an antibody response (usually including IgG) and / or a cell-mediated immune response. The polypeptide immunogen typically elicits an immune response that recognizes the corresponding pathogen (or allergen or tumor) polypeptide, but in some embodiments the polypeptide can act as a mimotope and elicit an immune response that recognizes a carbohydrate. The immunogen is typically a surface polypeptide, such as an adhesin, hemagglutinin, envelope glycoprotein, spike glycoprotein, etc. The RNA molecule can encode a single polypeptide immunogen or multiple polypeptides. Multiple immunogens can be presented as a single polypeptide immunogen (fusion polypeptide) or as separate polypeptides. When the immunogen is expressed as a separate polypeptide from the replicon, one or more of these can be provided with an upstream IRES or additional viral promoter element. Alternatively, multiple immunogens can be expressed as a polyprotein encoding individual immunogens fused to a short self-cleaving protease (e.g., foot-and-mouth disease virus 2A protein) or as an intein. In certain embodiments, the polypeptide immunogen (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more immunogens) can be used alone or together with an RNA molecule such as a self-replicating RNA encoding one or more immunogens (the same or different from the polypeptide immunogen).

[0110]

[0215] In some embodiments the immunogen is, without limitation, where the immunogen is SARS CoV-1, SARS-CoV-2 9Coronaviruses, including those derived from [[ID=]], (Roujian Lu, Xiang Zhao, Juan Li et al. "Genomic Characterisation and Epidemiology of 2019 Novel Coronavirus: Implications for Virus Origins and Receptor Binding". Lancet 2020 Feb 22; 395(10224): 565 - 574. doi: 10.1016 / S0140 - 6736(20)30251 - 8. Epub 2020 Jan 30.) useful immunogens for, but not limited to, Neisseria meningitidis include membrane proteins such as adhesins, autotransporters, toxins, iron acquisition proteins, and H factor binding proteins. Combinations of three useful polypeptides are disclosed by Giuliani et al. 11 (2006) Proc Natl Head Sci USA 103(29): 10834 - 9; useful polypeptide immunogens for Streptococcus pneumoniae are disclosed by Veja, Masignani et al. in International Publication No. WO 2009 / 016515 and include, for example, the RrgB fimbrial subunit, beta - N - acetyl - hexosaminidase precursor (spr0057), spr0096, general stress protein GSP - 781 (spr2021, SP2216), serine / threonine kinase StkP (SP1732), and pneumococcal surface adhesin PsaA;

[0111]

[0216] A hepatitis virus in which the immunogen can include hepatitis B virus surface antigen (HBsAg), hepatitis C virus, hepatitis D virus, hepatitis E virus, or hepatitis G virus antigen; a rhabdovirus in which the immunogen includes, but is not limited to, those derived from, for example, lyssavirus (such as rabies virus) and vesiculovirus (VSV); a calicivirus in which the immunogen includes, but is not limited to, those derived from, for example, norwalk virus (norovirus), and norwalk-like viruses such as hawaii virus and snow mountain virus; avian infectious bronchitis virus (IBV), mouse hepatitis virus (MHV), and porcine transmissible gastroenteritis virus (TGEV); a retrovirus in which the immunogen includes those derived from oncovirus, lentivirus (such as HIV-I or HIV-2) or spumavirus; a reovirus in which there is an immunogen derived from, but not limited to, orthoreovirus, rotavirus, orbivirus, or coltivirus; a parvovirus in which the immunogen includes those derived from parvovirus B19; a herpesvirus in which the immunogen includes those derived from human herpesvirus, such as herpes simplex virus (HSV) (such as HSV type I and type 2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), and human herpesvirus 8 (HHV8); a papovavirus in which the immunogen includes those derived from papillomavirus and adenovirus.

[0112]

[0217] In some embodiments, the immunogen elicits an immune response against a virus that infects fish.

[0113]

[0218] The immunogen of fungi can be derived from dermatophytes and other opportunistic fungi.

[0114]

[0219] In some embodiments, the immunogen elicits an immune response against a parasite of the genus Plasmodium, such as Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae or Plasmodium ovale. Thus, the present invention can be used to immunize against malaria. In some embodiments, the immunogen elicits an immune response against a parasite of the family Caligidae, particularly those derived from the genera Lepeophtheirus and Caligus, such as sea lice, such as Lepeophtheirus salmonis or Caligus rogercresseyi.

[0115]

[0220] In some embodiments, the immunogen is an mRNA specific for neoantigens of cancer cells or solid tumors. (7) Peng, M., Mo, Y., Wang, Y. et al. Neoantigen vaccine: an emerging tumor immunotherapy. Mol Cancer 18, 128 (2019).

[0116]

[0221] In some embodiments, the immunogen is (a) a cancer - testis antigen, such as NY - ESO - I, SSX2, SCPI, and polypeptides of the RAGE, BAGE, GAGE, and MAGE families, such as GAGE - 1, GAGE - 2, MAGE - 1, MAGE - 2, MAGE - 3, MAGE - 4, MAGE - 5, MAGE - 6, and MAGE - 12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, thoracic, gastrointestinal, and bladder tumors); (b) a mutant antigen, such as p53 (associated with various solid tumors, such as colorectal, lung, head and neck cancers), p21 / Ras (associated with, for example, melanoma, pancreatic cancer, and colorectal cancer), CDK4 (associated with, for example, melanoma), MUMI (associated with, for example, melanoma), caspase - 8 (associated with, for example, head and neck cancer), CIA 0205 (associated with, for example, bladder cancer), HLA - A2 - R1701, beta - catenin (associated with, for example, melanoma), TCR (associated with, for example, T - cell non - Hodgkin lymphoma), BCR - abl (associated with, for example, chronic myelogenous leukemia), triosephosphate isomerase, KIA 0205, CDC - 27, and LDLRFUT; (c) an overexpressed antigen, such as galectin 4 (associated with, for example, colorectal cancer), galectin 9 (associated with, for example, Hodgkin's disease), proteinase 3 (associated with, for example, chronic myelogenous leukemia), WT I (associated with, for example, various leukemias), carbonic anhydrase (associated with, for example, kidney cancer), aldolase A (associated with, for example, lung cancer), PRAME (associated with, for example, melanoma), HER - 2 / neu (associated with, for example, breast, colon, lung, and ovarian cancers), mammaglobin, alpha - fetoprotein (associated with, for example, liver cancer), KSA (associated with, for example, colorectal cancer), gastrin (associated with, for example, pancreatic and stomach cancers), telomerase catalytic protein, MUC - I (associated with, for example, breast and ovarian cancers), G - 250 (associated with, for example, renal cell cancer), p53 (associated with, for example, breast, colon cancer), and cancer - fetal antigen (associated with, for example, breast cancer, lung cancer, and gastrointestinal cancers, such as colorectal cancer);(d) Tumor antigens selected from common antigens, such as melanoma-melanocyte antigens, such as MART-1 / Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase-related protein-I / TRPI, and tyrosinase-related protein-2 / TRP2 (e.g., related to melanoma); (e) prostate-related antigens, such as PAP, PSA, PSMA, PSH-PI, PSM-PI, PSM-P2, for example, related to prostate cancer; (f) immunoglobulin idiotypes (e.g., related to myeloma and B-cell lymphoma). In certain embodiments, tumor immunogens include, but are not limited to, p15, Hom / Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens, such as E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigen, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23HI, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29&BCAA), CA 195, CA 242, CA-50, CAM43, CD68&KPI, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-I, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophilin C-related protein), TAAL6, TAG72, TLP, TPS, etc.;

[0117]

[0222] A pharmaceutical composition as a vaccine. The pharmaceutical composition of the present invention, particularly useful for immunization, may contain one or more small molecule immune enhancers. The pharmaceutical composition of the present invention may contain one or more preservatives, such as thiomersal or 2-phenoxyethanol. Vaccines without mercury and without preservatives can be prepared.

[0118]

[0223] The composition comprises an immunologically effective amount of the lipid composition (e.g., LNPs) described herein, and optionally other components. An immunologically effective amount means an amount effective for a treatment (e.g., a prophylactic immune response against a pathogen) that is administered to an individual as part of a single dosage or a series of dosages. This amount varies depending on the health and physical condition of the individual being treated, age, taxonomic group of the individual being treated (e.g., non-human primates, primates, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical condition by the treating physician, and other relevant factors. This amount is expected to fall within a relatively broad range that can be determined by routine testing.

[0119]

[0224] The compositions of the invention generally express in terms of the amount of RNA per dose. A preferred dose has about 100 pg of RNA (e.g., 10 - 100 pg, e.g., about 10 pg, 25 pg, 50 pg, 75 pg or 100 pg), although expression is seen at much lower levels, e.g., about 1 pg / dose, about 100 ng / dose, about 10 ng / dose, about 1 ng / dose, etc. The invention also provides delivery devices (e.g., syringes, nebulizers, atomizers, inhalers, skin patches, etc.) containing the pharmaceutical compositions of the invention. This device can be used to administer the composition to a vertebrate subject.

[0120]

[0225] The LNP-formulated RNA and pharmaceutical compositions described herein are used in vivo to induce an immune response against an immunogen of a subject. The invention provides a method of inducing an immune response in a vertebrate comprising administering an effective amount of the LNP-formulated RNA, or pharmaceutical composition, described herein. The immune response is preferably protective and preferably includes antibody and / or cell-mediated immunity. The composition can be used for both priming and boosting purposes. Alternatively, the prime-boost immunization schedule can be a mixture of RNA and the corresponding polypeptide immunogen (e.g., RNA prime, protein boost).

[0121]

[0226] The present invention also provides lipid nanoparticles (LNPs) or pharmaceutical compositions for use in inducing an immune response in vertebrates. The present invention also provides the use of LNPs or pharmaceutical compositions in the manufacture of a medicament for inducing an immune response in vertebrates. By inducing an immune response in vertebrates by these uses and methods, vertebrates can be protected against various diseases and / or infections, such as bacterial and / or viral diseases as described above. The vaccines according to the invention may be for prophylactic (i.e., to prevent an infection) or therapeutic (i.e., to treat an infection) use, but are typically for prophylactic use. The vertebrate is preferably a mammal, such as a human or a large veterinary mammal (e.g., horse, cow, deer, goat, pig).

[0122]

[0227] The compositions of the invention are generally administered directly to the patient. Direct delivery can be achieved by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal, or into the interstitial space of a tissue). Alternative delivery routes include rectal, oral (e.g., tablets, sprays), buccal, sublingual, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, aural, pulmonary, or other mucosal administration. Intradermal and intramuscular administrations are two preferred routes. The injection can be via a needle (e.g., a hypodermic needle), but needleless injection may be used instead. A typical intramuscular dose is 0.5 ml. The present invention can be used to induce systemic and / or mucosal immunity, preferably to cause enhanced systemic and / or mucosal immunity. The dosing can be by a single dosing schedule or a multiple dosing schedule. Multiple dosing can be used in a primary immunization schedule and / or a booster immunization schedule.

[0123]

[0228] In multiple dosing schedules, the various doses can be administered by the same or different routes, such as by parenteral prime and mucosal boost, mucosal prime and parenteral boost, etc. The multiple doses are typically administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). In one embodiment, the multiple doses can be administered at about 6 weeks, 10 weeks, and 14 weeks of age, for example, at about 6 weeks, 10 weeks, and 14 weeks of age, as often used in the World Health Organization's Expanded Program on Immunization ("EPI"). In alternative embodiments, two primary doses are administered about 2 months apart, for example, about 7, 8, or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the second primary dose, for example, about 6, 8, 10, or 12 months after the second primary dose. In a further embodiment, three primary doses are administered about 2 months apart, for example, about 7, 8, or 9 weeks apart, followed by one or more boosters administered about 6 months to 1 year after the third primary dose.

[0124]

[0229] Gene editing

[0230] Gene editing is a group of scientific techniques that can be used to change an organism's DNA by adding, removing, or modifying gene sequences at specific locations in the genome. Several approaches to genome editing, including CRISPR ("clustered regularly interspaced short palindromic repeats") and CRISPR-associated protein 9 ("Cas9"), have been developed. CRISPR-Cas9 was adapted from a genome editing system in bacteria that captures small pieces of DNA from invading viruses and creates DNA segments known as CRISPR arrays. The CRISPR array enables the bacteria to "remember" the virus, so that if the virus attacks again, the bacteria produce RNA segments from the CRISPR array to target the virus's DNA. The bacteria then use Cas9 or a similar enzyme to cut and isolate the DNA, disabling the virus.

[0125]

[0231] The CRISPR-Cas9 system adapted for gene editing functions similarly. A small piece of RNA with a short "guide" sequence that attaches (binds) to a specific target sequence of DNA within the genome is generated. This RNA also binds to the Cas9 enzyme. Similar to within bacteria, the modified RNA is used to recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted position. Cas9 is the enzyme used in most cases, but other enzymes (such as Cpf1) can also be used. Once the DNA is cut, researchers can use the cell's own DNA repair mechanism to add or delete pieces of genetic material, or change the DNA by replacing existing segments with customized DNA sequences.

[0126]

[0232] In embodiments of the present invention, the new ionizable lipid is utilized, for example, as part of the delivery of a CRISPER-Cas system chimeric RNA polynucleotide sequence for modifying an organism by manipulation of a target sequence of a genomic locus of interest. Other nucleic acid components can include guide sequences, tracr mate sequences, and tracr sequences that can hybridize to a target sequence within a eukaryotic cell, such as those described by CHZHAN, Fen et al. in International Publication No. 14204726.

[0127]

[0233] The CRISPR-Cas9 system is used in embodiments to target genes in live cells by delivery to an appropriate location of the CRISPR-Cas9 system (i.e., to cells within a target organ or tissue). Preferred tissues are within the following organs: the kidney; the digestive system including the stomach, pancreas, duodenum, ileum, and / or colon; the lung; the brain, particularly neurons, and / or generally the CNS; the eye, such as retinal tissue; the ear, such as the inner ear; the skin; muscle; bone; and / or generally the liver.

[0128]

[0234] The genes subjected to editing using the ionizable lipid and composition according to embodiments of the present invention are those associated with diseases.

[0129]

[0235] A pharmaceutical composition according to the present disclosure can be prepared, packaged, and / or sold in bulk as a single unit dose and / or as a plurality of single unit doses. As used herein, "unit dose" means an individual amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient administered to a subject and / or can be a convenient fraction of such dosage, such as, but not limited to, one-half or one-third of such dosage.

[0130]

[0236] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or additional ingredients in a pharmaceutical composition according to the present disclosure can vary depending on the type, size, and / or condition of the subject being treated and further on the route by which the composition is administered. For example, the composition can contain from 0.1 percent to 99 percent (w / w) of the active ingredient.

[0131]

[0237] The pharmaceutical formulation may further contain pharmaceutically acceptable excipients, which, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, etc. suitable for the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro, Lippincott, Williams and Wilkins, Baltimore, MD, 2006). The use of conventional excipient media is contemplated in the present invention, except when the conventional excipient media may not be compatible with a substance or its derivatives, for example, by producing some undesirable biological effect or interacting in a harmful manner with other component(s) of the pharmaceutical composition.

[0132]

[0238] In some embodiments, the particle size of the lipid particles may be increased and / or reduced. The change in particle size is not limited, but may be able to help counteract a biological reaction such as inflammation, or may increase the biological effect of the NAT delivered to the mammal by changing the in vivo distribution. Size can also be used to determine the target tissue, with larger particles being rapidly removed and smaller ones reaching different organ systems.

[0133]

[0239] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, diluents, viscosity reducing agents, antioxidants, solubility enhancing agents, bulking agents, fillers, surfactants and / or emulsifiers, preservatives, buffers, lubricants, and / or oily substances. Excipients may be available in a variety of grades and may be of natural, synthetic, or semi-synthetic origin, animal-derived, plant-derived, biotechnology-derived (recombinant), and / or mineral-derived, and may be solid, semi-solid, liquid, or gaseous. Such excipients may optionally be included in the pharmaceutical formulations of the present invention.

[0134]

[0240] In some embodiments, exemplary mRNAs, plasmids or other NATs encode a protein or element selected from human growth hormone, erythropoietin, ATP-binding cassette (ABC) transporters, alpha-1-antitrypsin, acid alpha-glucosidase, arylsulfatase A, carboxypeptidase N, a-galactosidase A, alpha-L-iduronidase, iduronic acid-2-sulfatase, iduronic acid sulfatase, N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase, alpha-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfate sulfatase, beta-galactosidase, beta-glucuronidase, BMPER-2, glucocerebrosidase, heparan sulfamidase, heparin-N-sulfatase, lysosomal acid lipase, hyaluronidase, galactocerebrosidase, ornithine transcarbamylase (OTC), carbamoyl-phosphate synthetase 1 (CPS 1), argininosuccinate synthetase (ASS 1), argininosuccinate lyase (ASL), arginase 1 (ARGI), cystic fibrosis transmembrane conductance regulator (CFTR), survival motor neuron (SMN), factor VIII, factor IX, transcription activator-like effector nucleases such as TALENS, zinc finger nucleases (ZFN), (CRISPR)-associated protein 9 (Cas9), and self-replicating RNAs, as well as low density lipoprotein receptor (LDLR).

[0135]

[0241] Other plasmids or nucleic acids can be applied to cell-based systems using the present invention in the context of research or screening settings. These involve the introduction of genetic material in cells for the purpose of inducing specific physiological or functional changes, for example, in the reprogramming process for the generation of induced pluripotent stem cells. In this case, specific genes (known as Yamanaka factors) are introduced into patient-derived somatic cells to cause the reversal of the cells to a hepatocyte-like state. This enables the cells to divide indefinitely and become pluripotent (able to differentiate into many other downstream cell types), which can be used for both research and clinical applications. These and similar genetic manipulation steps are enhanced by the lipid particles of the present invention to improve the efficiency of processes commonly used when functioning with induced hepatocytes.

[0136]

[0242] Hereinafter, representative lipid particles prepared with nucleic acids are described, including their preparation methods, evidence of advantages, and methods of delivering therapeutic effects using them.

[0137]

[0243] The lipid mix composition of the lipid particles was generated by rapidly mixing a lipid-ethanol solution with an aqueous buffer in a microfluidic mixer designed to induce chaotic advection and provide a mixed environment controlled at intermediate Reynolds numbers (24 < Re < 1000). The microfluidic flow path has a herringbone feature or is configured as shown by Wild, Leaver, and Taylor in International Publication No. 2017117647.

[0138]

[0244] The particle size and "polydispersity index" (PDI) of the lipid particles were measured by dynamic light scattering (DLS). The PDI indicates the width of the particle distribution. This is a parameter calculated from the cumulative analysis of the intensity autocorrelation function measured by (DLS), assuming a single particle size mode and a single exponential function fit to the autocorrelation function. From a biophysical perspective, a PDI of less than 0.1 indicates that the sample is monodisperse. Particles generated by mechanical micromixers, such as the NanoAssemblr® Spark™ and NanoAssemblr® Benchtop (Precision Nanosystems Inc.), are substantially uniform in size assuming all other variables are neutral. A lower PDI indicates a more uniform population of lipid particles. The Spark™ instrument is used in a screening environment to confirm the lead composition. Once the composition is selected, the lipid particles can be fine-tuned using the NanoAssemblr® Benchtop. Once the process parameters flow rate ratio and total flow rate are specified for a particular nanoparticle composition, the nanoparticle technology can be scaled up using the same process parameter values.

[0139]

[0245] In a preferred embodiment, the nucleic acid is a plasmid composed of double-stranded deoxyribonucleic acid. A plasmid is a genetic structure that is typically a small, circular DNA strand that exists in the cytoplasm of a cell (as opposed to the nucleus to which conventional cytogenetics applies) and can replicate independently of the chromosome. Plasmids can also be used to create new cell or animal models for medical research. Plasmids are important tools in molecular biology and as newly emerging therapeutics due to their i) ease of manipulation and isolation, ii) ability to self-replicate for large-scale production, iii) long-term stability, and iv) functionality in a range of organisms and applications. Genetically engineered plasmids have, in addition to an origin of replication (which may not be necessary depending on the purpose of use), restriction enzyme recognition sites that allow the circle to be broken to introduce new genetic material, and selectable markers, such as antibiotic resistance genes. Plasmids can be from about 1000 base pairs (bp) to about 20 kilobase pairs (bp).

[0140]

[0246] As used herein, the term "about" is defined to mean plus or minus 10% of the number indicated, and is used to indicate that while the desired target concentration may be, for example, 40 Mol%, due to inconsistencies in mixing, the actual percentage may vary by + / - 5 Mol%.

[0141]

[0247] As used herein, the term "substantially" is defined as plus or minus 5% of the number indicated, and is used to indicate that while the desired target concentration may be, for example, 40 Mol%, due to inconsistencies in mixing, the actual percentage may vary by + / - 5 Mol%.

[0142]

[0248] As used herein, the term "nucleic acid" is defined as a substance intended to have a direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have a direct effect in restoring, correcting or modifying a physiological function, or to serve as a research reagent. In preferred embodiments, the nucleic acid is an oligonucleotide. In preferred embodiments, the therapeutic agent is a nucleic acid therapeutic, such as an RNA polynucleotide. In preferred embodiments, the therapeutic agent is double-stranded circular DNA (plasmid), linearized plasmid DNA, minicircle or msDNA (multicopy single-stranded DNA).

