Sulfur-containing ionizable lipids for the delivery of nucleic acids and other therapeutic agents

Abstract

Embodiments disclosed herein relate to sulfur-containing lipids. Examples of lipids described herein have a structure of Formula (A) or a pharmaceutically acceptable salt thereof. The compounds may be formulated in a lipid nanoparticle for use in the delivery of charged cargo such as nucleic acid.

Description

SULFUR-CONTAINING IONIZABLE LIPIDS FOR THE DELIVERY OF NUCLEIC ACIDS AND OTHER THERAPEUTIC AGENTS TECHNICAL FIELD

[0001] Provided herein are sulfur-containing lipids that may be formulated in a delivery vehicle so as to facilitate the encapsulation of a wide range of therapeutic agents or prodrugs therein, such as, without limitation, nucleic acids (e.g., RNA or DNA), proteins, peptides, pharmaceutical drugs and salts thereof.BACKGROUND

[0002] Nucleic acid-based therapeutics have enormous potential in medicine. To realize this potential, however, the nucleic acid must be delivered to a target site in a patient. This presents challenges since nucleic acid is rapidly degraded by enzymes in the plasma upon administration. Even if the nucleic acid is delivered to a disease site, there still remains the challenge of intracellular delivery. To address these problems, lipid nanoparticles have been developed that protect nucleic acid from such degradation and facilitate delivery across cellular membranes to gain access to the intracellular compartment, where the relevant translation machinery resides.

[0003] A key component of a lipid nanoparticle (LNP) is an ionizable lipid. The ionizable lipid is typically positively charged at low pH, which facilitates association with the negatively charged nucleic acid. However, the ionizable lipid is neutral at physiological pH, making it more biocompatible in biological systems. Further, it has been suggested that after the LNPs are taken up by a cell by endocytosis, the ionizability of these lipids at low pH enables endosomal escape. This in turn enables the nucleic acid to be released into the intracellular compartment.

[0004] An earlier example of an LNP product approved for clinical use and reliant on ionizable lipid is Onpattro®. Onpattro® is a lipid nanoparticle-based short interfering RNA (siRNA) drug for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis. Onpattro® is reliant on an ionizable lipid referred to as “DLin- MC3-DMA” or more commonly “MC3”, 1 (Scheme 1), by investigators. Furthermore, MC3 represents an evolution of a structurally related ionizable lipid, referred to by investigators as “KC2”, 2 (Scheme 1). MC3 is considered a state-of-the art ionizable lipid for the delivery of siRNA, requiring about 3 times less siRNA than KC2. As a consequence, MC3 is often regarded as the benchmark against which the potency of new lipids is evaluated. Still, KC2 remains a valuable research tool.

[0005] Ionizable lipids are also crucial components of certain COVID-19 vaccines. To illustrate, the Pfizer / BioNTech and Modema vaccines rely on LNPs to deliver mRNA to the cytoplasm of liver cells. This mRNA encodes for the highly immunogenic Sars-Cov-2 spike protein. Once inside the host cell, the mRNA is transcribed to produce antigenic proteins. The Pfizer / BioNTech vaccine comprises an ionizable lipid referred to as “ALC- 0315”, 3 (Scheme 1), while the Modema vaccine comprises an ionizable lipid referred to as “SM-102”, 4.Scheme 1

[0006] All of the above lipids were optimized for delivery of therapeutic nucleic acids to the liver. However, there remains a need to develop new lipids for the delivery of charged cargo, such as nucleic acids, to other organs, such as the spleen, lungs, bone marrow, skin, etc. The delivery of therapeutics beyond the liver would expand the clinical utility of LNPs to target disease conditions that affect tissues and organs beyond the liver. There is also an ongoing need to develop LNPs with improved delivery of nucleic acid or other charged cargo to the liver.

[0007] The present disclosure seeks to address one or more of the above identified problems and / or provides useful alternatives to known products and / or compositions for the delivery of nucleic acid or other charged cargo.DEFINITIONS

[0008] As used herein, the term "ionizable lipid" refers to a lipid that, at a given pH, is in an electrostatically neutral form and that may either accept or donate protons, thereby becoming electrostatically charged, and for which the electrostatically neutral form has a calculated logarithm of the partition coefficient between water and 1-octanol (i.e., a CLogP) that is greater than 8, 9, 10 or 11.

[0009] As used herein, the term “lipophilic chain” refers to an alkyl group bonded to a nitrogen atom of the lipid, said alkyl group comprising at least 6 carbon atoms and optionally comprising one or more C=C double bonds, and / or ring structures, and / or carbonyl groups, and / or heteroatoms such as N, O, S, and such that the parent compound of said alkyl group has a CLogP of at least 6.

[0010] As used herein, the term "ionizable, cationic amino lipid" refers to a lipid that, at physiological pH, is in an electrostatically neutral form and comprises a central nitrogen atom in its head group that accepts a proton, thereby becoming electrostatically positively charged at a pH below its pKa.

[0011] The terms, “protonatable amino head group”, “ionizable head group” or “head group” are used interchangeably herein and refer to a moiety of the ionizable, cationic amino lipid that comprises the central nitrogen atom in its head group that accepts a proton, thereby becoming electrostatically positively charged at a pH below its pKa. The two lipophilic chains are directly bonded to the central nitrogen atom.