[0143]

[0249] In the present disclosure, the word "comprising" is used non-limitingly to mean that the matter preceding that word is included, but matters not specifically recited are not excluded. In embodiments that include or may include a specified feature or variable or parameter, it is understood that alternative embodiments may consist of or consist essentially of such feature, or variable or parameter. Reference to an element by the indefinite article "a" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the element.

[0144]

[0250] In the present disclosure, "transfection" means the movement of nucleic acids into cells for the purpose of inducing the expression of specific gene(s) in a subject in both laboratory and clinical settings, and typically includes nucleic acids and ionizable lipids for binding to structural lipids. LIPOFECTIN™ and LIPOFECTAMINE™ are established commercial transfection reagents sold by ThermoFisher Scientific. These research reagents contain permanently cationic lipids and are not suitable for in vivo or ex vivo use.

[0145]

[0251] In the present disclosure, a description of a range of numbers by endpoints includes all numbers included within that range, for example, all whole numbers, all integers, and all intermediate fractional numbers (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.). In the present disclosure, the singular forms "a" and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to a composition containing "a compound" includes a mixture of two or more compounds. In the present disclosure, the term "or" is generally used to include "and / or" unless the context clearly indicates otherwise.

[0146]

[0252] "Stabilizer" or "stabilizing agent" is a term used to identify substances added to ionizable lipids, structural lipids, and sterols that form the lipid compositions according to the present invention. Stabilizers are nonionic as described herein. Examples of nonionic stabilizers include polysorbates (Tween), Brij™ S20 (polyoxyethylene (20) stearyl ether), Brij™ 35 (polyoxyethylene lauryl ether, polyethylene glycol lauryl ether), Brij™ S10 (polyethylene glycol octadecyl ether, polyoxyethylene (10) stearyl ether), Myrj™ 52 (polyoxyethylene (40) stearate). Combinations of stabilizers, such as polysorbates and maltosides, alkyl polyglycosides (TBD), PEG-conjugated lipids or other polymer-conjugated lipids are also used in some embodiments.

[0147]

[0253] "Alkyl" means a straight-chain or branched, acyclic or cyclic saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight-chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, etc.; saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, etc. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.; unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, etc.

[0148]

[0254] "Alkenyl" means alkyl as defined above and containing at least one double bond between adjacent carbon atoms. Alkenyl includes both cis and trans isomers. Representative straight-chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutenylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, etc.

[0149]

[0255] "Alkynyl" means alkyl or alkenyl as defined above and further containing at least one triple bond between adjacent carbons. Representative straight-chain and branched alkynyls include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

[0150]

[0256] The term "acyl" refers to a carbonyl group substituted with hydrogen, alkyl, partially saturated or fully saturated cycloalkyl, partially saturated or fully saturated heterocyclic ring, aryl, and heteroaryl. For example, acyl includes (C1-C 20 ) alkanoyl (e.g., formyl, acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.), (C3-C 20 ) cycloalkylcarbonyl (e.g., cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl, pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl), and heteroaroyl (e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl, furanyl-3-carbonyl, 1H-pyrrolyl-2-carbonyl, 1H-pyrrolyl-3-carbonyl, benzo[b]thiophenyl-2-carbonyl, etc.) groups.

[0151]

[0257] The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, where the ring atoms can be substituted. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, anthracenyl, and pyrenyl.

[0152]

[0258] "Heterocyclic ring" means a 3- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic ring, which can be saturated, unsaturated, or aromatic, and contains 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Here, the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. Also included are bicyclic rings in which any of the above heterocyclic rings is condensed with a benzene ring. The heterocyclic ring can be bonded via any heteroatom or carbon atom. The heterocyclic ring includes heteroaryl as defined below. Examples of the heterocyclic ring include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, terahydrothiophenyl, tetrahydrothiopyranyl, etc.

[0153]

[0259] The term "heteroaryl" refers to an aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms for monocyclic, 1 to 6 heteroatoms for bicyclic, or 1 to 9 heteroatoms for tricyclic, where the heteroatoms are selected from O, N, or S (e.g., carbon atoms and 1 to 3 heteroatoms N, O, or S for monocyclic, 1 to 6 for bicyclic, or 1 to 9 for tricyclic respectively), and any of the ring atoms may be substituted. The heteroaryl groups described herein may also contain fused rings sharing a common carbon-carbon bond. The term "alkyl heterocyclic ring" means a heteroaryl in which at least one ring atom is substituted with alkyl, alkenyl, or alkynyl.

[0154]

[0260] The term "substitution" means the replacement of one or more hydrogen groups in a given structure with a defined substituent group including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, oxo, thioxy, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, arylcarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituents may be further substituted. Exemplary substituents include amino, alkylamino, dialkylamino, and cyclic amino compounds.

[0155]

[0261] "Halogen" means fluoro, chloro, bromo and iodo substituents.

[0156]

[0262] The terms "alkylamine" and "dialkylamine" mean -NH(alkyl) and -N(alkyl)2 radicals, respectively. The term "hydroxyalkyl" means -alkyl-OH radical. The term "alkyl heterocycle" refers to an alkyl in which at least one methylene is replaced by a heterocycle.

[0157]

[0263] In some embodiments, the methods of the present invention may require the use of protecting groups. Protecting group methodologies are well known to those skilled in the art (see, e.g., Protective Groups in Organic Synthesis, Greene, T.W. et al., Wiley-Interscience, New York City, 1999). Briefly, within the context of the present invention, a protecting group is a group that reduces or eliminates the unwanted reactivity of a particular functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is a group that reduces or eliminates the unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

[0158]

[0264] Furthermore, the present invention may be described as a compound represented by formula (I), (II) or (III), or a pharmaceutically acceptable salt thereof, wherein the experimental pKa of the nanoparticles ranges from 5.8 to 7.1.

[0159]

[0265] The compounds of the present invention can be prepared by known organic synthesis techniques, such as those described in more detail in the examples. The scope of the present invention can include various salts, hydrates, and solvates of the compounds. The compounds of the present invention can also include various pharmaceutically acceptable isotopes. The compounds of the present invention can include various pharmaceutically acceptable substitutions, such as fluorinated or iodinated derivatives.

[0160]

[0266] The compounds of the present invention can be synthesized using a variety of possible synthetic routes that can be readily selected by those skilled in organic synthesis.

[0161]

[0267] The phrase "pharmaceutically acceptable" is used to describe solutions or any other form, such as lyophilized, powder form, aerosol, or other dosage forms of a compound, material, composition, nanoparticle suspension, within the scope of sound medical judgment suitable for use in contact with human and animal tissues or cells at a reasonable benefit / risk ratio. The benefit / risk ratio can be derived from protecting a therapeutic entity, such as a small molecule, nucleic acid, peptide, or protein, from degradation in an in vivo or ex vivo biological environment.

[0162]

[0268] Compounds can also be evaluated in one or more preclinical models known to those skilled in the art to demonstrate the therapeutic validation of pharmaceutically viable cargos such as NAT, peptides, and proteins. These include, but are not limited to, rodent models and non-human primates.

[0163]

[0269] The features and advantages of the subject matter will become more apparent from the following detailed description of the selected embodiments illustrated in the accompanying drawings. As will be understood, the subject matter disclosed in the claims can be modified in various respects without departing from the scope of the claims. Accordingly, the drawings and the following description are to be regarded as illustrative in nature and not restrictive, and the full scope of the subject matter is set forth in the claims.

Examples

[0164]

[0270] Overview:

[0271] All solvents and reagents were commercial products and used as received unless otherwise noted. Temperatures are given in degrees Celsius. The structures of the final starting materials, intermediates, and final products were confirmed by standard analytical methods, e.g., MS or NMR. Unless otherwise noted, 1H NMR spectra were recorded at 298 K in CDCl3 solution using an AVANCE NEO NanoBay Bruker 400 MHz NMR spectrometer. Chemical shifts are reported in parts per million (ppm) relative to TMS (0.00), and coupling constants J are reported in hertz (Hz) for 1H. The following abbreviations are used to indicate signal patterns: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, dd = doublet of doublets, br s = broad singlet, dt = doublet of triplets. Unless otherwise noted, column purifications were performed using an Isolera™ Prime with an appropriate eluent of isocratic or gradient composition.

[0165]

[0272] All final compounds were determined to be purer than 85% by reverse-phase UHPLC-MS analysis using a Shimadzu Nexera UHPLC instrument, DAD and ELSD, and an Acquity Peptide BEH C18 2.1 mm × 50 mm, 1.7 μm column and gradient, 10 mM ammonium bicarbonate in water (A) and acetonitrile:methanol 80:20 ratio (B) (retention time RT, minutes). The gradient study was performed linearly from 80 - 100% B over 12 minutes at 0.8 mL / min. The injection volume was 2 μL and the column temperature was ambient temperature. Detection was performed in positive and negative modes in multi-mode by electrospray and atmospheric pressure chemical ionization (ESI and APCI) using a Shimadzu 2020 Single Quad mass spectrometer (Science Park, Singapore) and an evaporative light scattering detector (ELSD), except for PNI 76, 119, 120, 121, 122 and 127. Low-resolution MS data for PNI 76, 119, 120, 121, 122 and 127 were recorded using a Bruker micrOTOF™ Time-of-Flight mass spectrometer with a positive electrospray ionization source of an Agilent® 1200 HPLC. Sodium formate was used as a reference. Samples were introduced by flow injection via HPLC using acetonitrile / water (0.1% formic acid) as the mobile phase.

[0166] Abbreviations

[0273] ACD-A = anticoagulant citrate dextrose solution

[0274] AcOH = acetic acid

[0275] aq. = aqueous

[0276] cat. = catalyst

[0277] DCM = dichloromethane

[0278] DIPEA = N,N-diisopropylethylamine

[0279] DMAP = 4-dimethylaminopyridine

[0280] DMF = N,N-dimethylformamide

[0281] EDCI = 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0282] EPO = Erythropoietin

[0283] ESI = Electrospray ionization

[0284] EtOAc = Ethyl acetate

[0285] Et2O = Diethyl ether

[0286] g = Gram

[0287] h = Hour

[0288] Hz = Hertz

[0289] K = Kelvin

[0290] MC3 = DLin-MC3-DMA

[0291] MeOH = Methanol

[0292] mg = Milligram

[0293] MHz = Megahertz

[0294] min = Minute

[0295] mL = Milliliter

[0296] mmol = Millimole

[0297] MS = Mass spectrometry

[0298] NMR = Nuclear magnetic resonance

[0299] Pet. = Petroleum

[0300] ppm = Parts per million

[0301] Satd. = Saturated

[0302] TFA = Trifluoroacetic acid

[0303] THF = Tetrahydrofuran

[0304] TLC = Thin layer chromatography

[0305] TMS = Tetramethylsilane

[0306] TNS = Sodium 6-(p-toluidino)-2-naphthalenesulfonate

[0307] UHPLC = Ultra High Performance Liquid Chromatography

[0308] ℃ = Celsius temperature

[0167] Example 1 Synthesis of 1,4 - anhydroxylitol (1)

Chemical formula

[0168]

[0309] (±)-Xylitol (20.0 g, 131.4 mmol) was added to a 250 mL one - neck round - bottom flask equipped with a reflux condenser and dissolved in 10% aq. H2SO4 (10 mL). The reaction mixture was heated at 135 °C for 6 h. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with aqueous saturated NaHCO3 solution and lyophilized to obtain crude (±)-1,4 - anhydroxylitol (1, 14.5 g, 23.86 mmol, 82.0% yield) as a thick colorless oil and used in the next step without further purification. 1 H (400 MHz, MeOD) δ 4.16 - 4.03 (m, 4H), 3.82 (dd, 1H, J = 12.0, 4.0), 3.74 (dd, 1H, J = 12.0, 4.0), 3.66 (d, 1H, J = 8.0).

[0169] Example 2

[0310] Synthesis of PNI 121, 122, 127, 321, 325, 328, 329, 336, 538, 539 and 540

Chemical formula

[0170]

[0311] PNI 121:

Chemical formula

[0312] To a solution of (±)-1 (1.25 g, 9.3 mmol) in dry DMF (15 mL), DMAP (cat.) was added, followed by the addition of a solution of 4-(dimethylamino)butanoic acid hydrochloride (1.56 g, 9.3 mmol) in dry DMF (10 mL) under a N2 atmosphere. DCM (25 mL) was added to the reaction mixture. Finally, solid EDCI hydrochloride (3.56 g, 18.6 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 4 h. TLC analysis of the reaction mixture showed complete consumption of (±)-1. Further, DMAP (cat.) was added to the reaction mixture, followed by the addition of linoleic acid (7.2 mL, 23.3 mmol) and EDCI hydrochloride (7.13 g, 37.2 mmol). Next, dry DMF (20 mL) and dry CH2Cl2 (10 mL) were added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated on a rotary evaporator, diluted with EtOAc (50 mL), and washed with water (3 × 20 mL). The organic layer was washed with brine, dried over anhyd. Na2SO4, and evaporated to dryness on a rotary evaporator to obtain a crude mixture, which was purified by silica gel column chromatography using 5% MeOH / CH2Cl2 as the eluent. PNI 121 was obtained as a colorless oil (590 mg, 0.76 mmol) in 8% yield. 1 H (500 MHz, CDCl3) δ 5.42-5.31 (m, 9H), 5.13-5.11 (m, 1H), 4.33-4.23 (m, 3H), 4.20-4.16(m, 1H), 3.76 (dd, 1H, J = 15.0 Hz, 5.0 Hz), 2.78 (apparent t,4H, J = 5.0 Hz), 2.40-2.32 (m, 8H), 2.26 (s, 6H), 2.06 (q, 8H, J = 15.0 Hz, 5.0Hz), 1.85-1.79 (m, 2H), 1.65-1.60 (m, 4H), 1.39-1.26 (m, 28H), 0.90 (apparent t, 6H, J = 7.5 Hz). C 47 H 82 Molecular weight of NO7 [M + H] + Calculated value 772.6091. Found value 772.6126.

[0171]

[0313] PNI 122: [Chem.]

[0172]

[0314] To a solution of (±)-1 (1.25 g, 9.3 mmol) in dry DMF (15 mL) was added DMAP (cat.), followed by the addition of a solution of 5-(dimethylamino)pentanoic acid hydrochloride (1.68 g, 9.3 mmol) in dry DMF (10 mL) under a N2 atmosphere. DCM (25 mL) was added to the reaction mixture. Finally, solid EDCI hydrochloride (3.56 g, 18.6 mmol) was added, and the reaction was stirred at room temperature for 4 h. TLC analysis of the reaction mixture showed complete consumption of (±)-1. Further, DMAP (cat.) was added to the reaction mixture, followed by the addition of linoleic acid (7.2 mL, 23.25 mmol) and EDCI hydrochloride (7.13 g, 37.2 mmol). Next, dry DMF (20 mL) and dry DCM (10 mL) were added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated on a rotary evaporator, diluted with ethyl acetate (50 mL), and washed with water (3 × 20 mL). The organic layer was washed with brine, dried over anhyd. Na2SO4, and evaporated to dryness on a rotary evaporator to obtain a crude mixture, which was purified by silica gel column chromatography using 5% MeOH / DCM as the eluent. PNI 122 was obtained as a colorless oil (912 mg, 1.16 mmol) in 12% yield. 1 H (500 MHz, CDCl3) δ 5.42 - 5.31 (m, 9H), 5.13 - 5.11 (m, 1H), 4.35 - 4.23 (m, 3H), 4.17 (dd, 1H, J = 10.0 Hz, 5.0 Hz), 3.76 (dd, 1H, J = 10.0 Hz, 5.0 Hz), 2.78 (apparent t, 4H, J = 5.0 Hz), 2.39 - 2.30 (m, 8H), 2.25 (s, 6H), 2.06 (q, 8H, J = 15.0 Hz, 5.0 Hz), 1.69 - 1.60 (m, 6H), 1.52 (p, 2H, J = 7.5 Hz), 1.39 - 1.26 (m, 28H), 0.90 (apparent t, 6H, J = 7.5 Hz). C48 H 84 Molecular weight of NO7 [M+H] + Calculated value: 786.6248. Measured value: 786.6365.

[0173]

[0315] PNI 127:

Chem.

[0174]

[0316] (±)-1 (200 mg, 1.49 mmol) in dry DMF (2 mL) was added with DMAP (cat.), and then a solution of 1,4-dimethylpiperidine-4-carboxylic acid hydrochloride (289 mg, 1.49 mmol) in dry DMF (3 mL) was added at room temperature under N2 atmosphere. DCM (5 mL) was added to the reaction mixture. Finally, solid EDCI hydrochloride (571 mg, 2.98 mmol) was added, and the reaction was stirred overnight. TLC analysis of the reaction mixture showed complete consumption of (±)-1. DMAP (cat.) was added to the reaction mixture, followed by linoleic acid (1.2 mL, 3.73 mmol) and EDCI hydrochloride (1.14 g, 5.96 mmol). Next, dry DMF (5 mL) and dry DCM (5 mL) were added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated on a rotary evaporator, diluted with EtOAc (20 mL), and washed with water (3 × 10 mL). The organic layer was washed with brine, dried over anhyd. Na2SO4, and evaporated to dryness on a rotary evaporator to obtain a crude mixture, which was purified by silica gel column chromatography using 5% MeOH / DCM as the eluent. PNI 127 was obtained as a colorless oil (225 mg, 0.28 mmol) in 19% yield. 1H (500 MHz, CDCl3) δ 5.42 - 5.31 (m, 9H), 5.14 - 5.12 (m, 1H), 4.34 - 4.27 (m, 2H), 4.24 (apparent d, 2H, J = 5.0 Hz), 3.76 (dd, 1H, J = 15.0 Hz, 5.0 Hz), 2.78 (apparent t, 4H, J = 5.0 Hz), 2.69 (brs, 2H), 2.36 - 2.26 (m, 8H), 2.20 - 2.12 (m, 3H), 2.06 (q, 8H, J = 15.0 Hz, 5.0 Hz), 1.66 - 1.60 (m, 6H), 1.38 - 1.29 (m, 28H), 1.22 (s, 3H), 0.90 (apparent t, 6H, J = 7.5 Hz). C 49 H 84 Molecular weight of NO7 [M + H] + Calculated value: 798.6248. Measured value: 798.6157.

[0175]

[0317] PNI 321:

Chem.

[0176]

[0318] A solution of DMAP (0.046 g, 0.373 mmol) and 3-(dimethylamino)propionic acid hydrochloride (0.573 g, 3.73 mmol) in dry DMF (2.5 mL) was added to a stirred solution of (±)-1,4-anhydroxylitol (1, 0.50 g, 3.73 mmol) in dry DMF (10 mL). Dry DCM (10 mL) was added to this stirred solution, followed by EDCI hydrochloride (1.429 g, 7.46 mmol) and DIPEA (1.628 ml, 9.32 mmol). The reaction mixture was stirred at room temperature for 4 h, and 1 completely disappeared as indicated by TLC. DMAP (0.091 g, 0.746 mmol) was added to the reaction mixture, followed by linoleic acid (2.61 g, 9.32 mmol) in dry DMF (7.5 mL). Then EDCI hydrochloride (2.86 g, 14.91 mmol) and DIPEA (3.26 ml, 18.64 mmol) were added, followed by dry DCM (10 mL). The reaction mixture was stirred at room temperature for 16 h. After completion of the reaction as indicated by TLC, the solvent was evaporated under reduced pressure. The residue was dissolved in EtOAc (60 mL) and washed with water (2 × 50 mL). The aq. layer was extracted with EtOAc (2 × 30 mL), and the combined organic layers were washed with brine (2 × 50 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorela (trademark)) using 80% EtOAc in Pet. ether to give PNI 321 (0.265 g, 0.327 mmol, 8.78% yield) as a pale yellow oil. 1H (400 MHz, CDCl3) δ 5.42 - 5.30 (m, 9H), 5.15 - 5.11 (m, 1H), 4.34 - 4.16 (m, 4H), 3.76 (dd, 1H, J = 8.0, 4.0), 2.78 (apparent t, 4H, J = 6.0), 2.63 (apparent t, 2H, J = 6.0), 2.52 (apparent t, 2H, J = 6.0), 2.36 - 2.31 (m, 4H), 2.25 (s, 6H), 2.06 (q, 8H, J = 8.0), 1.64 - 1.61 (m, 4H), 1.38 - 1.26 (m, 28H), 0.91 - 0.88 (m, 6H). RT = 3.61 min. Purity 93.7%. C 46 H 80 ESI-MS of NO7: m / z = 759 [M+H] + .

[0177] PNI 325:

Chem.

[0178]

[0319] A solution of (±)-1,4-anhydroxylitol (1,700 mg, 5.22 mmol) and DMAP (63.8 mg, 0.522 mmol) in dry DMF (5 mL) at 25 °C was stirred well, and a solution of 1-methylpiperidine-4-carboxylic acid (747 mg, 5.22 mmol) in dry DMF (2.5 mL) was added thereto under a nitrogen atmosphere. Subsequently, dry DCM (10 mL) and EDCI hydrochloride (2 g, 10.44 mmol) were added. The reaction mixture was stirred at room temperature for 16 h under a nitrogen atmosphere, and TLC analysis showed complete consumption of 1. A solution of DMAP (63.8 mg, 0.522 mmol) and linoleic acid (3,659 mg, 13.05 mmol) in DMF (5 mL) was added to the reaction mixture. Then EDCI hydrochloride (2 g, 10.44 mmol) was added, followed by dry DCM (10 mL). The reaction mixture was stirred at room temperature for 16 h under a nitrogen atmosphere. The completion of the reaction was confirmed by TLC analysis, and the reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (60 mL) and washed with water (2 × 50 mL). The aqueous layer was extracted with EtOAc (2 × 30 mL), and the combined organic layers were washed with brine (2 × 50 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorela (trademark)) using 70% EtOAc in Pet. ether to obtain PNI 325 (0.600 g, 0.765 mmol, 14.66% yield) as a pale yellow oil. 1 H (400 MHz, CDCl3) δ 5.42 - 5.30 (m, 9H), 5.11 (apparent s, 1H), 4.33 - 4.15 (m, 4H), 3.76 (d, 1H, J = 12.0), 2.83 (br s, 2H), 2.78 (apparent t, 4H, J = 6.0), 2.35 - 2.29 (m, 5H), 2.26 (s, 3H), 2.06 (q, 8H, J = 8.0), 2.01 - 1.89 (m, 4H), 1.81 - 1.75 (m, 2H), 1.68 - 1.59 (m, 4H), 1.40 - 1.26 (m, 28H), 0.90 (apparent t, 6H, J = 6.0). RT = 3.63 min. Purity 97.4%. C 48 H82 ESI-MS of NO7: m / z = 785 [M+H] + .

[0179] PNI 328:

Chem.

[0180]

[0320] To a well-stirred solution of (±)-1,4-anhydroxylitol (1,700 mg, 5.22 mmol) and DMAP (63.8 mg, 0.522 mmol) in dry DMF (5 mL) at 25 °C, a solution of 1-methylpyrrolidine-3-carboxylic acid (674 mg, 5.22 mmol) in dry DMF (2.5 mL) was added under a nitrogen atmosphere. Then, dry DCM (10 mL) and subsequently EDCI hydrochloride (2 g, 10.44 mmol) were added. The reaction mixture was stirred at room temperature for 4 h, and TLC analysis showed complete consumption of 1. Then, a solution of DMAP (63.8 mg, 0.522 mmol) and linoleic acid (3,659 mg, 13.05 mmol) in dry DMF (5 mL) was added to the above reaction mixture. Then, EDCI hydrochloride (4,002 mg, 20.88 mmol) and dry DCM (10 mL) were added. The reaction mixture was stirred at room temperature for 16 h under a nitrogen atmosphere. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under reduced pressure. The residue was dissolved in EtOAc (60 mL) and washed with water (2 × 50 mL). The aq. layer was extracted with EtOAc (2 × 30 mL), and the combined organic layers were washed with brine (2 × 50 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorel (trademark)) using 70% EtOAc in Pet. ether to give PNI 328 (0.290 g, 0.377 mmol, 7.22% yield) as a pale yellow oil. 1H (400 MHz, CDCl3) δ 5.41 - 5.30 (m, 9H), 5.12 (apparent s, 1H), 4.34 - 4.19 (m, 4H), 3.76 (d, 1H, J = 12.0), 3.09 - 3.05 (m, 1H), 2.82 - 2.76 (m, 5H), 2.71 - 2.51 (m, 3H), 2.36 - 2.32 (m, 7H), 2.14 - 2.03 (m, 10H), 1.68 - 1.60 (m, 4H), 1.36 - 1.26 (m, 28H), 0.90 (apparent t, 6H, J = 6.0). RT = 3.62 min. Purity 93.1%. C 47 H 80 ESI-MS of NO7: m / z = 771 [M+H] + .

[0181] PNI 329:

Chem.

[0182]

[0321] A stirred solution of (±)-1,4-anhydroxylitol (1, 0.7 g, 5.22 mmol) and DMAP (0.064 g, 0.522 mmol) in dry DMF (15 mL) at 25 °C was added a solution of 1,3-dimethylpyrrolidine-3-carboxylic acid (0.747 g, 5.22 mmol) in dry DMF (5 mL) under a nitrogen atmosphere. Subsequently, dry DCM (5 mL), EDCI hydrochloride (4.00 g, 20.88 mmol) and DIPEA (2.279 ml, 13.05 mmol) were successively added. The reaction mixture was stirred at room temperature for 4 h, and TLC analysis showed complete consumption of 1. A solution of DMAP (0.064 g, 0.522 mmol) and linoleic acid (3.66 g, 13.05 mmol) in dry DMF (5 mL) was added to the reaction mixture. Then EDCI hydrochloride (4.00 g, 20.88 mmol) and DIPEA (3.37 g, 26.1 mmol) were added, followed by dry DCM (5 mL). The reaction mixture was stirred at room temperature for 16 h under a nitrogen atmosphere. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under reduced pressure. The residue was dissolved in EtOAc (60 mL) and washed with water (2 × 50 mL). The aq. layer was extracted with EtOAc (2 × 30 mL), and the combined organic layers were washed with brine (2 × 50 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 80% EtOAc in pet. ether to give PNI 329 (0.090 g, 0.115 mmol, 2.199% yield) as a pale yellow oil. 1H (400 MHz, CDCl3) δ 5.42 - 5.29 (m, 9H), 5.14 - 5.12 (m, 1H), 4.35 - 4.22 (m, 4H), 3.76 (dd, 1H, J = 8.0, 4.0), 3.01 (apparent d, 1H, J = 8.0), 2.78 (t, 4H, J = 6.0), 2.71 - 2.57 (m, 2H), 2.47 - 2.39 (m, 2H), 2.39 - 2.31 (m, 7H), 2.09 - 1.97 (m, 9H), 1.79 - 1.67 (m, 4H), 1.37 - 1.26 (m, 31H), 0.88 (apparent t, 6H, J = 6.0). RT = 3.78 min. Purity 91.6%. C 48 H 82 ESI-MS of NO7: m / z = 785 [M+H] + .

[0183]

[0323] PNI 336:

Chem.

[0184]

[0324] A stirred solution of (±)-1,4-anhydroxylitol (1, 0.7 g, 5.22 mmol) in dry DMF (10 mL) was added with DMAP (0.064 g, 0.522 mmol) at room temperature under a nitrogen atmosphere. To this mixture was added a solution of 2-(1-methyl-1H-imidazol-4-yl)acetic acid (0.731 g, 5.22 mmol) in DMF (5 mL), followed by dry DCM (5 mL). Then, EDCI hydrochloride (2.001 g, 10.44 mmol) and DIPEA (2.329 ml, 13.05 mmol) were added. The reaction mixture was stirred at room temperature for 4 h, and TLC analysis showed complete consumption of 1. To the above mixture was added a solution of DMAP (0.064 g, 0.522 mmol) and linoleic acid (3.66 g, 13.05 mmol) in DMF (5 mL). Then, EDCI hydrochloride (4.00 g, 20.88 mmol) and DIPEA (3.37 g, 26.1 mmol) were added. Finally, dry DCM (5 mL) was added, and the reaction mixture was stirred at room temperature for 16 h. TLC analysis showed completion of the starting material. The solvent was evaporated under reduced pressure, and the residue was dissolved in EtOAc (60 mL). The EtOAc layer was washed with water (2 × 30 mL), and the combined aqueous layers were extracted with EtOAc (2 × 30 mL). The combined organic layers were washed with brine (2 × 50 mL) and dried over anhydrous Na2SO4. The organic layer was filtered and concentrated under reduced pressure. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorela (trademark)) using 70% EtOAc in Pet. ether to give PNI 336 (0.37 g, 0.474 mmol, 9.08% yield) as a pale yellow liquid. 1H (400 MHz, CDCl3) δ 7.45 (s, 1H), 6.88 (s, 1H), 5.42 - 5.30 (m, 9H), 5.11 - 5.09 (m, 1H), 4.37 - 4.20 (m, 4H), 3.75 (dd, 1H, J = 8.0, 4.0), 3.70 - 3.69 (m, 2H), 3.68 (s, 3H), 2.77 (t, 4H, J = 6.0), 2.33 (t, 4H, J = 8.0), 2.05 (q, 8H, J = 8.0), 1.64 - 1.59 (m, 4H), 1.38 - 1.26 (m, 28H), 0.89 (apparent t, 6H, J = 6.0). RT = 3.44 min. Purity 89.4%. C 47 H 77 ESI-MS of N2O7: m / z = 782 [M+H] + .

[0185]

[0325] PNI 538:

Chem.

[0186]

[0326] To a stirred solution of (±)-1,4-anhydroxylitol (1, 0.5 g, 3.73 mmol) in dry DMF (10 mL), DMAP (0.046 g, 0.373 mmol) was added at room temperature under a nitrogen atmosphere. To this mixture, a solution of 2-(1-methylpyrrolidin-3-yl)acetic acid (0.587 g, 4.10 mmol) in DMF (5 mL) was added, followed by dry DCM (10 mL). EDCI hydrochloride (1.429 g, 7.46 mmol) and DIPEA (1.664 mL, 9.32 mmol) were added to the reaction flask contents. The reaction mixture was stirred at room temperature for 4 h, and TLC analysis showed complete consumption of 1. Then, a solution of DMAP (0.455 g, 3.73 mmol) and linoleic acid (2.61 g, 9.32 mmol) in DMF (5 mL) was added. To the reaction mixture, EDCI hydrochloride (2.86 g, 14.91 mmol) and DIPEA (2.409 g, 18.64 mmol) were added. Finally, dry DCM (10 mL) was added, and the reaction mixture was stirred at room temperature for 16 h. TLC analysis showed consumption of the starting material. The solvent was evaporated under reduced pressure, and the residue was dissolved in EtOAc (60 mL). The EtOAc layer was washed with water (2 × 30 mL), and the combined aqueous layers were extracted with EtOAc (2 × 30 mL). The combined organic layers were washed with brine (2 × 50 mL) and dried over anhydrous Na2SO4. The organic layer was filtered and concentrated under reduced pressure. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 70% EtOAc in Pet. ether to give PNI 538 (0.165 g, 0.210 mmol, 5.64% yield) as a pale yellow oil. 1H (400 MHz, CDCl3) δ 5.43 - 5.30 (m, 9H), 5.12 - 5.11 (m, 1H), 4.32 - 4.14 (m, 4H), 3.75 (dd, 1H, J = 12.0, 4.0), 2.83 - 2.73 (m, 5H), 2.65 - 2.50 (m, 3H), 2.46 - 2.44 (m, 2H), 2.37 - 2.82 (m, 8H), 2.18 - 2.03 (m, 10H), 1.55 - 1.42 (m, 4H), 1.40 - 1.24 (m, 28H), 0.90 (apparent t, 6H, J = 6.0). RT = 3.23 min. Purity 87.0%. C 48 H 82 ESI-MS of NO7: m / z = 785 [M+H] + .

[0187]

[0327] PNI 539:

Chem.

[0188]

[0328] To a well-stirred solution of (±)-1,4-anhydroxylitol (1, 0.6 g, 4.47 mmol) and DMAP (0.055 g, 0.447 mmol) in dry DMF (10 mL) at 25 °C, a solution of 4-(pyrrolidin-1-yl)butanoic acid hydrochloride (0.866 g, 4.47 mmol) in dry DMF (2.5 mL) was added under a nitrogen atmosphere. Then, EDCI hydrochloride (1.715 g, 8.95 mmol) and dry DCM (10 mL) were added. The reaction mixture was stirred at room temperature for 4 h, and TLC analysis showed complete consumption of 1. To the reaction mixture, a solution of DMAP (0.109 g, 0.895 mmol) and linoleic acid (3.14 g, 11.18 mmol) in dry DMF (7.5 mL) was added. Then, EDCI hydrochloride (3.43 g, 17.89 mmol) and dry DCM (10 mL) were added. The reaction mixture was stirred at room temperature for 16 h under a nitrogen atmosphere. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under reduced pressure. The residue was dissolved in EtOAc (70 mL) and washed with water (2 × 40 mL). The aq. layer was extracted with EtOAc (2 × 35 mL), and the combined organic layers were washed with brine (2 × 50 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (Isolera (trademark)) using silica gel (100 - 200 mesh) with 70% EtOAc in Pet. ether to give PNI 539 (0.5 g, 0.608 mmol, 13.58% yield) as a pale yellow oil. 1 H (400 MHz, CDCl3) δ 5.42 - 5.30 (m, 9H), 5.13 - 5.11 (m, 1H), 4.33 - 4.14 (m, 4H), 3.75 (dd,1H, J = 12.0, 4.0), 2.78 (t, 4H, J = 6.0), 2.50 - 2.31 (m, 12H), 2.06 (q, 8H, J =8.0), 1.84 (p, 2H, J = 8.0), 1.78 - 1.75 (m, 4H), 1.67 - 1.60 (m, 4H), 1.40 - 1.25(m, 28H), 0.90 (apparent t, 6H, J = 6.0). RT = 3.71 min. Purity 90.6%. C 49 H 84ESI-MS of NO7: m / z = 799 [M+H] + .

[0189] PNI 540:

Chem.

[0190]

[0329] To a well-stirred solution of (±)-1,4-anhydroxylitol (1, 0.6 g, 4.47 mmol) and DMAP (0.055 g, 0.447 mmol) in dry DMF (10 mL) at 25 °C, 2-(1-methylpiperidin-2-yl)acetic acid hydrochloride (0.866 g, 4.47 mmol) in dry DMF (2.5 mL) was added under a nitrogen atmosphere. Subsequently, EDCI (1.715 g, 8.95 mmol) and dry DCM (10 mL) were added. The reaction mixture was stirred at room temperature for 4 h, and TLC analysis showed complete consumption of 1. To this reaction mixture, a solution of DMAP (0.109 g, 0.895 mmol) and linoleic acid (3.14 g, 11.18 mmol) in DMF (7.5 mL) was added. Then, EDCI hydrochloride (3.43 g, 17.89 mmol) and dry DCM (10 mL) were added. The reaction mixture was stirred at 25 °C for 16 h under a nitrogen atmosphere. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under reduced pressure. The residue was dissolved in EtOAc (70 mL) and washed with water (2 × 40 mL). The aq. layer was extracted with EtOAc (2 × 35 mL), and the combined organic layers were washed with brine (2 × 50 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 75% EtOAc in Pet. ether to give PNI 540 (0.41 g, 0.500 mmol, 11.18% yield) as a pale yellow oil. 1H (400 MHz, CDCl3) δ 5.42 - 5.30 (m, 9H), 5.13 - 5.11 (m, 1H), 4.33 - 4.17 (m, 4H), 3.75 (dd, 1H, J = 12.0, 4.0), 2.86 (br s, 2H), 2.78 (t, 4H, J = 6.0), 2.60 (br s, 1H), 2.46 - 2.27 (m, 9H), 2.06 (q, 8H, J = 8.0), 1.77 - 1.61 (m, 10H), 1.40 - 1.25 (m, 28H), 0.89 (apparent t, 6H, J = 6.0). RT = 3.79 min. Purity 97.4%. C 49 H 84 ESI-MS of NO7: m / z = 799 [M + H] + .

[0191]

[0330] Example 3 Synthesis of PNI 342 and 541

Chem.

[0192]

[0331] Compound 2:

[0332] To a stirred solution of oleic acid (58 g, 205 mmol) in dry EtOH (580 mL) was slowly added conc. H2SO4 (1.094 mL, 20.53 mmol), and the reaction mixture was refluxed for 16 h. After completion of the reaction as indicated by TLC, the solvent was evaporated under reduced pressure. The residue was cooled to 0 °C, neutralized with satd. aq. NaHCO3 solution, and extracted with DCM (3 × 250 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated to give ethyl oleate (2, 61.6 g, 198 mmol, 97% yield) as a colorless oil. This ester was used directly in the next step without further purification. 1H (400 MHz, CDCl3) δ 5.39 - 5.31 (m, 2H), 4.13 (q, 2H, J = 8.0), 2.29 (t, 2H, J = 8.0), 2.02 (apparent q, 4H, J = 8.0), 1.62 (p, 2H, J = 8.0), 1.35 - 1.24 (m, 23H), 0.89 (t, 3H, J = 6.0). RT = 3.73 min. Purity 99.5%. C 20 H 39 ESI-MS of O2: m / z = 311 [M+H] + .

[0193]

[0333] Compound 3:

[0334] A solution of diiodomethane (31.2 mL, 386 mmol) in toluene (120 mL) was stirred at -15 °C under a nitrogen atmosphere. Diethylzinc (129 mL, 193 mmol, 1.5 M solution in toluene) was added dropwise to the reaction mixture at -15 °C over 30 min. Note: The internal temperature of the reaction mixture should be maintained below 0 °C. Subsequently, a solution of 2 (30 g, 97 mmol) in toluene (30 mL) was added dropwise to the above reaction mixture while maintaining the internal temperature below 0 °C. The reaction mixture was stirred at the same temperature for 15 min and then gradually warmed to room temperature over 30 min. After stirring at room temperature for 7 h, TLC analysis indicated completion of the reaction. The reaction mixture was cooled to 0 °C and quenched with satd. aq. NH4Cl solution (100 mL). The organic layer was separated and the aqueous layer was extracted with toluene (2 × 75 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 6% EtOAc in Pet. ether to obtain 3 (28.05 g, 86 mmol, 89% yield) as a pale yellow oil. 1H (400 MHz, CDCl3) δ 4.13 (q, 2H, J = 8.0), 2.29 (t, 2H, J = 8.0), 1.63 (p, 2H, J = 8.0), 1.38 - 1.24 (m, 25H), 1.19 - 1.08 (m, 2H), 0.89 (t, 3H, J = 6.0), 0.68 - 0.62 (m, 2H), 0.59 - 0.54 (m, 1H), -0.34 (apparent q, 1H, J = 4.0). RT = 4.01 min. Purity 98.7%. C 21 H 41 ESI-MS of O2: m / z = 325 [M + H] + .

[0194]

[0335] Compound 4:

[0336] LiOH (1.660 g, 69.3 mmol) was added to a stirred solution of 3 (15 g, 46.2 mmol) in EtOH (105 mL) and water (45 mL), and the reaction mixture was stirred at ambient temperature for 16 h. At the completion of the reaction as indicated by TLC, the reaction mixture was concentrated in vacuo to give a residue. The residue was diluted with water (100 mL) and washed with MTBE (2 × 100 mL). The aq. layer was cooled to 0 °C, acidified with 6N HCl, and extracted with EtOAc (3 × 300 mL). The combined organic layers were dried over anhyd. Na2SO4, filtered, and concentrated to give 4 (13.05 g, 43.0 mmol, 93% yield) as a white solid. 1 H (400 MHz, CDCl3) δ 2.36 (t, 2H, J = 6.0), 1.64 (p, 2H, J = 8.0), 1.39 - 1.28 (m, 22H), 1.19 - 1.10 (m, 2H), 0.89 (t, 3H, J = 6.0), 0.68 - 0.62 (m, 2H), 0.59 - 0.54 (m, 1H), -0.33 (apparent q, 1H, J = 4.0). RT = 2.91 min. Purity 97.7%. C 19 H 35 ESI-MS of O2: m / z = 295 [M - H] - .

[0195]

[0337] PNI 342: [Chem.]

[0196]

[0338] To a stirred solution of (±)-1,4-anhydroxylitol (1, 0.8 g, 5.96 mmol) in dry DMF (10 mL) was added DMAP (0.073 g, 0.596 mmol) at room temperature under a nitrogen atmosphere. To this mixture was added a solution of 3-(dimethylamino)propanoic acid hydrochloride (0.916 g, 5.96 mmol) in dry DMF (3 mL), followed by dry DCM (10 mL) and then EDCI hydrochloride (2.287 g, 11.93 mmol). The reaction mixture was stirred at room temperature for 4 h and the consumption of 1 was confirmed by TLC analysis. To the above reaction mixture was added a solution of DMAP (0.146 g, 1.193 mmol) and 4 (4.42 g, 14.91 mmol) in dry DMF (10 mL). Then, EDCI hydrochloride (4.57 g, 23.86 mmol) and then dry DCM (10 mL) were added and the reaction mixture was stirred at room temperature for 16 h. TLC analysis showed complete consumption of the starting material and the reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (60 mL) and washed with water. The aq. layer was extracted with EtOAc (2 × 30 mL) and the combined organic layers were washed with brine (2 × 50 mL). The organic layer was separated, dried over anhyd. Na2SO4, filtered and concentrated. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorel®) using 50% EtOAc in Pet. ether. The product was re-purified using 10% acetone in DCM (Isorel®) to give PNI 342 (0.19 g, 0.240 mmol, 4.03% yield) as a pale yellow oil. 1H (400 MHz, CDCl3) δ 5.38 - 5.35 (m, 1H), 5.16 - 5.12 (m, 1H), 4.34 - 4.14 (m, 4H), 3.80 - 3.74 (m, 1H), 2.66 - 2.58 (m, 2H), 2.55 - 2.50 (m, 2H), 2.36 - 2.31 (m, 4H), 2.25 (s, 6H), 1.62 (p, 4H, J = 8.0), 1.39 - 1.26 (m, 44H), 1.19 - 1.09 (m, 4H), 0.89 (apparent t, 6H, J = 8.0), 0.68 - 0.62 (m, 4H), 0.59 - 0.54 (m, 2H), -0.33 (apparent q, 2H, J = 4.0). RT = 5.03 min. Purity 94.7%. C 48 H 88 ESI-MS of NO7: m / z = 791 [M + H] + .

[0197]

[0339] PNI 541:

Chem.

[0198]

[0340] A well-stirred solution of (±)-1,4-anhydroxylitol (1, 0.6 g, 4.47 mmol) and DMAP (0.055 g, 0.447 mmol) in dry DMF (10 mL) at 25 °C was added to a solution of 1,4-dimethylpiperidine-4-carboxylic acid hydrochloride (0.866 g, 4.47 mmol) in dry DMF (2.5 mL). Subsequently, EDCI hydrochloride (1.715 g, 8.95 mmol) and dry DCM (10 mL) were added. The reaction mixture was stirred at room temperature for 4 h, and TLC analysis showed complete consumption of 1. To this reaction mixture was added a solution of DMAP (0.109 g, 0.895 mmol) and 4 (3.32 g, 11.18 mmol) in dry DMF (7.5 mL). Subsequently, EDCI hydrochloride (3.43 g, 17.89 mmol) and dry DCM (10 mL) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 16 h under a nitrogen atmosphere. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under reduced pressure. The residue was dissolved in EtOAc (70 mL) and washed with water (2 × 40 mL). The aq. layer was extracted with EtOAc (2 × 35 mL), and the combined organic layers were washed with brine (2 × 50 mL). The organic layer was separated, dried over anhyd. Na2SO4, filtered, and concentrated. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorela (trademark)) using 65% EtOAc in Pet. ether to give PNI 541 (0.68 g, 0.819 mmol, 18.31% yield) as a colorless oil. 1H (400 MHz, CDCl3) δ 5.35 (d, 1H, J = 4.0), 5.15 - 5.13 (m, 1H), 4.33 - 4.23 (m, 4H), 3.76 (d, 1H, J = 12.0), 2.92 (br s, 2H), 2.61 - 2.39 (m, 4H), 2.36 (td, 4H, J = 8.0, 4.0), 2.22 - 2.19 (m, 3H), 1.68 - 1.57 (m, 6H), 1.39 - 1.25 (m, 47H), 1.19 - 1.11 (m, 4H), 0.89 (apparent t, 6H, J = 6.0), 0.71 - 0.62 (m, 4H), 0.59 - 0.54 (m, 2H), -0.33 (q, 2H, 4.0). RT = 5.03 min. Purity 96.2%. C 51 H 92 ESI-MS of NO7: m / z = 831 [M+H] + .

[0199] Example 4 Synthesis of PNI 535

Chem.

[0200]

[0341] To a stirred solution of (±)-1,4-anhydroxylitol (1, 0.70 g, 5.22 mmol) in dry DMF (5 mL), DMAP (0.064 g, 0.522 mmol) was added at room temperature under a nitrogen atmosphere. To this mixture, a solution of 3-(dimethylamino)propanoic acid hydrochloride (0.673 g, 5.74 mmol) in dry DMF (2.5 mL) was added, followed by dry DCM (5 mL). Then, EDCI hydrochloride (2.001 g, 10.44 mmol) and DIPEA (2.329 ml, 13.05 mmol) were added. The reaction mixture was stirred at room temperature for 4 h, and TLC analysis showed the consumption of 1. To the reaction flask, a solution of DMAP (0.064 g, 0.522 mmol) and 2-hexyldecanoic acid 5 (3.35 g, 13.05 mmol) in DMF (5 mL) was added. Then, EDCI hydrochloride (4.00 g, 20.88 mmol) and DIPEA (3.37 g, 26.1 mmol) were added. Finally, dry DCM (5 mL) was added, and the reaction mixture was stirred at room temperature for 16 h. TLC analysis showed the completion of the starting material. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in EtOAc (60 mL). The EtOAc layer was washed with water (2 × 30 mL), and the combined aq. layer was extracted with EtOAc (2 × 30 mL). The combined organic layers were washed with brine (2 × 50 mL) and dried over anhydrous Na2SO4. The organic layer was separated, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorel (trademark)) using 60% EtOAc in Pet. ether to give PNI 535 (0.43 g, 0.606 mmol, 11.60% yield) as a colorless liquid. 1H (400 MHz, CDCl3) δ 5.35 - 5.32 (m, 1H), 5.12 - 5.08 (m, 1H), 4.34 - 4.20 (m, 4H), 3.80 - 3.73 (m, 1H), 2.64 - 2.60 (m, 2H), 2.53 - 2.49 (m, 2H), 2.40 - 2.31 (m, 2H), 2.24 (s, 6H), 1.60 - 1.53 (m, 4H), 1.50 - 1.40 (m, 4H), 1.34 - 1.16 (m, 40H), 0.88 (apparent t, 12H, J = 8.0). RT = 3.08 min. Purity 97.9%. C 42 H 80 ESI-MS of NO7: m / z = 711 [M+H] + .

[0201] Example 5 Synthesis of PNI 119, 120 and 344

Chem.

[0202]

[0342] PNI 119:

Chem.

[0203]

[0343] A solution of (±)-1 (1.25 g, 9.3 mmol) in dry DMF (15 mL) was added with DMAP (cat.), followed by the addition of a solution of 4-(dimethylamino)butanoic acid hydrochloride (1.56 g, 9.3 mmol) in dry DMF (10 mL) under a N2 atmosphere. CH2Cl2 (25 mL) was added to the reaction mixture at room temperature. Finally, solid EDCI hydrochloride (3.56 g, 18.6 mmol) was added, and the reaction was stirred at room temperature for 4 h. TLC analysis of the reaction mixture indicated complete consumption of (±)-1. Further, DMAP (cat.) was added to the reaction mixture, followed by the addition of myristic acid (5.32 g, 23.3 mmol) and EDCI hydrochloride (7.13 g, 37.2 mmol) dissolved in dry DMF (20 mL) as solids. Next, dry CH2Cl2 (10 mL) was added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated on a rotary evaporator, diluted with EtOAc (50 mL), and washed with water (3 × 20 mL). The organic layer was washed with brine, dried over anhyd. Na2SO4, and evaporated to dryness on a rotary evaporator to obtain a crude mixture, which was purified by column chromatography using 5% MeOH / DCM as the eluent. PNI 119 was obtained as a colorless oil (780 mg, 1.17 mmol) in 13% yield. 1 H (500 MHz, CDCl3) δ 5.36 (d, 1H, J = 5.0 Hz), 5.13 - 5.11 (m, 1H), 4.33 - 4.28 (m, 2H),4.25 (dd, 1H, J = 10.0 Hz, 5.0 Hz), 4.18 (dd, 1H, J = 10.0 Hz, 7.5 Hz), 3.75(d, 1H, J = 10.0 Hz), 2.39 - 2.32 (m, 6H), 2.28 (apparent t,2H, J = 5.0 Hz), 2.21 (s, 6H), 1.79 (p, 2H, J = 7.5 Hz), 1.65 - 1.59 (m, 4H),1.33 - 1.26 (m, 40H), 0.89 (apparent t, 6H, J = 7.5 Hz). C 39 H 74 Molecular weight of NO7 [M + H] + Calculated value 668.5465. Found value 668.5466.

[0204]

[0344] PNI 120:

Chem.

[0205]

[0345] (±)-1 (1.25 g, 9.3 mmol) in dry DMF (15 mL) was added with DMAP (cat.), followed by the addition of a solution of 5-(dimethylamino)pentanoic acid hydrochloride (1.68 g, 9.3 mmol) in dry DMF (10 mL) under a N2 atmosphere at room temperature. CH2Cl2 (25 mL) was added to the reaction mixture. Finally, solid EDCI hydrochloride (3.56 g, 18.6 mmol) was added to the reaction mixture and stirred at room temperature for 4 h. TLC analysis of the reaction mixture showed complete consumption of (±)-1. Further, DMAP (cat.) was added to the reaction mixture, followed by the addition of myristic acid (5.31 g, 23.3 mmol) dissolved in dry DMF (20 mL) and EDCI hydrochloride (7.13 g, 37.2 mmol) as a solid. Next, dry DCM (10 mL) was added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated on a rotary evaporator, diluted with EtOAc (50 mL), and washed with water (3 × 20 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4, and evaporated to dryness on a rotary evaporator to obtain a crude mixture, which was purified by silica gel column chromatography using 5% MeOH / DCM as an eluent. PNI 120 was obtained as a colorless oil (1.03 g, 1.51 mmol) in a 16% yield. 1 H (500 MHz, CDCl3) δ 5.35 (d, 1H, J = 5.0 Hz), 5.12 - 5.11 (m, 1H), 4.32 - 4.23 (m, 3H), 4.16 (dd, 1H, J = 10.0 Hz, 7.5 Hz), 3.75 (d, 1H, J = 10.0 Hz), 2.38 - 2.28 (m, 8H), 2.23 (s, 6H), 1.68 - 1.59 (m, 6H), 1.51 (p, 2H, J = 7.5 Hz), 1.32 - 1.25 (m, 40H), 0.88 (apparent t, 6H, J = 7.5 Hz). C40 H 76 Molecular weight of NO7 [M+H] + Calculated value: 682.5622. Measured value: 682.5589.

[0206]

[0346] PNI 344:

Chem.

[0207]

[0347] To a stirred solution of (±)-1,4-anhydroxylitol (1, 0.500 g, 3.73 mmol) in dry DMF (5 mL) was added DMAP (0.046 g, 0.373 mmol) at room temperature under a nitrogen atmosphere. To this mixture was added a solution of 3-(dimethylamino)propanoate hydrochloride (0.437 g, 3.73 mmol) in dry DMF (2.5 mL) and dry DCM (10 mL). EDCI hydrochloride (1.429 g, 7.46 mmol) and DIPEA (1.623 ml, 9.32 mmol) were added, and the reaction mixture was stirred at room temperature for 4 h. TLC analysis showed complete consumption of 1. Then, a solution of DMAP (0.046 g, 0.373 mmol) and myristic acid (2.128 g, 9.32 mmol) in DMF (5 mL) was added to the above reaction mixture. Further, EDCI hydrochloride (2.86 g, 14.91 mmol) and DIPEA (3.25 ml, 18.64 mmol) were added. Finally, dry DCM (10 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 16 h. Completion of the reaction was confirmed by TLC analysis, and the reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (60 mL) and washed with water (2 × 30 mL). The aq. layer was extracted with EtOAc (2 × 30 mL), and the combined organic layers were washed with brine (2 × 50 mL). The organic layer was separated, dried over anhyd. Na2SO4, filtered, and concentrated. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorel (trademark)) using 70% EtOAc in Pet. ether to give PNI 344 (0.220 g, 0.336 mmol, 9.02% yield) as a colorless oil. 1H (400 MHz, CDCl3) δ 5.38 - 5.34 (m, 1H), 5.13 - 5.11 (m, 1H), 4.34 - 4.16 (m, 4H), 3.76 (d, 1H, J = 12.0), 2.65 - 2.61 (m, 2H), 2.54 - 2.50 (m, 2H), 2.36 - 2.31 (m, 4H), 2.25 (s, 6H), 1.62 (p, 4H, J = 8.0), 1.35 - 1.22 (m, 40H), 0.88 (apparent t, 6H, J = 6.0). RT = 3.57 min. Purity 91.8%. C 38 H 72 ESI-MS of NO7: m / z = 655 [M + H] + .

[0208] Example 6 Synthesis of PNI 534

Chemical Structure

[0209]

[0348] To a stirred solution of (±)-1,4-anhydroxylitol (1, 1.0 g, 7.46 mmol) in dry DMF (5 mL) was added DMAP (0.091 g, 0.746 mmol) at room temperature under a nitrogen atmosphere. To this mixture was added a solution of 3-(dimethylamino)propanoic acid hydrochloride (0.961 g, 8.20 mmol) in DMF (2.5 mL), followed by dry DCM (10 mL). Then, EDCI hydrochloride (2.86 g, 14.91 mmol) and DIPEA (2.409 g, 18.64 mmol) were added. The reaction mixture was stirred at room temperature for 4 h, whereupon TLC analysis indicated consumption of 1. To the reaction mixture was added a solution of DMAP (0.091 g, 0.746 mmol) and dodecanoic acid (3.73 g, 18.64 mmol) in dry DMF (5 mL). Then, EDCI hydrochloride (5.72 g, 29.8 mmol) and DIPEA (4.82 g, 37.3 mmol) were added. Finally, dry DCM (10 mL) was added and the mixture was stirred at room temperature for 16 h. TLC analysis indicated complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure and the residue was dissolved in EtOAc (60 mL). The EtOAc layer was washed with water (2 × 30 mL) and the combined aq. layers were extracted with EtOAc (2 × 30 mL). The combined organic layers were washed with brine (2 × 50 mL) and dried over anhyd. Na2SO4. The organic layer was filtered and concentrated under reduced pressure. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera™) using 70% EtOAc in Pet. ether to give PNI 534 (0.230 g, 0.385 mmol, 5.16% yield) as a colorless oil. 1 H (400 MHz, CDCl3) δ 5.38 - 5.34 (m, 1H), 5.16 - 5.11 (m, 1H), 4.34 - 4.15 (m, 4H), 3.80 - 3.74(m, 1H), 2.64 - 2.59 (m, 2H), 2.53 - 2.49 (m, 2H), 2.36 - 2.31 (m, 4H), 2.24 (s, 6H),1.62 (p, 4H, J = 8.0), 1.36 - 1.21 (m, 32H), 0.88 (apparent t,6H, J = 6.0). RT = 1.85 min. Purity 85.6%.C 34 H 64ESI-MS of NO7: m / z = 599 [M+H] + .

[0210] Example 7 Synthesis of PNI 532

Chem.

[0211]

[0349] Compound 8:

[0350] DMAP (0.524 g, 4.29 mmol) was added to a stirred solution of 8-(tert-butoxy)-8-oxooctanoic acid (6, 11.36 g, 49.3 mmol) in dry DCM (100 mL) at room temperature under a nitrogen atmosphere. (Z)-Non-2-en-1-ol (7, 6.1 g, 42.9 mmol) was added to the above mixture and stirring was continued for 15 min. The reaction was cooled to 0 °C and DCC (9.73 g, 47.2 mmol) was added. The reaction mixture was allowed to reach room temperature and stirred for 16 h. TLC analysis showed complete consumption of the starting material. The precipitated urea was filtered through a Celite pad and washed with DCM (2 × 50 mL). The combined filtrates were concentrated and the residue was washed with satd. aq. NaHCO3 solution (2 × 50 mL). The organic layer was separated and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure and the residue was purified by silica gel (100 - 200 mesh) column chromatography (Isorel (trademark)) using 3% EtOAc in Pet. ether to give (Z)-1-(tert-butyl) 8-(non-2-en-1-yl) octanedioate (8, 11.35 g, 29.8 mmol, 69.4% yield). 1H (400 MHz, CDCl3) δ 5.68 - 5.61 (m, 1H), 5.56 - 5.49 (m, 1H), 4.63 (d, 2H, J = 4.0), 2.31 (t, 2H, J = 8.0), 2.20 (t, 2H, J = 8.0), 2.10 (apparent q, 2H, J = 8.0), 1.67 - 1.57 (m, 4H), 1.44 (s, 9H), 1.40 - 1.25 (m, 12H), 0.89 (t, 3H, J = 6.0). RT = 1.40 min. Purity 89.2%. C 21 H 38 ESI-MS of O4Na: m / z = 377 [M + Na] + .

[0212]

[0351] Compound 9:

[0352] TFA (10.00 mL, 130 mmol) was added dropwise to a stirred solution of 8 (5 g, 14.10 mmol) in dry DCM (50 mL) at room temperature under a nitrogen atmosphere, and stirring was continued for 16 h. TLC analysis showed the presence of 8. Again, TFA (10.00 mL, 130 mmol) was added, and the reaction mixture was refluxed for 16 h. The reaction did not reach completion, and TLC analysis showed the presence of 8. The reaction was stopped, and the mixture was evaporated under reduced pressure. The crude material was purified by column silica gel (100 - 200 mesh) chromatography (Isorella (trademark)) using 30% EtOAc in Pet. ether to obtain (Z)-8-(non-2-en-1-yloxy)-8-oxooctanoic acid (9, 3.41 g, 11.43 mmol, 81% yield) as a brown oil, and 600 mg of 8 was also recovered. 1 H (400 MHz, CDCl3) δ 5.68 - 5.61 (m, 1H), 5.56 - 5.49 (m, 1H), 4.63 (d, 2H, J = 4.0), 2.37 - 2.30 (m, 4H), 2.10 (apparent q, 2H, J = 8.0), 1.68 - 1.60 (m, 4H), 1.40 - 1.24 (m, 12H), 0.89 (t, 3H, J = 6.0).

[0213]

[0353] PNI 532 [Chem.]

[0214]

[0354] To a 100 mL two-necked round-bottom flask containing a well-stirred solution of (±)-1,4-anhydroxylitol (1, 0.5 g, 3.73 mmol) in dry DMF (5 mL) was added a solution of DMAP (0.046 g, 0.373 mmol) and 3-(dimethylamino)propanoic acid hydrochloride (0.480 g, 4.10 mmol) in dry DMF (2.5 mL) at room temperature under a nitrogen atmosphere. To this mixture was added dry DCM (10 mL), followed by EDCI (1.429 g, 7.46 mmol) and DIPEA (1.204 g, 9.32 mmol). The reaction mixture was stirred at ambient temperature for 4 h, and TLC analysis indicated the consumption of 1. DMAP (0.046 g, 0.373 mmol) was added to the above reaction mixture. A solution of 9 (2.78 g, 9.32 mmol) in dry DMF (5 mL) was added, followed by EDCI hydrochloride (2.86 g, 14.91 mmol) and DIPEA (2.409 g, 18.64 mmol). Finally, dry DCM (10 mL) was added, and the reaction mixture was stirred at room temperature for 16 h. After completion of the reaction as indicated by TLC, the solvent was evaporated under reduced pressure, and the residue was dissolved in EtOAc (60 mL). The organic layer was washed with water (2 × 30 mL), and the aq. layer was extracted with EtOAc (2 × 30 mL). The combined organic layers were washed with brine (2 × 50 mL) and dried over anhyd. Na2SO4. The organic layer was separated, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 70% EtOAc in Pet. ether to give PNI 532 (0.270 g, 0.340 mmol, 9.12% yield) as a pale yellow oil. 1H (400 MHz, CDCl3) δ 5.68 - 5.61 (m, 2H), 5.55 - 5.49 (m, 2H), 5.37 - 5.34 (m, 1H), 5.16 - 5.10 (m, 1H), 4.62 (d, 4H, J = 8.0), 4.33 - 4.10 (m, 4H), 3.79 - 3.73 (m, 1H), 2.65 - 2.60 (m, 2H), 2.53 - 2.50 (m, 2H), 2.36 - 2.28 (m, 8H), 2.25 (s, 6H), 2.10 (q, 4H, J = 8.0), 1.67 - 1.59 (m, 8H), 1.39 - 1.24 (m, 24H), 0.89 (apparent t, 6H, J = 6.0). RT = 2.24 min. Purity 97.2%. C 44 H 76 NO 11 The ESI-MS of: m / z = 795 [M + H] + .

[0215] Example 8 Synthesis of PNI 127, 573, 574 and 575

Chemical Structure

[0216]

[0355] Compound 10:

[0356] A well-stirred solution of (±)-1,4-anhydroxylitol (1, 5.1 g, 38.0 mmol) in dry pyridine (25 mL) and trityl chloride (10.07 g, 36.1 mmol) were added to a 100 mL one-necked round-bottom flask under a nitrogen atmosphere at room temperature, and stirring was continued for 16 h. After completion of the reaction as indicated by TLC, the excess solvent was concentrated under reduced pressure. The residue was dissolved in DCM (100 mL) and water (200 mL). The organic layer was separated, and the aq. layer was extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine (3 × 100 mL), dried over anhyd. Na2SO4, filtered, and concentrated. The crude product was purified by silica gel (100 - 200 mesh) column chromatography using 50% EtOAc in Pet. ether to give (±)-10 (8.65 g, 22.93 mmol, 60.3% yield) as a white sticky solid. 1 H (400 MHz, CDCl3) δ 7.47 - 7.43 (m, 6H), 7.34 - 7.30 (m, 6H), 7.28 - 7.23 (m, 3H), 4.32 (brs, 1H), 4.28 - 4.23 (m, 3H), 3.77 (dd, 1H, J = 12.0, 4.0), 3.50 (dd, 1H, J =12.0, 4.0), 3.43 (dd, 1H, J = 8.0, 4.0), 3.14 (br s, 1H). RT = 2.66 min. Purity 99.9%. C 24 H 23 ESI-MS of C - .

[0217]

[0357] Compound 11:

[0358] A stirred solution of linoleic acid (6.55 g, 23.36 mmol) in dry DCM (50 mL), DMAP (2.85 g, 23.36 mmol), EDCI hydrochloride (17.91 g, 93 mmol) and DIPEA (9.38 ml, 53.7 mmol) was added to a 250 mL three-necked round-bottom flask at room temperature under a nitrogen atmosphere. The reaction mixture was stirred for 15 min and a solution of (±)-10 (3.52 g, 9.34 mmol) in dry DCM (20 mL) was added. After stirring for 16 h, the reaction mixture was quenched with water (150 mL) and extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine (3 × 75 mL), dried over anhyd. Na2SO4, filtered and concentrated. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 5% EtOAc in Pet. ether to give (±)-11 (7.21 g, 7.84 mmol, 33.6% yield) as a pale yellow oil. 1 H (400 MHz, CDCl3) δ 7.44 - 7.42 (m, 6H), 7.32 - 7.28 (m, 6H), 7.26 - 7.22 (m, 3H), 5.43 - 5.31(m, 9H), 5.12 - 5.10 (m, 1H), 4.35 - 4.31 (m, 1H), 4.26 (dd, 1H, J = 8.0, 4.0),3.74 (dd, 1H, J = 12.0, 4.0), 3.35 (apparent t, 1H, J =8.0), 3.16 (dd, 1H, J = 8.0, 6.0), 2.79 (t, 4H, J = 6.0), 2.36 (t, 2H, J =8.0), 2.16 - 1.99 (m, 10H), 1.65 (p, 2H, J = 8.0), 1.40 - 1.22 (m, 30H), 0.90 (t,6H, J = 6.0). RT = 3.95 min. Purity 98.1%.C 60 H 84 ESI-MS of O6Na: m / z = 923 [M+Na] + .

[0218]

[0359] Compound 12

[0360] TFA (0.427 ml, 5.55 mmol) was added to a solution of (±)-11 (2.0 g, 2.219 mmol) and triethylsilane (1.772 ml, 11.09 mmol) in dry DCM (50 ml) at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 1 h, and TLC analysis showed complete consumption of 11. The mixture was quenched with satd. aq. NaHCO3 solution (50 mL) and extracted with DCM (2 × 50 mL). The organic layer was dried over anhyd. Na2SO4, filtered, and concentrated. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 10% EtOAc in Pet. ether to give (±)-12 (1.15 g, 1.745 mmol, 79% yield) as a colorless oil. 1 H (400 MHz, CDCl3) δ 5.43 - 5.29 (m, 9H), 5.11 - 5.09 (m, 1H), 4.51 (dd, 1H, J = 12.0, 8.0), 4.30 (dd, 1H, J = 8.0, 4.0), 4.20 - 4.16 (m, 2H), 4.11 - 4.07 (m, 1H), 3.76 (dd, 1H, J = 12.0, 4.0), 2.78 (t, 4H, J = 6.0), 2.37 - 2.30 (m, 4H), 2.06 (q, 8H, J = 8.0), 1.69 - 1.62 (m, 4H), 1.40 - 1.25 (m, 28H), 0.90 (t, 6H, J = 6.0). RT = 2.29 min. Purity 92.5%. C 41 H 71 ESI-MS of C + .

[0219]

[0361] PNI 127 (Alternative method)

[0362] A 100 mL two-necked round-bottom flask containing a well-stirred solution of 1,4-dimethylpiperidine-4-carboxylic acid hydrochloride (0.970 g, 5.01 mmol) in dry DCM (30 mL) was charged with DMAP (0.556 g, 4.55 mmol) and EDCI hydrochloride (1.745 g, 9.10 mmol) under a nitrogen atmosphere. The reaction mixture was stirred at 25 °C for 10 min, a solution of (±)-12 (3 g, 4.55 mmol) in dry DCM (15 mL) was added, and the reaction mixture was stirred at 25 °C for 16 h. After completion of the reaction as indicated by TLC, the solvent was evaporated under reduced pressure. The residue was dissolved in EtOAc (150 mL), washed with water (2 × 100 mL), brine (2 × 100 mL), dried over anhyd. Na2SO4, filtered, and concentrated. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 3% MeOH in DCM to afford PNI 127 (2.96 g, 3.71 mmol, 81% yield) as a pale yellow oil.

[0220]

[0363] PNI 574:

Chem.

[0221]

[0364] A 100 mL two-necked round-bottom flask containing a stirred solution of 2-(4-methylpiperazin-1-yl)acetic acid (0.125 g, 0.789 mmol) in dry DCM (25 mL) was charged with DMAP (0.102 g, 0.789 mmol) and EDCI hydrochloride (0.348 g, 1.821 mmol) under a nitrogen atmosphere. The reaction mixture was stirred at 25 °C for 10 minutes, and a solution of (±)-12 (0.4 g, 0.607 mmol) in dry DCM (5 mL) was added. The mixture was stirred at 25 °C for 16 hours, and the completion of the reaction was confirmed by TLC analysis. The reaction mixture was quenched with water (120 mL) and extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine (3 × 75 mL), dried over anhyd.Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 5% MeOH in DCM to afford PNI 574 (0.090 g, 0.101 mmol, 16.70% yield) as a thick red oil. 1 H (400 MHz, CDCl3) δ 5.42 - 5.30 (m, 9H), 5.13 (s, 1H), 4.33 - 4.14 (m, 4H), 3.74 (d, 1H, J = 4.0), 3.27 (s, 2H), 2.78 (t, 4H, J = 6.0), 2.73 - 2.46 (m 8H), 2.39 - 2.26 (m,7H), 2.06 (q, 8H, J = 8.0), 1.66 - 1.58 (m, 4H), 1.49 - 1.26 (m, 28H), 0.90 (apparent t, 6H, J = 6.0). RT = 2.34 min. Purity 93.3%. C 48 H 83 ESI-MS of N2O7: m / z = 800 [M+H] + .

[0222]

[0365] PNI 573:

Chem.

[0223]

[0366] PNI 573 was synthesized using a method similar to the one used for the synthesis of PNI 574. The synthesis of PNI 573 was carried out using quinuclidin-4-carboxylic acid hydrochloride (0.092 g, 0.592 mmol), DMAP (0.076 g, 0.592 mmol), EDCI hydrochloride (0.304 g, 1.593 mmol) in dry DCM (25 mL), and 12 (0.3 g, 0.455 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 5% MeOH in DCM to obtain PNI 573 (0.18 g, 0.226 mmol, 49.7% yield) as a pale yellow oil. 1 H (400 MHz, CDCl3) δ 5.42 - 5.30 (m, 9H), 5.14 - 5.07 (m, 1H), 4.32 - 4.23 (m, 3H), 4.11 (dd,1H, J = 12.0, 8.0), 3.76 (dd, 1H, J = 12.0, 4.0), 3.24 - 3.05 (m, 6H), 2.77 (t,4H, J = 6.0), 2.33 (q, 4H, J = 8.0), 2.08 - 1.98 (m, 10H), 1.92 (apparent t, 4H, J = 8.0), 1.66 - 1.58 (m, 4H), 1.42 - 1.26 (m, 28H), 0.89 (t, 6H,J = 6.0). RT = 2.34 min. Purity 92.4%.C 49 H 82 ESI-MS of NO7: m / z = 796 [M+H] + .

[0224]

[0367] PNI 575:

Chem.

[0225]

[0368] PNI 575 was synthesized using a method similar to the one used for the synthesis of PNI 574. The synthesis of PNI 143 was carried out using 1-methylpiperidine-3-carboxylic acid (0.085 g, 0.592 mmol), DMAP (0.076 g, 0.592 mmol), EDCI hydrochloride (0.304 g, 1.593 mmol) in dry DCM (25 mL), and 12 (0.3 g, 0.455 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 5% MeOH in DCM to obtain PNI 575 (0.265 g, 0.338 mmol, 74.2% yield) as a pale yellow oil. 1 H (400 MHz, CDCl3) δ 5.42 - 5.10 (m, 9H), 5.11 (s, 1H), 4.34 - 4.12 (m, 4H), 3.77 (d, 1H, J = 12.0), 2.99 - 2.59 (m, 6H), 2.40 - 2.13 (m, 7H), 2.11 - 1.84 (m, 11H), 1.80 - 1.52(m, 8H), 1.42 - 1.26 (m, 28H), 0.90 (apparent t, 6H, J = 6.0). RT = 2.48 min. Purity 93.2%. C 48 H 82 ESI-MS of C + .

[0226] Example 9 Synthesis of PNI 576, 577 and 578

Chem.

[0227]

[0369] Compound 13:

[0370] Compound 13 was synthesized using a method similar to the one used for the synthesis of 11. The synthesis of 13 was carried out using 4 (9.06 g, 30.5 mmol), DMAP (4.05 g, 33.2 mmol), EDCI hydrochloride (10.15 g, 53.1 mmol), DIPEA (14.28 mL, 80 mmol) in dry DCM (70 mL), and (±)-10 (5.0 g, 13.28 mmol) in dry DCM (30 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 5% EtOAc in Pet. ether to obtain 13 (11.1 g, 11.89 mmol, 90% yield) as a colorless oil. 1 H (400 MHz, CDCl3) δ 7.46 - 7.44 (m, 6H), 7.34 - 7.24 (m, 9H), 5.44 (d, 1H, J = 4.0), 5.14 - 5.12 (m, 1H), 4.37 - 4.33 (m, 1H), 4.28 (dd, 1H, J = 12.0, 4.0), 3.76 (dd, 1H, J = 12.0, 4.0), 3.38 (dd, 1H, J = 12.0, 8.0), 3.19 (dd, 1H, J = 12.0, 8.0), 2.39 (apparent t, 2H, J = 6.0), 2.18 - 2.02 (m, 2H), 1.67 (p, 2H, J = 8.0), 1.50 - 1.24 (m, 46H), 1.19 - 1.13 (m, 4H), 0.92 (t, 6H, J = 6.0), 0.71 - 0.65 (m, 4H), 0.62 - 0.57 (m, 2H), -0.30 (q, 2H, J = 6.0).

[0228]

[0371] Compound 14:

[0372] Compound 14 was synthesized using a method similar to that used for the synthesis of 12. The synthesis of 14 was carried out using (±)-13 (11.0 g, 11.78 mmol), Et3SiH (9.41 mL, 58.9 mmol), and TFA (1.489 mL, 11.78 mmol) in dry DCM (100 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 10% EtOAc in Pet. ether to give 14 (5.5 g, 7.96 mmol, 67.5% yield) as a colorless oil. 1 H (400 MHz, CDCl3) δ 5.28 - 5.10 (m, 2H), 4.53 - 4.07 (m, 3H), 3.78 - 3.56 (m, 2H), 2.40 - 2.31(m, 4H), 1.68 - 1.59 (m, 4H), 1.38 - 1.10 (m, 44H), 1.17 - 1.10 (m, 4H), 0.89 (t, 6H,J = 6.0), 0.68 - 0.62 (m, 4H), 0.59 - 0.54 (m, 2H), -0.33 (q, 2H, J = 6.0). C 43 H 79 ESI-MS of C + .

[0229]

[0373] PNI 576:

Chem.

[0230]

[0374] PNI 576 was synthesized using a method similar to the one used for the synthesis of PNI 574. The synthesis of PNI 576 was carried out using 1-cyclopropylpiperidine-4-carboxylic acid (0.108 g, 0.637 mmol), DMAP (0.085 g, 0.695 mmol), EDCI hydrochloride (0.222 g, 1.158 mmol) in dry DCM (15 mL), and 14 (0.4 g, 0.579 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorel® (trademark)) using 3% MeOH in DCM to obtain PNI 576 (0.45 g, 0.534 mmol, 92% yield) as a yellow oil. 1 H (400 MHz, CDCl3) δ 5.35 (s, 1H), 5.10 (s, 1H), 4.31 - 4.18 (m, 4H), 3.76 (d, 1H, J = 12.0), 3.05 - 2.95 (m, 1H), 2.40 - 2.12 (m, 7H), 1.93 - 1.81 (m, 2H), 1.76 - 1.50 (m, 8H), 1.42 - 1.26 (m, 44H), 1.19 - 1.05 (m, 4H), 0.89 (t, 6H, J = 6.0), 0.69 - 0.61 (m, 4H), 0.59 - 0.54 (m, 2H), 0.48 - 0.33 (m, 4H), -0.33 (q, 2H, J = 6.0). RT = 3.35 min. Purity 99.1%. C 52 H 92 ESI-MS of C + .

[0231]

[0375] PNI 577:

Chem.

[0232]

[0376] PNI 577 was synthesized using a method similar to the one used for the synthesis of PNI 574. The synthesis of PNI 577 was carried out using 1-ethylpiperidine-4-carboxylic acid (0.104 g, 0.658 mmol), DMAP (0.085 g, 0.658 mmol), EDCI hydrochloride (0.290 g, 1.519 mmol) in dry DCM (20 mL), and 14 (0.35 g, 0.506 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorel®) using 5% MeOH in DCM to obtain PNI 577 (0.29 g, 0.349 mmol, 69.0% yield) as a thick colorless oil. 1 H (400 MHz, CDCl3) δ 5.37 - 5.35 (m, 1H), 5.13 5.09 (m, 1H), 4.33 - 4.16 (m, 4H), 3.76 (d,1H, J = 12.0), 2.95 - 2.90 (m, 2H), 2.51 - 2.25 (m, 7H), 2.14 - 1.75 (m 6H), 1,70 - 1.55(m, 4H), 1.42 - 1.28 (m, 44H), 1.17 - 1.09 (m, 7H), 0.89 (t, 6H, J = 6.0),0.68 - 0.62 (m, 4H), 0.59 - 0.54 (m, 2H), -0.33 (q, 2H, J = 6.0). RT = 3.59 min. Purity 95.1%. C 51 H 92 ESI-MS of C + .

[0233]

[0377] PNI 578:

Chem.

[0234]

[0378] PNI 578 was synthesized using a method similar to the one used for the synthesis of PNI 574. The synthesis of PNI 578 was carried out using 2-(1-methylpiperidin-2-yl)acetic acid (0.096 g, 0.608 mmol), DMAP (0.078 g, 0.608 mmol), EDCI hydrochloride (0.290 g, 1.519 mmol) in dry DCM (20 mL), and 14 (0.35 g, 0.506 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorela (trademark)) using 5% MeOH in DCM to obtain PNI 578 (0.32 g, 0.385 mmol, 76% yield) as a pale yellow oil. 1 H (400 MHz, CDCl3) δ 5.35 (s, 1H), 5.13 (s, 1H), 4.32 - 4.16 (m, 4H), 3.76 (dt, 1H, J = 8.0, 4.0), 2.81 - 2.18 (m, 12H), 1.77 - 1.51 (m, 10H), 1.45 - 1.21 (m, 44H), 1.19 - 1.10 (m, 4H), 0.89 (t, 6H, J = 6.0), 0.68 - 0.61 (m, 4H), 0.59 - 0.54 (m, 2H), -0.33 (q, 2H, J = 4.0). RT = 3.61 min. Purity 99.7%. C 51 H 91 ESI-MS of C + .

[0235] Example 10 Synthesis of PNI 579 and 580

Chemical formula

[0236]

[0379] Compound 15:

[0380] Compound 15 was synthesized using a method similar to the one used for the synthesis of 11. The synthesis of 15 was carried out using 2-hexyldecanoic acid (3.92 g, 15.27 mmol), DMAP (1.866 g, 15.27 mmol) in dry DCM (40 mL), followed by EDCI hydrochloride (6.37 g, 33.2 mmol), DIPEA (5.80 ml, 33.2 mmol), and (±)-10 (2.5 g, 6.64 mmol) in dry DCM (10 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 2% EtOAc in Pet. ether to obtain 15 (5.12 g, 6.00 mmol, 90% yield) as a pale yellow oil. 1 H (400 MHz, CDCl3) δ 7.47 - 7.45 (m, 6H), 7.33 - 7.22 (m, 9H), 5.29 (d, 1H, J = 4.0), 5.09 - 5.07 (m, 1H), 4.29 - 4.24 (m, 2H), 3.75 (dd, 1H, J = 8.0, 4.0), 3.42 (dd, 1H, J = 12.0, 8.0), 3.22 (dd, 1H, J = 12.0, 4.0), 2.43 - 2.34 (m, 1H), 2.22 - 2.15 (m, 1H), 1.66 - 1.38 (m, 8H), 1.36 - 1.12 (m, 40H), 0.92 - 0.86 (m, 12H).

[0237]

[0381] Compound 16:

[0382] Compound 16 was synthesized using a method similar to the one used for the synthesis of 12. The synthesis of 16 was carried out using (±)-15 (5.1 g, 5.98 mmol), triethylsilane (4.77 mL, 29.9 mmol), and TFA (1.151 mL, 14.94 mmol) in dry DCM (70 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 2% EtOAc in Pet. ether to obtain (±)-16 (3.23 g, 5.29 mmol, 88% yield) as a colorless oil. 1H (400 MHz, CDCl3) δ 5.26 (d, 1H, J = 4.0), 5.16 (s, 1H), 4.29 (dd, 1H, J = 12.0, 4.0), 4.21 - 4.17 (m, 1H), 3.80 - 3.74 (m, 2H), 3.58 (dd, 1H, J = 12.0, 4.0), 2.43 - 2.32 (m, 2H), 2.15 (br s, 1H), 1.67 - 1.41 (m, 8H), 1.34 - 1.19 (m, 40H), 0.89 (t, 12H, J = 6.0). RT = 2.18 min. Purity 99.9%. C 37 H 71 ESI-MS of O6: m / z = 611 [M+H] + .

[0238]

[0383] PNI 579

Chem.

[0239]

[0384] PNI 579 was synthesized using a method similar to the one used for the synthesis of PNI 574. The synthesis of PNI 579 was carried out using 1,4-dimethylpiperidine-4-carboxylic acid hydrochloride (0.152 g, 0.786 mmol), DMAP (0.096 g, 0.786 mmol), EDCI hydrochloride (0.301 g, 1.571 mmol) in dry DCM (20 mL), and 16 (0.4 g, 0.655 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 4% MeOH in DCM to obtain PNI 579 (0.444 g, 0.592 mmol, 90% yield) as a pale yellow oil. 1H (400 MHz, CDCl3) δ 5.34 (d, 1H, J = 4.0), 5.11 - 5.10 (m, 1H), 4.32 - 4.27 (m, 3H), 4.21 - 4.16 (m, 1H), 3.74 (dd, 1H, J = 12.0, 4.0), 2.69 (br s, 2H), 2.41 - 2.31 (m, 5H), 2.22 - 2.14 (m, 4H), 1.63 - 1.41 (m, 10H), 1.30 - 1.22 (m, 43H), 0.88 (t, 12H, J = 8.0). RT = 2.66 min. Purity 97.4%. C 45 H 84 ESI-MS for NO7: m / z = 751 [M+H] + .

[0240]

[0385] PNI 580:

Chem.

[0241]

[0386]

[0387] PNI 580 was synthesized using a method similar to the one used for the synthesis of PNI 574. The synthesis of PNI 580 was carried out using 4-(dimethylamino)butanoic acid hydrochloride (0.165 g, 0.982 mmol), DMAP (0.120 g, 0.982 mmol), EDCI hydrochloride (0.377 g, 1.964 mmol) in dry DCM (20 mL), and 16 (0.4 g, 0.655 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 4% MeOH in DCM to give PNI 580 (0.39 g, 0.539 mmol, 82% yield) as a colorless oil. 1H (400 MHz, CDCl3) δ 5.33 (d, 1H, J = 4.0), 5.10 (d, 1H, J = 4.0), 4.33 - 4.21 (m, 4H), 3.75 (dd, 1H, J = 8.0, 4.0), 2.42 - 2.29 (m, 12H), 1.91 - 1.79 (m, 2H), 1.64 - 1.41 (m, 8H), 1.34 - 1.20 (m, 40H), 0.88 (t, 12H, J = 6.0). RT = 2.49 min. Purity 98.7%. C 43 H 82 ESI-MS of NO7: m / z = 724 [M+H] + .

[0242] Example 11 Synthesis of PNI 581, 582 and 583

Chemical Structure

[0243]

[0388] Compound 17:

[0389] Under a nitrogen atmosphere, to a stirred solution of 10 (2.0 g, 5.31 mmol) in dry DMF (100 mL) and dry THF (100 mL) in a 500 mL three-necked RBF at 0 °C was slowly added NaH (1.062 g, 26.6 mmol). The reaction mixture was stirred at the same temperature for 15 minutes, then linoleyl bromide (5.25 g, 15.94 mmol) was added. The reaction mixture was slowly warmed to 25 °C and stirred for 7 h. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with ice-cold water (250 mL) and extracted with EtOAc (3 × 120 mL). The combined organic layers were washed with brine (3 × 150 mL), dried over Na2SO4, filtered, and concentrated to give a crude product. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorela (trademark)) using 3% EtOAc in Pet. ether to give 17 (2.4 g, 2.75 mmol, 51.7% yield) as a pale yellow oil. 1H (400 MHz, CDCl3) δ 7.49 - 7.45 (m, 6H), 7.30 - 7.20 (m, 9H), 5.42 - 5.31 (m, 8H), 4.22 - 4.18 (m, 1H), 4.04 (dd, 1H, J = 8.0, 4.0), 3.91 - 3.86 (m, 1H), 3.84 (d, 1H, J = 4.0), 3.73 (dd, 1H, J = 12.0, 4.0), 3.50 - 3.43 (m, 2H), 3.39 - 3.28 (m, 3H), 3.22 (dd, 1H, J = 12.0, 8.0), 2.78 (t, 4H, J = 6.0), 2.06 (q, 8H, J = 8.0), 1.59 (p, 4H, J = 4.0), 1.43 - 1.23 (m, 32H), 0.91 - 0.88 (m, 6H). RT = 3.86 min. Purity 98.1%. C 60 H 88 ESI-MS of O4Na: m / z = 896 [M+Na] + .

[0244]

[0390] Compound 18:

[0391] A solution of 17 (2.35 g, 2.69 mmol) and Et3SiH (2.149 ml, 13.45 mmol) in DCM (40 mL) was stirred in a 250 mL two-necked RBF under a nitrogen atmosphere, and TFA (0.427 mL, 5.55 mmol) was added at 0 °C. The reaction mixture was stirred at 0 °C for 1 h. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with satd. NaHCO3 solution (60 mL) and extracted with DCM (2 × 50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to give a crude product. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 8% EtOAc in Pet. ether to give 18 (1.46 g, 2.314 mmol, 86% yield) as a colorless oil. 1H (400 MHz, CDCl3) δ 5.42 - 5.30 (m, 8H), 4.13 (dd, 1H, J = 8.0, 4.0), 4.09 - 4.05 (m, 1H), 3.96 - 3.92 (m 2H), 3.90 - 3.81 (m, 2H), 3.75 (dd, 1H, J = 12.0, 4.0), 3.64 - 3.59 (m, 1H), 3.47 - 3.42 (m, 3H), 2.78 (t, 4H, J = 6.0), 2.06 (q, 8H, J = 8.0), 1.62 - 1.48 (m, 4H), 1.40 - 1.26 (m, 32H), 0.90 (t, 6H, J = 6.0). RT = 2.63 min. Purity 99.9%. C 41 H 75 ESI-MS of O4: m / z = 631 [M + H] + .

[0245]

[0392] PNI 581:

Chem.

[0246]

[0393] A stirred solution of 1,4-dimethylpiperidine-4-carboxylic acid (0.120 g, 0.762 mmol) in dry DCM (20 mL) in a 100 mL two-necked RBF under a nitrogen atmosphere was added with DMAP (0.072 g, 0.586 mmol) and EDCI hydrochloride (0.281 g, 1.466 mmol), and the reaction mixture was stirred at 25 °C for 10 minutes. Then, 18 (0.37 g, 0.586 mmol) in dry DCM (5 mL) was added to the reaction mixture, and the mixture was stirred at 25 °C for 16 h. TLC showed a majority of unreacted starting material. Again, a stirred solution of 1,4-dimethylpiperidine-4-carboxylic acid hydrochloride (0.148 g, 0.762 mmol) in dry DCM (10 mL) in a 100 mL two-necked RBF under a nitrogen atmosphere was added with DMAP (0.072 g, 0.586 mmol) and EDCI hydrochloride (0.281 g, 1.466 mmol), and the reaction mixture was stirred at 25 °C for 10 minutes. Then, the first reaction mixture was added to this mixture, and the reaction mixture was stirred at room temperature for 16 h. After completion of the reaction as indicated by TLC, the solvent was evaporated to obtain a residue. This residue was dissolved in EtOAc (130 mL) and washed with water (2 × 100 mL) and brine (2 × 100 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 4% MeOH in DCM to obtain PNI 581 (0.395 g, 0.501 mmol, 86% yield) as a brown oil. 1H (400 MHz, CDCl3) δ 5.42 - 5.31 (m, 8H), 4.44 - 4.38 (m, 1H), 4.30 - 4.26 (m, 1H), 4.22 - 4.18 (m, 1H), 4.07 (dd, 1H, J = 8.0, 4.0), 3.93 (s, 1H), 3.85 (d, 1H, J = 4.0), 3.75 (d, 1H, J = 8.0), 3.60 - 3.54 (m, 1H), 3.45 (t, 2H, J = 6.0), 3.41 - 3.36 (m, 1H), 3.26 (br s, 2H), 2.84 - 2.71 (m, 6H), 2.65 (s, 3H), 2.32 - 2.22 (m, 2H), 2.06 (q, 8H, J = 8.0), 1.68 - 1.48 (m, 6H), 1.38 - 1.26 (m, 35H), 0.90 (t, 6H, J = 6.0). RT = 4.06 min. Purity 97.8%. C 49 H 88 ESI-MS of NO5: m / z = 771.2 [M+H] + .

[0247]

[0394] PNI 582:

Chem.

[0248]

[0395] PNI 582 was synthesized using a method similar to the one used for the synthesis of PNI 574. The synthesis of PNI 582 was carried out using 1-methylpiperidine-3-carboxylic acid (0.113 g, 0.792 mmol), DMAP (0.097 g, 0.792 mmol), EDCI hydrochloride (0.304 g, 1.585 mmol) in dry DCM (20 mL), and 18 (0.4 g, 0.634 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorel®) using 5% MeOH in DCM to obtain PNI 582 (0.43 g, 0.569 mmol, 90% yield) as a pale yellow oil. 1H (400 MHz, CDCl3) δ 5.42 - 5.30 (m, 8H), 4.38 - 4.34 (m, 1H), 4.23 - 4.14 (m, 2H), 4.08 (dd, 1H, J = 12.0, 4.0), 3.92 (s, 1H), 3.83 (s, 1H), 3.77 (d, 1H, J = 12.0), 3.58 - 3.53 (m, 1H), 3.45 (t, 2H, J = 8.0), 3.42 - 3.36 (m, 1H), 3.17 (br s, 1H), 2.93 (br s, 1H), 2.78 (t, 4H, J = 8.0), 2.60 - 2.26 (m, 4H), 2.17 - 1.96 (m, 10H), 1.87 - 1.74 (m, 2H), 1.67 - 1.49 (m, 6H), 1.38 - 1.26 (m, 32H), 0.90 (t, 6H, J = 6.0). RT = 2.99 min. Purity 99.2%. C 48 H 86 ESI-MS of NO5: m / z = 756.6 [M+H] + .

[0249]

[0396] PNI 583:

Chem.

[0250]

[0397] PNI 583 was synthesized using a method similar to the one used for the synthesis of PNI 574. The synthesis of PNI 583 was carried out using 2-(4-methylpiperazin-1-yl)acetic acid (0.125 g, 0.792 mmol), DMAP (0.097 g, 0.792 mmol), EDCI hydrochloride (0.304 g, 1.585 mmol) in dry DCM (20 mL), and 18 (0.4 g, 0.634 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isorel (trademark)) using 4% MeOH in DCM to obtain PNI 583 (0.415 g, 0.527 mmol, 83% yield) as a pale yellow oil. 1H (400 MHz, CDCl3) δ 5.41 - 5.30 (m, 8H), 4.41 (dd, 1H, J = 12.0, 4.0), 4.25 - 4.16 (m, 2H), 4.09 (dd, 1H, J = 8.0, 4.0), 3.92 (s, 1H), 3.83 (d, 1H, J = 4.0), 3.76 (dd, 1H, J = 12.0, 4.0), 3.58 - 3.52 (m, 1H), 3.44 (t, 2H, J = 8.0), 3.41 - 3.36 (m, 1H), 3.28 (s, 2H), 2.84 - 2.58 (m, 12H), 2.43 (s, 3H), 2.06 (q, 8H, J = 8.0), 1.57 - 1.51 (m, 4H), 1.40 - 1.26 (m, 32H), 0.92 - 0.86 (m, 6H). RT = 2.68 min. Purity 97.9%. C 48 H 87 ESI-MS of N2O5: m / z = 771.6 [M+H] + .

[0251] Example 12 Synthesis of PNI 76

Chem.

[0252]

[0398] PNI 76:

[0399] A solution of (±)-19 (600 mg, 3.65 mmol) in dry DMF (10 mL) was added with DMAP (cat.), followed by the addition of a solution of 4-(dimethylamino)butanoic acid hydrochloride (1.56 g, 9.3 mmol) in dry DMF (10 mL) under a N2 atmosphere. CH2Cl2 (10 mL) was added to the reaction mixture. Then, solid EDCI hydrochloride (1.39 g, 7.30 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 4 h. TLC analysis of the reaction mixture showed complete consumption of (±)-19. Further, DMAP (cat.) was added to the reaction mixture, followed by the addition of myristic acid (2.73 g, 11.98 mmol) and EDCI hydrochloride (3.50 g, 18.25 mmol) dissolved in dry DMF (10 mL) as solids. Next, dry DCM (10 mL) was added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated on a rotary evaporator, diluted with EtOAc (20 mL), and washed with water (3 × 10 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4, and evaporated to dryness on a rotary evaporator to obtain a crude mixture, which was purified by silica gel column chromatography using 5% MeOH / DCM as the eluent. PNI 76 was obtained as a colorless oil (294 mg, 0.32 mmol) in 9% overall yield.

[0253]

[0400] HCl salt of PNI 76

[0401] PNI 76 was dissolved in the minimum amount of anhyd. diethyl ether. HCl in ether was added until the pH reached 3 - 5 (checked with pH test paper). The solvent was evaporated, and CH2Cl2 was added and evaporated 2 - 3 times to remove trace amounts of HCl. Next, the compound was dissolved in a small amount of deionized water and freeze-dried to obtain the HCl salt of PNI 76. 1H (500 MHz, CDCl3) δ 5.38 (d, 1H, , J = 3.0 Hz), 5.22 - 5.19 (m, 1H), 5.07 (d, 1H, J = 5.0 Hz), 4.61 (d, 1H, J = 10.0 Hz), 4.26 (dd, 1H, J = 10.0 Hz, 5.0 Hz), 4.21 (dd, 1H, J = 10.0 Hz, 5.0 Hz), 4.13 (dd, 1H, J = 15.0 Hz, 5.0 Hz), 3.79 (d, 1H, J = 10.0 Hz), 2.42 - 2.29 (m, 14H), 2.23 (apparent t, 2H, J = 7.5 Hz), 1.84 (p, 2H, J = 6.3 Hz), 1.65 - 1.54 (m, 6H), 1.30 - 1.27 (m, 60H), 0.89 (apparent t, 9H, J = 7.5 Hz). C 54 H 102 Molecular weight of NO9 [M + H] + Calculated value: 908.7555. Measured value: 908.7511.

[0254] Example 13 Synthesis of PNI 369

Chem.

[0255]

[0402] Compound 21:

[0403] To a stirred solution of 2-(dodecyloxy)ethanol (20, 3.7 g, 16.06 mmol) and triphenylphosphine (10.53 g, 40.1 mmol) in dry toluene (70 mL) was slowly added carbon tetrabromide (13.31 g, 40.1 mmol), and the reaction mixture was stirred at 60 °C for 16 h. After completion of the reaction as indicated by TLC analysis, the reaction mixture was concentrated under reduced pressure, and the crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 5% EtOAc in Pet. ether to give 1-(2-bromoethoxy)dodecane (21, 4.55 g, 15.20 mmol, 95% yield) as a colorless oil. 1H (400 MHz, CDCl3) δ 3.74 (d, 2H, J = 6.0), 3.50 - 3.45 (m, 4H), 1.59 (p, 2H, J = 8.0), 1.38 - 1.21 (m, 18H), 0.89 (t, 3H, J = 6.0). RT = 3.40 min. Purity 96.6%.

[0256]

[0404] Compound 22:

[0405] Compound 22 was synthesized using a method similar to the method used for the synthesis of 17. The synthesis of 22 was carried out using 10 (1.0 g, 2.66 mmol), sodium hydride (0.531 g, 13.28 mmol, 60% dispersion in mineral oil), and 1-(2-bromoethoxy)dodecane (21, 2.337 g, 7.97 mmol) in dry DMF (10 mL) and dry THF (10 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 5% EtOAc in Pet. ether to obtain 22 (1.1 g, 1.373 mmol, 51.7% yield) as a colorless oil. 1 H (400 MHz, CDCl3) δ 7.46 (d, 6H, J = 8.0), 7.30 - 7.20 (m, 9H), 4.25 - 4.21 (m, 1H), 4.06 - 4.02 (m, 2H), 4.00 (d, 1H, J = 4.0), 3.79 - 3.75 (m, 1H), 3.72 - 3.58 (m, 5H), 3.52 - 3.46 (m, 3H), 3.41 - 3.28 (m, 5H), 3.22 (d, 1H, J = 8.0, 4.0), 1.60 (t, 2H, J = 6.0), 1.48 (p, 2H, J = 8.0), 1.35 - 1.21 (m, 36H), 0.89 (t, 6H, J = 6.0). RT = 2.63 min. Purity 97.8%. C 52 H 80 ESI-MS of C + .

[0257]

[0406] Compound 23:

[0407] Compound 23 was synthesized using a method similar to the one used for the synthesis of 18. The synthesis of 23 was carried out using 22 (1.1 g, 1.373 mmol), Et3SiH (1.096 mL, 6.86 mmol), and TFA (0.264 mL, 3.43 mmol) in dry DCM (30 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 20% EtOAc in Pet. ether to obtain 23 (0.58 g, 1.038 mmol, 76% yield) as a colorless oil. 1 1H (400 MHz, CDCl3) δ 4.15 - 4.03 (m, 4H), 3.87 - 3.76 (m, 4H), 3.66 - 3.54 (m, 7H), 3.47 - 3.43(m, 4H), 1.61 - 1.54 (m, 4H), 1.36 - 1.20 (m, 36H), 0.90 - 0.87 (m, 6H). RT = 1.97 min. Purity 99.1%. C 33 1H 67 ESI - MS of C28H52O6: m / z = 559.5 [M + H] + .

[0258]

[0408] PN

Chem.

[0259]

[0409] PNI 369 was synthesized using a method similar to the one used for the synthesis of PNI 574. The synthesis of PNI 369 was carried out using 1,4 - dimethylpiperidine - 4 - carboxylic acid (0.110 g, 0.698 mmol), DMAP (0.086 g, 0.698 mmol), EDCI hydrochloride (0.359 g, 1.879 mmol) in dry DCM (25 mL), and 23 (0.3 g, 0.537 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100 - 200 mesh) column chromatography (Isolera (trademark)) using 5% MeOH in DCM to obtain PNI 369 (0.21 g, 0.301 mmol, 56.0% yield) as a pale yellow oil. 1H (400 MHz, CDCl3) δ 4.37 - 4.28 (m, 2H), 4.21 - 4.17 (m, 1H), 4.11 - 4.07 (m, 2H), 3.99 (d, 1H, J = 4.0), 3.81 - 3.76 (m, 1H), 3.73 - 3.69 (m, 1H), 3.66 - 3.59 (m, 3H), 3.57 - 3.52 (m, 4H), 3.46 - 3.41 (m, 4H), 2.89 (br s, 2H), 2.48 - 2.35 (m, 5H), 2.21 (d, 2H, J = 12.0), 1.86 - 1.71 (m, 2H), 1.60 - 1.52 (m, 4H), 1.37 - 1.20 (m, 39H), 0.88 (t, 6H, J = 8.0). RT = 2.13 min. Purity 98.5%. C 41 H 80 ESI-MS of NO7: m / z = 698.6 [M + H] + .

[0260]

[0410] Example 14 Synthesis Scheme of Representative Compounds

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

[0261] Example 15 Isolation and Proliferation of Primary T Cells Derived from Human Whole Blood

[0411] Unless otherwise specified, all reagents were purchased from STEMCELL Technologies, Vancouver, Canada.

[0262]

[0412] Lyophilized human IL-2 was reconstituted at a concentration of 0.1 mg / ml in sterile calcium- and magnesium-free 1×PBS in a biological safety cabinet. 50 μl of this human IL-2 was added to 50 mL of ImmunoCult-XF™ T cell expansion medium to generate a medium for T cells. 7 - 30 mL of human whole peripheral blood containing ACDA anticoagulant was placed in a sterile 50 mL polypropylene conical tube in a biological safety cabinet.

[0263]

[0413] Negative selection protocol. Blood was collected from healthy human donors and mixed with ACD-A anticoagulant. CD4+ and CD8+ T cells were isolated using the EasySep™ Direct Human T Cell Isolation Kit, a pan T cell negative selection kit. Cells were maintained in ImmunoCult-XF™ T Cell Exp Medium supplemented with human recombinant IL2 (Peprotech). Cells were activated on the day of isolation with the triple activator ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator.

[0264]

[0414] Positive selection protocol. Blood was collected from healthy human donors and mixed with ACDA anticoagulant. A PBMC suspension was prepared using density gradient centrifugation with Lymphoprep™. T cells were then positively selected from the PBMC suspension using the EasySep™ Human CD3 Pos Selection Kit II. Cells were maintained in ImmunoCult-XF™ T Cell Exp Medium supplemented with human recombinant IL2 (Peprotech). On the day of isolation, cells were activated with the triple activator ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator.

[0265]

[0415] T cells were isolated from blood using the EasySep™ Direct Human T Cell Isolation Kit. First, 50 μl / mL of Isolation Cocktail™ and then 50 μl / mL of EasySep™ RapidSpheres™ were added to the blood tube. The blood was gently mixed and incubated for 5 minutes at room temperature (RT). The tube was placed in an EasySep™ 50 Magnet™ device and incubated for 10 minutes at RT. The enriched cell suspension was pipetted into a new sterile 50 mL polypropylene tube and the RapidSpheres™ process was repeated.

[0266]

[0416] This doubly enriched cell suspension was pipetted into a new sterile 50 mL polypropylene conical tube and centrifuged for 10 min at 300 g at RT.

[0267]

[0417] The supernatant was removed and the cell pellet was resuspended in 10 mL of PBS and recentrifuged at 300 g for 10 min to wash the remaining supernatant from the cells. The supernatant was removed again and the cells were resuspended in pre-warmed complete T cell medium. Samples were taken and a trypan blue exclusion test for cell viability was performed (Thermo Fisher).

[0268] Example 16 Activation / Proliferation of T Cells

[0418] The scientific background of T cell activation can be found in the 2003 paper by Trickett A. et al., paper 6. The cell suspension of Example 8 was diluted to 1E6 cells / ml in Complete T Cell Medium (ThermoFisher), and T cells were activated by adding either 25 μl of ImmunoCult™ Human CD3 / CD28 / CD2 Tri T Cell Activator™ or ImmunoCult™ Human CD3 / CD28 Dual T Cell Activator™ per 1 mL of T Cell Medium. Cell growth was monitored by daily cell counting under expansion. The cells were diluted in Complete T Cell Medium to maintain a concentration of approximately 1E6 cells / mL. Around day 5, 6, or 7, the T cells entered the logarithmic phase of growth and rapid proliferation occurred.

[0269]

[0419] To confirm that the T cells were in the logarithmic phase, CD25 expression was evaluated and needed to be greater than 80% by flow cytometry, and cell proliferation was monitored by graphing the total number of T cells over time (not shown).

[0270] Example 17 Downstream processing and analysis of treated T cells: Flow cytometry and ELISA

[0420] Reagents were obtained from Stemcell Technologies unless otherwise noted. T cells were isolated from a single donor. 48H after lipid particle mRNA exposure, the cell suspension was transferred to pre-labeled 1.5 mL tubes, and the treated T cells were collected by centrifugation at 300×G for 10 minutes at 4°C. The supernatant was removed, and the pellet was resuspended in PBS. 0.5 ul of BD Horizon™ Fixable Viability Stain 575V™ (BD Biosciences) was added, and the mixture was incubated in the dark for 10 minutes at RT. This dye binds to amines.

[0271]

[0421] The cells were centrifuged again as before, then washed twice with 1 mL of dye buffer (BSA, BD Pharminigen), and the washed pellet was diluted in 100 μl of BSA. 2 μl volumes of the following antibodies were added to each tube of cells treated as follows: CD25, CD8, CD4, (PerCP-Cy™5.5 mouse anti-human CD25, BV786 mouse anti-human CD8 Clone RPA-T8, APC-Cy™7 mouse anti-human CD4 Clone SK3 (all from BD Pharmingen)). However, controls were excluded: no antibodies were added to samples of eGFP only and viability controls. In single dye compensation tubes, only one antibody was added per tube.

[0272]

[0422] When the tubes were incubated at 4 °C for 30 min, 400 μl of dye buffer was added and the cells were centrifuged again. The cells were washed once with 1 mL of dye buffer and spun down again as in step 1. The cell pellet was resuspended in 1 mL of dye buffer and added to pre-labeled flow tubes (Corning Falcon) with cell strainer caps.

[0273]

[0423] Negative selection protocol. Blood was collected from healthy human donors and mixed with ACDA anticoagulant. CD4+ and CD8+ T cells were isolated using the EasySep™ Direct Human T Cell Isolation Kit, a pan-T cell negative selection kit. The cells were maintained in ImmunoCult-XF™ T Cell Experimental Medium supplemented with human recombinant IL2 (Peprotech). On the day of isolation, the cells were activated with the triple activator ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator.

[0274]

[0424] Freezing and thawing of human T cells. Blood was collected from healthy human donors and mixed with ACDA anticoagulant. Both CD4+ and CD8+ T cells were isolated using the EasySep Direct Human T Cell Isolation Kit, a pan T cell negative selection kit. The cells were cryopreserved using CryoStor® CS10 and stored in liquid nitrogen. At thawing, the cells were maintained in ImmunoCult-XF™ T Cell Exp Medium supplemented with human recombinant IL2 (Peprotech). On the day of thawing, the cells were activated with a triple activator. After transfection, flow cytometry was used for general eGFP expression levels and median fluorescence intensity values for research purposes.

[0275] Example 18 Microfluidic mixing of nucleic acid therapeutic (NAT) into lipid particles (LNP)

[0425] Unless otherwise specified, N / P = 10 was used for all of these experiments. The lipid mix composition solution was prepared in ethanol by mixing a specified amount of lipids (see Table 3) from individual lipid stocks in ethanol. A lipid mix solution concentration of 37.5 mM was used for NanoAssembl® SPARK™, and a 12.5 mM lipid mix solution was typically used for NanoAssembl® Benchtop or Ignite™.

Table 3-A

Table 3-B

Table 3-C

[0276]

[0426] IL = Ionizable lipid; Tween80 = Polysorbate 80; BRIJ™ L4 = Polyoxyethylene(4) lauryl ether; BRIJ™ S10 = Polyoxyethylene(10) stearyl ether; BRIJ™ S20 = Polyoxyethylene(20) stearyl ether; BRIJ™ S35 = Polyoxyethylene(23) lauryl ether; TPGS1000 = D-α-tocopherol polyethylene glycol 1000 succinate; Lipid H = equimolar Tween20 / Polysorbate 80 / tridecyl-D-maltoside; Stabilizer = PEG-DMG or any stabilizer defined above and falling within this category.

[0277]

[0427] The components of the lipid mix include an ionizable lipid, a structural lipid, cholesterol, and a stabilizer. A low pH buffer (3 - 6) may be used. In the case of an ionizable amino lipid, the pH of the buffer is typically lower than the pKa of the lipid.

[0278]

[0428] The preparation of siRNA, messenger RNA, or plasmid NAT is described below. The observed particle attributes were generally in the range of 50 - 200 nm for mRNA depending on the lipid composition.

[0279]

[0429] Messenger RNA or plasmid NAT was diluted to the required concentration using sodium acetate buffer. Next, a lipid nucleic acid particle sample was prepared by flowing both fluids using a NanoAssemblr® Spark instrument. Briefly, 10 - 20 μg of nucleic acid in 3 μL total volume of 100 mM sodium acetate buffer was mixed with 16 μL of 37.5 mM lipid mix solution required by the N / P ratio (4, 6 or 10 in the exemplified embodiments). The lipid nucleic acid particles created within the instrument were immediately diluted in 48 μL of Ca- and Mg-free pH 7.4 1×PBS in the aqueous output well. These nucleic acid lipid particles were immediately collected into microcentrifuge tubes containing 96 μL of Ca- and Mg-free pH 7.4 1×PBS. The encapsulation efficiency was measured by the modified Ribogreen® assay (QuAnti-iT RiboGreen® RNA Assay Kit, Fisher). The observed particle attributes were generally 60 - 200 nm in size for mRNA depending on the lipid composition and production method.

[0280]

[0430] Lipid-based formulations were also manufactured with the larger instrument NanoAssemblr® Ignite® for testing. Briefly, 350 μL of mRNA was diluted to the required concentration of 0.2 - 0.3 mg / mL using 100 mM sodium acetate buffer. Next, a lipid particle sample was prepared by flowing both fluids, namely the nucleic acid in an aqueous solvent and the lipid mix in ethanol, at a flow rate ratio of 3:1 and a total flow rate of 12 ml / min. After mixing in the microfluidic device, the lipid nucleic acid particle (LNAP) sample was diluted into tubes containing 3 - 40 volumes of phosphate buffered saline (PBS), pH 7.4, free of RNase. Finally, ethanol was removed by dialysis in PBS, pH 7, or using an Amicon® centrifugal filter (Millipore, USA) at 3000 RPM, or using a TFF system. Once the required concentration was achieved, the lipid nucleic acid particles were filter sterilized using a 0.2 μm filter under aseptic conditions. The final encapsulation efficiency was measured by the Ribogreen® assay.

[0281]

[0431] Nucleic acid reagent. The messenger RNA or plasmid nucleic acid therapeutic (NAT) described below was diluted to the required concentration using sodium acetate buffer. Then, an LNAP sample was prepared by flowing both fluids using a NanoAssemblr® Spark instrument. Briefly, 10-20 μg of nucleic acid in 32 μL of 100 mM sodium acetate buffer was mixed with 16 μL of 37.5 mM lipid mix solution required by the N / P ratio (4, 6, 8, 10 in the exemplified embodiments). The microfluidically mixed LNAP created in the instrument was immediately diluted in the aqueous output well with 48 μL of Ca++ and Mg++ free pH 7.4 1×PBS. These LNAPs were immediately collected in microcentrifuge tubes containing 96 μL of the same buffer (pH 7.4). The encapsulation efficiency was measured by a modified Ribogreen® assay (Quanti-iT RiboGreen® RNA Assay Kit, Fisher). This information was used to establish the desired dosage.

[0282]

[0432] The nucleic acid therapeutic model reagents used in the following experiments were as follows:

[0433] Trilink Cleancap® eGFP mRNA: Cat. L-7601 (Trilink Biotechnologies, San Diego, CA); Trilink Cleancap® EPO mRNA: Cat. L-7209 (Trilink Biotechnologies); Millipore Sigma TagRFP Simplicon RNA Kit: Cat. SCR712 (containing both TagRFP RNA & B18R RNA) (Millipore Sigma Canada, Oakville Ontario); The CD19 CAR plasmid with EGFP reporter was purchased from Creative Biolabs (Shirley, NY) and contains a T7 promoter (Mut)-signal peptide-scFv-CD8 hinge transmembrane-4-1BB-CD3zeta-T2A-eGFP reporter gene CAR cassette (2353 bp) within pcDNA. The overall size of this custom CD19 CAR plasmid DNA template was approximately 7649 - 7661 bp.

[0283]

[0434] Unmodified CAR messenger RNA (mRNA) transcripts encoding the CD19 scFv-h(BB±-eGFP reporter gene cassette were synthesized by in vitro transcription using wild-type bases and capped using the Cleancap® AG method by Trilink Biotechnologies Inc. (cap 1). This unmodified CAR mRNA transcript was enzymatically polyadenylated, followed by DNase and phosphatase treatment. The final mRNA transcription product was purified by silica membrane and packaged in a solution of 1 mM sodium citrate buffer (pH 6.4) at a concentration of 1 mg / mL. This custom CD19 CAR plasmid vector and the mRNA encoding CD19 CAR were purchased from Creative Biolab and Trilink Biotechnologies Inc., respectively.

[0284]

[0435] OVA antigen has been used as a model antigen to stimulate immune responses 10Therefore, it is useful for vaccine research. Usually, OVA antigen is used together with a sensitizing substance such as alum. However, when the OVA antigen is delivered in the form of mRNA, the premise is that a sensitizing substance such as alum is not necessary, and the mRNA itself can generate a person who can generate a reaction against the antigen by immune cells. Cleancap® OVA mRNA L-7610 and Cleancap® OVA mRNA(5moU) L-7210 were used.

[0285]

[0436] Plasmid preparation, a custom-made 5514 nt pCX-EGFP plasmid containing ampicillin resistance and restriction enzyme HINDIII in ddH2O by GenScript USA Inc, Piscataway, NJ, was used for this evaluation. The plasmid contained a GFP expression component that produced the target protein only when the plasmid was expressed intracellularly.

[0286] Example 19

[0437] Comparative data of lipids showing activity with eGFP mRNA LNP in primary human T cells

[0438] Reagents were obtained from Abcam, Cambridge, UK unless otherwise stated. An eGFP SimpleStep™ ELISA® Kit was used to show mRNA delivery and activity in vitro. The assay was performed as instructed by the eGFP SimpleStep ELISA® Kit protocol. Briefly, cryopreserved human T cells isolated from fresh human whole blood using a negative selection protocol in advance were thawed and activated using a triple activator. Ten days after activation, the T cells were administered an N / P 10 mRNA LNP encoding eGFP at 2 μg of mRNA per 500,000 cells. The T cells were collected 48 hours after treatment with the mRNA LNP and lysed for total eGFP content.

[0287]

[0439] To expose isolated and activated T cells (day 0) to formulated mRNA, 2 μg of CleanCap™ eGFP (Trilink Biotechnologies, San Diego, CA) mRNA-containing LNP was added to 500,000 T cells in 1 mL of complete T cell medium together with 1 μg / mL of recombinant human ApoE4 (“ApoE”) (Peprotech Inc., Montreal, Canada).

[0288]

[0440] The positive control was a standard lipid (DLin-MC3-DMA or “MC3” lipid data) using the Trio activation protocol.

[0289]

[0441] The LNP was calculated based on the previous Ribogreen™ assay results. T cells were counted by trypan blue (Sigma) exclusion and diluted to 500,000 cells / mL. Briefly, in a 12-well plate, 1 mL aliquots were placed in each well. ApoE was added to each well to a final concentration of 1 μg / mL. Based on the calculations in step 1, the required amount of mRNA LNP was added (day 7) and the plates were incubated for 48 h.

[0290]

[0442] The lipid mix compositions were tested for their ability to induce transfection as measured by the median fluorescence intensity of labeled mRNA in T cells (measured by flow cytometry). Primary human T cells freshly isolated from human blood were selected by a negative selection method and treated with a dose of 2 μg of mRNA per 500,000 cells.

[0291]

[0443] Ionizable lipids, PNI 76, 119, 121 and MC3, were compared at a composition of 40 Mol% ionizable lipid, 20 Mol% structural lipid DSPC, 37.5 Mol% cholesterol, and 2.5 Mol% BRIJ™ S10, N / P ratio 10.

[0292]

[0444] The transfection efficiency and amount of eGFP expressed for each lipid are shown in the 5th and 6th columns of Table 4, respectively. Figure 1 shows the performance of LNPs containing either PNI 76 or PNI 121 for ionizable lipid Dlin-MC3-DMA (MC3) in a CT10 composition with an N / P ratio of 10, and gene expression was analyzed by flow cytometry 48 hours after treatment for eGFP mRNA expression. It was found that PNI 76 had the same transfection efficiency as MC3, while PNI 121 was greater than MC3.

[0293]

[0445] In related experiments, primary human T cells from 6 different donors were isolated from fresh whole blood using a negative selection protocol, activated using a triple activator, and then treated with mRNA LNPs 7 days after activation. The results of analyzing gene expression by flow cytometry 48 hours after treatment are shown in Figure 2. It was found that the transfection efficiency of LNPs containing PNI 121 was greater than that of those containing MC3, which was clinically confirmed in various donors. The viability of T cells was not affected by its treatment with PNI 121 compared to untreated cells (not shown).

[0294]

[0446] Furthermore, the level of eGFP expression was quantified by eGFP ELISA as shown in Figure 3. Human T cells isolated from fresh human whole blood and frozen using a negative selection protocol in advance were thawed and activated using a triple activator. On the 10th day after activation, T cells were administered with CT10 mRNA LNPs containing DLin-MC3-DMA, PNI 76, PNI 119, PNI 120, PNI 121, and PNI 122 encoding eGFP at 2 μg of mRNA per 500,000 cells and an N / P of 10. Protein expression was measured by eGFP ELISA 48 hours later. It was found that all PNI novel lipids mediated eGFP expression. Most notably, eGFP expression mediated by PNI 121 was greater than that of MC3.

[0295]

[0447] In related experiments, GFP expression in previously cryopreserved isolated primary human T cells treated with mRNA encapsulated by mRNA-LNP containing DLin-MC3-DMA, PNI 127, PNI 328, PNI 329, or PNI 541 in a CT10 composition with an N / P ratio of 8 at 500 ng per 125,000 cells 4 days after activation. GFP MFI (first column), transfection efficiency (middle column), and viability (third column) were measured by flow cytometry 48 h after LNP addition in Figure 4.

[0296]

[0448] GFP MFI was measured by flow cytometry 48 hours after LNP addition. T cells were isolated from whole blood using a negative isolation procedure (EasySep™ Human T Cell Isolation Kit, Stemcell Technologies). mRNA-LNP containing 500 ng of encapsulated mRNA per 125,000 cells was administered to the T cells 3 days after activation. The LNP was a CT10 composition with an N / P of 8. The results are shown in Figure 5. Transfection efficiency (upper graph) and MFI (lower graph) were measured by flow cytometry 48 h after LNP addition. PNI 328, 329, and 541 showed higher levels of protein expression (MFI) than MC3.

[0297]

[0449] Table 4 shows the quantitative results for additional experiments with MC3, PNI 76, PNI 119, and PNI 121 eGFP mRNA LNPs in CT10 with an N / P of 8.

Table 4

[0298] Example 20 Erythropoietin mRNA Delivery and Expression

[0450] The in vitro mRNA delivery and activity were demonstrated using the Quantikine® IVD Human Epo ELISA 2 - antibody sandwich assay. The reagents were obtained from Quantikine, Minneapolis, MN. The assay was performed as instructed in the Quantikine® IVD® ELISA Human Erythropoietin Immunoassay protocol REF DEP00 Package Insert. Briefly, primary human T cells were isolated from fresh whole blood using a negative - selection protocol and activated with a triple activator. Seven days after activation, the T cells were administered 2 μg of mRNA per 500,000 cells and mRNA LNP encoding EPO with N / P 10. Quantikine® Human Serum Control was used. T cells were collected 48 hours after treatment with mRNA LNP, lysed to obtain cytoplasmic EPO, and secreted EPO samples were collected from the culture supernatant. The results are shown in Figure 6 in mIU / mL. Table 5 shows the data in Figure 6 and the increment (fold) of EPO relative to MC3 for PNI 76 and 121. It was found that EPO expression mediated by LNP containing PNI 121 was higher than that containing MC3. [Table 5]

[0299]

[0451] C56BL / 6 mice were administered an i.v. dose of 0.5, 1, or 3 mg / Kg of mRNA LNP encoding recombinant human EPO containing the ionizable lipids MC3, PNI 76, PNI 121, and PNI 127 using the LM02 composition with N / P 6, and hEPO protein was measured using the Quantikine® IVD® ELISA Human Erythropoietin Immunoassay protocol. The results for the 0.5 mg / Kg dose are shown in the scatter plot of Figure 7. The results for the 1 and 3 mg / Kg doses are shown in Figure 8 as scatter plots of hEPO expression levels at 6 h (left) and 24 h (right).

[0300]

[0452] In another study, 5-moU EPO mRNA encapsulated in LM02 LNPs with an N / P ratio of 6 and a dose of 0.5 mg / Kg, along with MC3 as a control, was administered, containing 15 different ionizable lipids (PNI 336, PNI 534, PNI 535, PNI 342, PNI 321, PNI 532, PNI 538, PNI 541, PNI 325, PNI 328, PNI 329, PNI 539, PNI 540, PNI 127). hEPO protein was measured using the ProteinSimple® Ella platform and rhEPO microfluidic cartridge provided by the manufacturer. The results are shown in Figure 9, where some of the PNI compounds (PNI 328, 329, 539, 540, and 127) are comparable to MC3. PNI-127, PNI-329, PNI 541, and PNI-565 are better than MC3 with respect to the amount of protein produced (higher MFI than MC3). Example 21 Characterization and Encapsulation of Lipid Nucleic Acid Particles or "LNPs"

[0453] After creating the lipid particles as described above, the particle size (hydrodynamic diameter of the particles) was determined by dynamic light scattering (DLS) using a Zetasizer™ Nano ZS™ (Malvern Instruments, UK). A He / Ne laser adjusted to a wavelength of 633 nm was used as the light source. The data was measured from the scattered intensity data in the backscattering detection mode (measurement angle = 173). The measurements were the average of 10 times for each of 2 cycles per sample. The Z-average size was reported as the particle size and defined as the harmonic intensity average particle diameter.

[0301]

[0454] The physical characteristics of lipid nanoparticle (LNP) compositions containing compounds according to formula (I) and manufactured on a NanoAssemblr® Spark and benchtop are shown in Tables 6 and 7 below. Size, size variation of the LNP (PDI), encapsulation efficiency, and pKa are shown. These physical characteristics of the LNPs are important from the viewpoints of stability, biodistribution, and cell membrane crossing. Encapsulation was good in all formulations, and the polydispersity (PDI) was below 0.3. Size and PDI were measured using dynamic light scattering technology, and the nucleic acid encapsulation efficiency was calculated using a modified QuantiT RiboGreen RNA assay.

[0302]

[0455]

Table 6

[0456]

Table 7

[0303] Example 22 hEPO / Cytokine Measurement by Simplex® Automated ELISA

[0457] Blood from mice treated with hEPO mRNA LNP was analyzed using the automated ELISA platform "ELLA" with a Simplex (trademark) antibody panel cartridge. EPO specific to humans and IL-5, IL-6, TNF-α, and IFN-γ specific to mice were measured (reagents from Bio-techne). Briefly, 50 μL of sample reagent (diluted biological sample, quality control, or calibration point sample) was placed into each sample inlet, and 1 mL of wash buffer was placed into the corresponding inlet of the cartridge. All immunoassay operations (e.g., starting the system, splitting the sample into channels, incubating the sample, washing, rehydrating and flowing the secondary antibody, washing, rehydrating and flowing the streptavidin dye conjugate, incubating, washing, scanning) were performed automatically. Raw signal levels (relative fluorescence units, RFU), mean signal values, standard deviations, and coefficients of variation (CV) relative to each glass nanoreactor (GNR) value were provided. The RFU values were automatically backfit by ELLA to generate analyte concentrations corresponding to the analyte / sample using the manufacturer's predefined calibration method. The results are shown in Figure 10.

[0304]

[0458] The delivery and activity of mRNA were demonstrated in vivo using the Quantikine® IVD Human Epo ELISA dual antibody sandwich assay. The reagent was obtained from Quantikine, Minneapolis, MN. The assay was performed as instructed in the Quantikine® IVD® ELISA Human Erythropoietin Immunoassay protocol REF DEP00 Package Insert. Briefly, the EPO expression analysis of serum samples at 6 h and / or 24 h after intravenous administration of 0.5, 1, or 3 mg / kg doses of mRNA encoding EPO was analyzed and expressed in mIU / mL. Standard appropriately diluted serum, as well as horseradish peroxidase (HRP)-conjugated rabbit anti-EPO polyclonal antibody and TMB (tetramethylbenzidine) as a substrate, were added to a 96-well microplate pre-coated with the EPO antibody provided by the manufacturer, and incubated for 20 minutes. A stop solution (sulfuric acid) was added to the wells, and the plate was read at 450 nm to calculate the concentration of EPO. The amount of color generated was directly proportional to the amount of conjugate bound to the EPO antibody, which was directly proportional to the amount of EPO in the sample or standard. The standard curve was created by plotting the absorbance against the concentration of the provided standards. Quantikine® Human Serum Control (CEP 01, CEP 03) was used as an internal control.

[0305] Example 23 mRNA-LNP-mediated CD19 CAR expression in isolated primary human T cells

[0459] mRNA containing IL with the CT10 composition of N / P8 was assayed after in vitro exposure. The CAR vector pcDNA3.1 anti-CD19-h(BB lambda)-EGFP-2nd-CAR(T7 Mut) 7661 bp was purchased from Creative BioLabs, NY, USA.

[0306]

[0460] T cells were isolated from whole blood using negative isolation procedures and T cell activation, and expansion was performed by triple activation with ImmunoCult™ Human T Cell Expansion Media as described above. As seen in Figure 11, CD19 CAR expression was greater than PBS in transfected T cells in vitro with respect to PNI 328, PNI 329, and PNI 127 LNP.

[0307] Example 24 In vivo delivery by intramuscular administration of mRNA LNP encoding Ova

[0461] CleanCap® OVA WT mRNA [TriLink L7610] or CleanCap® OVA 5-moU mRNA [TriLink L7210] encoding the OVA antigen was encapsulated using LNP of composition LM02b. A lipid mixture of ionizable lipid, DOPE, cholesterol, and PEG-DMG2000 in ethanol was mixed with a low pH buffer solution of OVA mRNA at an N / P ratio of 8 using a NanoAssemblr® Ignite™ microfluidic mixer. The LNP was filtered using Amicon® Ultra filtration technology and characterized for size, PDI, and encapsulation efficiency. Size and PDI were measured using dynamic light scattering technology, and nucleic acid encapsulation efficiency was calculated from the QuantiT RiboGreen RNA assay. The hydrodynamic diameter of these OVA mRNA-LNP was approximately 83 nm, the polydispersity index was approximately 0.1, and the encapsulation efficiency was approximately 97%.

[0308]

[0462] All animals were handled in accordance with the animal experimentation committee ethical protocol. For the experiments, C57BL / 6 mice (n = 4) aged 6 - 8 weeks were purchased from Envigo. On days 1 and 10, the mice were immunized intramuscularly with 50 μL of Ova LNP. Each mouse was vaccinated with a dose of 5 μg of OVA mRNA encapsulated in LNP. A dose of 50 μg of OVA antigen was used as a positive control. At defined time intervals, 110 - 130 μL of blood was collected by retro-orbital bleeding and processed into serum. Two weeks after the second immunization, the animals were euthanized and blood was collected by cardiac puncture. The blood samples were immediately processed into serum and stored at -80 °C. Aliquots of the serum samples were thawed and Ova expression and IgG measurements were analyzed using standard ELISA techniques with appropriate dilutions.

[0309]

[0463] For serum preparation, after whole blood collection, the blood was allowed to clot at room temperature for 15 - 30 minutes. The clotted blood was removed by centrifuging the tubes at 1000 - 2000 × g for 10 min at 4 °C. The supernatant was carefully removed and transferred to a sterile clear polypropylene tube with a screw cap on ice.

[0310]

[0464] OVA ELISA of serum samples: Mouse serum was collected by retro-orbital bleeding 6 h after vaccination. OVA antigen was measured using a standard sandwich ELISA with the components of the Ovalbumin (OVA)-ELISA Kit abx150365 (Abbexa Biologics, Inc., Arlington, TX, USA) according to the manufacturer's recommendations. Briefly, standard appropriately diluted serum and biotin-conjugated reagent were added to wells of a 96-well Abrexxa (trademark) microplate pre-coated with antibody and incubated for 1 h. Horseradish peroxidase (HRP)-conjugated reagent was added and incubated for 20 min. Stop solution was added to each well and the absorbance at 450 nm was measured using a plate reader, from which the concentration of OVA protein was calculated.

[0311]

[0465] Anti-OVA-IgG ELISA of serum samples: Serum from immunized mice was collected 2 weeks after the second immunization. Specific production of IgG against OVA in response to mRNA encoding OVA in various LNPs was measured by ELISA as described above. Briefly, OVA protein at a concentration of 2 μg protein per well in 100 mM sodium bicarbonate buffer (pH 9.6) was pre-coated overnight at 4 °C on 96-well ELISA plates. The pre-coated plates were then blocked with 10% FBS (BSA) in 7.4 buffered PBS-Tween-20™ (0.05%) v / v and incubated at 37 °C for 2 h. Serum samples and immunoglobulin standards were appropriately diluted in 1% BSA-PBS (1:8 - 1:1000), added to the 96-well plates, and incubated at 2 h RT. For labeling, (HRP) conjugated goat anti-mouse IgG (Catalog #7076, Cell Signaling) was used at a dilution of 1:5,000 in PBS-Tween-10% FBS and incubated for 1 h. After the incubation period, horseradish peroxidase substrate (3,3’,5,5’-tetramethylbenzide - TMB) was added. After 30 min incubation, 2N sulfuric acid stop solution was added and absorbance at 450 nm was determined using a plate reader. Results for PNI ID325 and 539 are shown in Figure 12. Results for PNI 127 are shown in Figure 13.

[0312]

[0466] Referring to Table 7 and the figures, PNI 121, PNI 127, PNI 328, PNI 329, and PNI 540 are equivalent to MC3 in in vivo protein expression. PNI 121, PNI 127, PNI 328, PNI 329, and PNI 541 are better than MC3 (higher MFI) with respect to the amount of protein produced in protein expression in human T cells.

[0313]

[0467] LNPs carrying OVA antigen, including PNI 127, PNI 325, and PNI 539, have been shown to effectively elicit an immune response similar to that of OVA antigen, indicating the usefulness of PNI lipids for vaccine applications. [Table 8]

[0314] Example 25 pKa studies

[0468] The effect of surface pKa on transgene expression following LNP transfection in LNPs derived from different ionizable lipids was examined.

[0315]

[0469] The pKa of each cationic lipid was determined using fluorescent probes for the conformational state of the protein. 5 The lipid nanoparticles were determined using a fluorescence-based assay of 6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt (TNS, Sigma Aldrich). Empty lipid nanoparticles containing ionizable lipids of LM02 composition in distilled water at a concentration of 3.125 mM total lipid were formulated using a NanoAssemblr® Spark™ and then characterized on a Zetasizer™ using a 30x dilution with 1x PBS in a low volume cuvette. The lipid nanoparticles were further diluted 4x with distilled water to 0.781 mM total lipid. TNS was prepared as a 25 μM stock solution in distilled water. 6.4 μL of diluted LNP sample at 0.781 mM total lipid was then mixed with 10 μL of diluted TNS to a final volume of 250 μL containing a buffer containing 10 mM HEPES, 10 mM MES, 10 mM NH4OAc, and 130 mM NaCl, where the pH ranged from 3 to 9 in 0.5 pH increments. Each well had a final concentration of 20 μM total lipid, 1 μM TNS, and 233.4 μL of buffer solution (total volume 250 μL).

[0316]

[0470] The samples were thoroughly mixed, and fluorescence intensity was measured at room temperature using a BioTek™ Synergy™ H1 Hybrid Multi-Mode Monochromator™ Fluorescence Microplate Reader with excitation and emission wavelengths of 321 nm and 445 nm, respectively. Sigmoidal best-fit analysis was applied to the fluorescence data using GraphPad Prism™ software, and the pKa was measured as the pH at half-maximal fluorescence intensity.

[0317]

[0471] The results are shown in Figure 14, which illustrates the surface pKa measurements of LNPs incorporating ionizable lipid compounds PNI 119, PNI 121, PNI 321, PNI 329, PNI 336, PNI 535, PNI 538, and PNI 540 (LM02 composition). Lipids with lower pKa values were found to be inactive in transgene expression in T cells. Lipids with higher pKa values were found to be toxic to T cells. Lipids with pKa in the range of 5.5 - 6.9 were found to be active in promoting transgene expression in T cells.

[0318] Example 26 Plasmid encapsulation

[0472] The 5514 nt pCX-EGFP plasmid, custom-ordered from GenScript USA Inc, Piscataway, NJ, was used. Preparation of lipid particles was as described above. The PNI ionizable lipids PNI 121, PNI 127, PNI 328, PNI 329, and PNI 541 were tested for their ability to encapsulate plasmids suitable for mammalian expression (described above) using the NanoAssemblr® Ignite™ system with an LM02 composition of N / P6. All lipids tested yielded acceptable LNPs with an average hydrodynamic diameter of 110 nm and a PDI of 0.2 with a greater than 80% encapsulation efficiency.

[0319]

[0473] Preferred embodiments have been described above and illustrated in the accompanying drawings. However, as will be apparent to those skilled in the art, modifications can be made without departing from the present disclosure. Such modifications are considered possible variations within the scope of the present disclosure.

[0320] References 1. Garg, S.; Heuck, G.; Ip, S.; Ramsay, E., Microfluidics: a transformational tool for nanomedicine development and production. J Drug Target 2016, 24 (9), 821 - 835. 2. Zhang, S.-h.; Shen, S.-c.; Chen, Z.; Yun, J.-x.; Yao, K.-j.; Chen, B.-b.; Chen, J.-z., Preparation of solid lipid nanoparticles in co-flowing microchannels. Chemical Engineering Journal 2008, 144 (2), 324 - 328. 3. JEFFS, L. B., et al., A Scalable, Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA. Pharmaceutical Research 2005, 22 (3), 362 - 372. 4. Gaj, T.; Gersbach, C. A.; Barbas, C. F., 3rd, ZFN, TALEN, and CRISPR / Cas-based methods for genome engineering. Trends in biotechnology 2013, 31 (7), 397 - 405. 5. McClure, W. O.; Edelman, G. M., Fluorescent Probes for Conformational States of Proteins. I. Mechanism of Fluorescence of 2-p-Toluidinylnaphthalene-6-sulfonate, a Hydrophobic Probe*. Biochemistry 1966, 5 (6), 1908-1919. 6. Trickett, A.; Kwan, Y. L., T cell stimulation and expansion using anti-CD3 / CD28 beads. J Immunol Methods 2003, 275 (1-2), 251-5. 7. Lundstrom, K. Nanoparticle-based delivery of self-amplifying RNA. Gene Ther 27, 183-185 (2020). 8. Peng, M., Mo, Y., Wang, Y. et al. Neoantigen vaccine: an emerging tumor immunotherapy. Mol Cancer 18, 128 (2019). 9. Roujian Lu 1, Xiang Zhao 1, Juan Li 2, et al. “Genomic Characterisation and Epidemiology of 2019 Novel Coronavirus: Implications for Virus Origins and Receptor Binding” Lancet 2020 Feb 22;395(10224):565-574. 10. Morokata T, et.al, Immunology (1999) 345-351.Giuliani et al. (2006) Proc Natl Head Sci USA103(29):10834-9

Claims

1. An agent for use in treating cancer, for use in regulating T cells, for use in preparing a vaccine, or for use in protein regulation in vivo or ex vivo, which comprises a therapeutic agent and lipid nanoparticles comprising a lipid mix composition comprising (i) a compound of formula (I) or a pharmaceutically acceptable salt thereof, or (ii) a compound of formula (I) in combination with one or more structural lipids, sterols, or stabilizers, wherein Formula (I) is 【Chemical 1】 [wherein p is 0 or 1; E １ is -O-δ １ , -OC(O)O-δ １ , -OC(O)-δ １ , -OC(O)N(Q)-δ １ , -OC(O)S-δ １ , -C(O)N(Q)-δ １ , -C(O)O-δ １ , -N(Q)C(O)-δ １ , -N(Q)C(O)O-δ １ , -N(Q)C(O)S-δ １ , and -N(Q)C(O)N(Q)-δ １ is selected from; Q is H or C １ ~C ５ alkyl; δ １ is a bond connected to the R １ group; R １ is 【Chemical Formula 2】 is selected from, where R ３ and R ４ are each independently selected from the group consisting of C １ to C ６ alkyl, C ２ to C ６ alkenyl, and C ２ to C ６ alkynyl; or R ３ and R ４ together may form a 4- to 6-membered ring optionally substituted with 1 to 2 substituents independently selected from oxygen (O) or up to two nitrogens (N), C １ to C ６ alkyl, cyclopropyl, OH, and C １ to C ３ alkoxy groups; R ５ is selected from C １ to C ６ alkyl, C ２ to C ６ alkenyl, C ２ to C ６ alkynyl, C ３ to C ６ cycloalkyl, and 2-hydroxyethyl group; R ６ is selected from H, and C １ ~C ６ alkyl groups; a is 1, 2, 3, 4 or 5; b and c are independently 0, 1, or 2; c' is 1, 2, 3, 4, or 5; d is 1, or 2; e is 0, 1, or 2; E ２ is -OC(O)-δ ２ 、 -OC(O)O-δ ２ 、 -OC(O)N(Q)-δ ２ 、 -O-δ ２ 、 -OCH ２ CH ２ O-δ ２ 、 and -OC(O)(CH ２ ) ６ C(O)O-δ ２ selected from, Q is H or C １ ~C ５ alkyl; δ ２ represents a bond linked to the R ２ group; R ２ is [Chemical 3] selected from, or the formula -(CH ２ ), ｇ -[L ３ -(CH ２ )] ｈ -R ９ having, where L １ and L ２ are each independently a direct bond, -O-δ ３ , -CH ２ OC(O)-δ ３ , and -CH ２ O-δ ３ where δ ３ represents a bond linked to the R ７ and R ８ groups; R ７ and R ８ are each independently C ４ to C １０ alkyl, C ４ to C １０ alkenyl or C ４ to C １０ alkynyl; f is 0, 1, 2, 3, 4, or 5; L ３ is 【Chemical Formula 4】 is selected from R ９ is selected from H and C ４ ~C ８ alkyl groups, g is an integer in the range of 1 to 18, h is 0, 1, 2, or 3]] is the agent.

2. An agent for use in treating cancer, for use in regulating T cells, for use in preparing a vaccine, or for use in protein regulation in vivo or ex vivo, which comprises a therapeutic agent and lipid nanoparticles comprising a lipid mix composition comprising (i) a compound of formula (II) or a pharmaceutically acceptable salt thereof, or (ii) a compound of formula (II) in combination with one or more structural lipids, sterols, or stabilizers, wherein Formula (II) is [Chemical Formula 5] [Wherein, E １ is -OC(O)O-δ １ , -OC(O)-δ １ , -OC(O)N(Q)-δ １ , and -OC(O)S-δ １ is selected from; Q is H or C １ ~C ５ alkyl; δ １ represents a bond linked to the R １ group; R １ is 【Chemical Formula 6】 selected from; where R ３ and R ４ are each independently selected from C １ -C ６ alkyl groups; or R ３ and R ４ together contain up to two nitrogens (N) and are optionally substituted with 1 to 2 substituents selected from C １ -C ６ alkyl groups to form a 5- to 6-membered ring which may be optionally substituted; R ５ is C １ to C ６ alkyl, and C ３ to C ６ selected from cycloalkyl groups; R ６ is selected from H, and C １ ~C ６ alkyl groups; a is 1, 2, 3, or 4; b and c are independently 0, 1, or 2; c' is 2, 3, or 4; d is 2; e is 0, or 1; E ２ is -O-δ ２ , -OC(O)-δ ２ , -OCH ２ CH ２ O-δ ２ , and -OC(O)(CH ２ ) ６ C(O)O-δ ２ selected from, where δ ２ is a bond linked to the R ２ group; R ２ is 【Chemical Formula 7】 selected from, or the formula -(CH ２ ), ｇ -[L ３ -(CH ２ )] ｈ -R ９ having, where L １ and L ２ are each independently directly bonded, -O-δ ３ , -CH ２ OC(O)-δ ３ , and -CH ２ O-δ ３ ; δ ３ represents a bond linked to the R ７ and R ８ groups; R ７ and R ８ each independently is C ４ ~C １０ alkyl, C ４ ~C １０ alkenyl or C ４ ~C １０ alkynyl; f is 0, 1, 2, 3, 4, or 5; L ３ is 【Chemical 8】 is selected from; R ９ is selected from H and C ４ ~C ８ alkyl groups; g is an integer in the range of 1 to 18; h is 0, 1, or 2]] is the agent.

3. An agent for use in treating cancer, for use in regulating T cells, for use in preparing a vaccine, or for use in protein regulation in vivo or ex vivo, which comprises a therapeutic agent and lipid nanoparticles comprising a lipid mix composition comprising (i) a compound of formula (II) or a pharmaceutically acceptable salt thereof, or (ii) a compound of formula (II) in combination with one or more structural lipids, sterols, or stabilizers, wherein Formula (II) is 【Chemical Formula 9】 [wherein, E １ is -OC(O)O-δ １ , -OC(O)-δ １ , -OC(O)N(Q)-δ １ , and -OC(O)S-δ １ is selected from; Q is H or C １ to C ５ alkyl; δ １ represents a bond linked to the R １ group; R １ is 【Chemical 10】 selected from; where R ３ and R ４ each independently selected from C １ -C ６ alkyl groups; or R ３ and R ４ together contain up to two nitrogens (N) and are optionally substituted with 1 to 2 substituents selected from C １ -C ６ alkyl groups to form a 5- to 6-membered ring; R ５ is selected from C １ to C ６ alkyl, and a cyclopropyl group; R ６ is selected from H, and C １ ~C ６ alkyl groups; a is 1, 2, 3, or 4; b is 0 or 1; c is 0, 1, or 2; c' is 2, 3, or 4; d is 2; e is 1; E ２ is -O-δ ２ , -OC(O)-δ ２ , -OCH ２ CH ２ O-δ ２ , and -OC(O)(CH ２ ) ６ C(O)O-δ ２ selected from, where δ ２ is a bond connected to the R ２ group; R ２ is 【Chemical 11】 selected from, or the formula -(CH ２ ) ｇ -[L ３ -(CH ２ )] ｈ -R ９ having, wherein L １ and L ２ are each direct connections; R ７ and R ８ are each independently selected from C ４ to C １０ alkyl groups; f is 0 or 1; L ３ is 【Chemical Formula 12】 selected from; R ９ is selected from H and C ４ -C ８ alkyl groups; g is an integer in the range of 1 to 18; h is 0, 1, or 2]] is an agent.

4. An agent for use in treating cancer, for use in regulating T cells, for use in preparing a vaccine, or for use in protein regulation in vivo or ex vivo, which comprises a therapeutic agent and lipid nanoparticles comprising a lipid mix composition comprising (i) a compound of formula (III) or a pharmaceutically acceptable salt thereof, or (ii) a compound of formula (III) combined with one or more structural lipids, sterols, or stabilizers, for ex vivo administration of the therapeutic agent to cells, wherein formula (III) is 【Chemical Formula 13】 [R １ is 【Chemical 14】 selected from; where R ３ and R ４ are each independently selected from C １ -C ６ alkyl groups; or R ３ and R ４ together may contain up to two nitrogens (N) and be optionally substituted with one or two substituents selected from C １ -C ６ alkyl groups to form a 5- to 6-membered ring; R ５ is C １ to C ６ selected from alkyl and cyclopropyl groups; R ６ is selected from H, and C １ ~C ６ alkyl groups; a is 1, 2, 3, or 4; b is 0 or 1; c is 0, 1, or 2; c' is 2, 3, or 4; d is 2; e is 1; E ２ is -O-δ ２ , -OC(O)-δ ２ , -OCH ２ CH ２ O-δ ２ , and -OC(O)(CH ２ ) ６ C(O)O-δ ２ is selected from; wherein δ ２ is R ２ represents a bond linked to the group; R ２ is 【Chemical Formula 15】 selected from, or the formula -(CH ２ ), ｇ -[L ３ -(CH ２ )] ｈ -R ９ having, where L １ and L ２ are each direct connections; R ７ and R ８ are each independently selected from C ４ to C １０ alkyl groups; f is 0 or 1; L ３ is 【Chemical 16】 selected from; R ９ is selected from H and C ４ ~C ８ alkyl groups; g is an integer in the range of 1 to 18; h is 0, 1, or 2]] is an agent.

5. An agent for use in treating cancer, for use in regulating T cells, for use in preparing a vaccine, or for use in protein regulation in vivo or ex vivo, which comprises a therapeutic agent and lipid nanoparticles comprising a lipid mix composition comprising (i) a compound of any one of the following structures, or (ii) a lipid mix composition comprising the compound combined with one or more structural lipids, sterols, or stabilizers, for ex vivo administration of the therapeutic agent to cells: 【Chemical 17-A】 【Chemical 17-B】 【Chemical 17-C】 【Chemical 17-D】 【Chemical 17-E】 or a pharmaceutically acceptable salt thereof.

6. An agent for use in treating cancer, for use in regulating T cells, for use in preparing a vaccine, or for use in protein regulation in vivo or ex vivo, which comprises a therapeutic agent and lipid nanoparticles comprising a lipid mix composition comprising (i) a compound of any one of the following structures, or (ii) a lipid mix composition comprising the compound combined with one or more structural lipids, sterols, or stabilizers, for ex vivo administration of the therapeutic agent to cells: 【Chemical Formula 18-A】 【Chemical 18-B】 【Chemical 18-C】 【Chemical 18-D】 【Chemical 18-E】 【Chemical Formula 18-F】 【Chemical 18-G】 【Chemical 18-H】 【Chemical Formula 18-I】 【Chemical 18-J】 【Chemical Formula 18-K】 【Chemical Formula 18-L】 【Chemical 18-M】 【Chemical Formula 18-N】 【Chemical Formula 18-O】 【Chemical 18-P】 【Chemical 18-Q】 【Chemical 18-R】 【Chemical 18-S】 【Chemical 18-T】 【Chemical Formula 18-U】 【Chemical 18-V】 【Chemical 18-W】 or a pharmaceutically acceptable salt thereof.

7. The agent according to any one of claims 1 to 6, wherein the stabilizer comprises PEG-DMG 2000, polyoxyethylene (10) stearyl ether, polyoxyethylene (40) stearate, polysorbate 80, polyoxyethylene (4) lauryl ether, polyoxyethylene (20) stearyl ether, polyoxyethylene (23) lauryl ether, D-α-tocopherol polyethylene glycol 1000 succinate, polyethylene glycol conjugated lipid, or a combination thereof.

8. The agent according to any one of claims 1 to 7, wherein the lipid mixture composition comprises a compound present at 10 Mol% to 90 Mol%, a structured lipid present at 0 to 50 Mol%, a sterol present at 0 to 45 Mol%, and a stabilizer present at 0 to 10 Mol%, and the total Mol% of the components of the lipid mixture composition is 100 mol%.

9. The agent according to any one of claims 1 to 7, wherein the lipid mixture composition comprises a compound present at 40 Mol% to 60 Mol% and a structured lipid present at 11 to 40 Mol%, and the total Mol% of the components of the lipid mixture composition is 100 mol%.

10. The agent according to any one of claims 1 to 9, wherein the experimental pKa of the lipid mixture composition comprising the compound defined in any one of claims 1 to 6 is in the range of 4.2 to 7.8, 4.2 to 5.9, or 6.9 to 7.

8.

11. The agent according to any one of claims 1 to 9, wherein the experimental pKa of the lipid mixture composition comprising the compound defined in any one of claims 1 to 6 is in the range of 5.6 to 7.

1.

12. The agent according to any one of claims 1 to 11, wherein the lipid mixture composition comprises a compound present at 40 Mol%, DSP C (distearoyl phosphatidylcholine) present at 20 Mol%, cholesterol present at 37.5 Mol%, and polyethylene glycol octadecyl ether present at 2.5 Mol%.

13. The agent according to any one of claims 1 to 11, wherein the lipid mixture composition comprises a compound present at 40 to 47.5 Mol%, DSP C (distearoyl phosphatidylcholine) or DOPE (dioleoyl-phosphatidylethanolamine) present at 12.5 Mol%, cholesterol present at 38.5 to 46 Mol%, and PEG-DMG 2000 present at 1.5 Mol%.

14. The agent according to any one of claims 1 to 11, wherein the lipid mix composition comprises a compound present at 40 to 50 Mol%, DSPPC (distearoyl phosphatidylcholine) present at 10 to 20 Mol%, cholesterol present at 39.5 to 40 Mol%, and D-α-tocopherol polyethylene glycol 1000 present at 0.5 to 2.5 Mol%.

15. The agent according to any one of claims 1 to 14, wherein the therapeutic agent further comprises one or more nucleic acids, polypeptides, oligonucleotides, proteins, a mixture of nucleic acids and proteins, or ribonucleoproteins.

16. The agent according to any one of claims 1 to 14, wherein the one or more nucleic acids comprise mRNA, self-replicating RNA, siRNA, miRNA, guide RNA, synthetic guide RNA, artificial chromosomes, circular or linearized DNA, DNA minicircles, peptidic or msDNA.

17. The agent according to any one of claims 1 to 16, wherein the vaccine relates to the prevention of viral diseases or to the prevention of coronavirus infection.

18. The agent according to any one of claims 1 to 16, wherein the vaccine is a cancer vaccine.

19. The agent according to any one of claims 1 to 16, wherein the use in the regulation of T cells comprises human T cells, CAR-T, TCR, gene editing of T cells, or regulation of allogeneic T cells.

20. The agent according to claim 19, wherein the T cells comprise T cells isolated from a patient, or T cells that have been genetically engineered, or allogeneic T cells.

Images