[0012] For example, ALC-0315, 3, has a central nitrogen atom and a pair of lipophilic chains derived from hexyl 2-hexyldecanoate, which has a CLogP of 10.01 :lipiphilic chain(s)

[0013] Lipid SM-102, 4, has one lipophilic chain derived from undecyl hexanoate, which has a CLogP of 7.59, and one lipophilic chain derived from heptadecane-9-yl octanoate, which has a CLogP of 11.6: yl hexanoategP: 7.588 lipiphilic chain(s) heptadecan-9-yl octanoate CLogP: 11.6

[0014] As used herein, “imparts an apparent pKa of between 6 and 7.5 to a lipid nanoparticle when formulated therein” with reference to an ionizable lipid, refers to the apparent pKa of the lipid nanoparticle when the ionizable lipid is formulated therein. The apparent pKa is measured in a lipid nanoparticle having a composition of ionizable lipid / DSPC / cholesterol / PEG2ooo-DMG (50:10:38.5:1.5 mol:mol) and prepared as described in WO 2023 / 184038, which is incorporated herein by reference.

[0015] The apparent pKais measured using a 6-( / ?-Toluidino)-2-naphthalenesul Ionic acid (TNS) assay adapted from previous studies from other groups (Shobaki et al., 2018, International Journal of Nanomedicine, 13:8395-8410; Jayaraman et al., 2012, Angew. Chem Int. Ed., 51 :8529-8533, which are incorporated herein by reference for the purposes of determining apparent pKa). In the adapted method, a series of buffers are prepared spanning a pH range of 2.5-10.9 in varying pH unit increments consisting of 130 mMNaCl, 10 mM ammonium acetate, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 10 mM HEPES. 0.15-0.2 mM of the LNP. A solution of 0.12 mM TNS is subsequently mixed with 175 pL of the LNP at each buffered pH in triplicate in a black, polystyrene 96-well plate, to yield a final concentration of 6.25 and 12 pM of lipid and TNS in each well, respectively. Fluorescence is subsequently measured using a SpectraMax M5™ microplate reader at Zex=321 nm, Zcm=445 nm. The fluorescence is subsequently plotted against pH using a sigmoidal curve fit through Prism™, in which the pKa is determined to be the pH value with 50% of maximal fluorescent intensity.

[0016] As used herein, the term “alkyl” or “alkyl group” is a carbon-containing chain that is linear or branched and that optionally comprises C=C double bonds (e.g., 0-2 double bonds) and / or comprises or consists of ring structure(s), and that is optionally substituted. A carbon-containing chain or ring structure having double bonds may be referred to as an alkene.

[0017] As used herein, the term “Cmto Cnalkyl” or “Cmto Cnalkyl group” refers to a linear or branched carbon chain having a total minimum of m carbon atoms and up to n carbon atoms, and that is optionally unsaturated and optionally substituted. For example, a “Ci to C3 alkyl” or “Ci to C3 alkyl group” is an alkyl having between 1 and 3 carbon atoms.

[0018] The term “ring structure”, “ring” or “cyclic alkyl group” is a 3- to 22 -membered monocyclic or polycyclic alkyl ring that is optionally substituted and optionally unsaturated. In some non-limiting examples, the ring structure, ring or cyclic alkyl group is a 3- to 16-membered monocyclic or polycyclic alkyl ring that is optionally substituted. In further examples, the ring structure is a 3- to 8-membered monocyclic or polycyclic alkyl ring that is optionally substituted.

[0019] The term “monocyclic” is an optionally substituted alkyl group that is a single ring or that comprises a single ring substituent.

[0020] The term “polycyclic” is optionally substituted alkyl group that is, or comprises as a substituent(s), two or more ring structures that are chemically bonded to each other or two or more discrete ring structures.

[0021] The term “optionally substituted” with reference to an alkyl or alkyl group means that at least one hydrogen atom of the alkyl group can be replaced by a non-hydrogen atom or group of atoms (i.e., a “substituent”), and / or the alkyl group is interrupted (i.e., a- (CH)2- group replaced) by a non-carbon atom or one or more substituents, including butnot limited to those comprising heteroatoms selected from O, S and / or NR', wherein R' is as defined below. Non-limiting examples of atoms or substituents that may replace a hydrogen atom include halogen; deuterium, an alkyl group; a cycloalkyl group (mono or polycyclic); an oxo group (=0); a hydroxyl group (-0H); — (C=O)OR'; — O(C=O)R'; — C(=O)R'; O(C=O)OR'-; —OR'; — S(O)XR'; —SR', — S— SR'; — C(=O)SR'; — SC(=O)R'; — NR'R'; — NR'C(=0)R'; — C(=0)NR'R'; — NR'C(=0)NR'R'; — 0C(=0)NR'R'; — NR'C(=0)0R'; — NR'S(O)XNR'R'; — NR'S(O)XR'; and — S(O)XNR'R', wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2. Non-limiting examples of atoms or substituents that may replace a carbon atom (interrupt the alkyl) include cycloalkyl groups (e.g., mono or polycyclic); — O— ; — (C=0)0— ; — 0(C=0)-; — C(=0); — 0(C=0)0-; — S(0)x-; — S— ; — S— S— ; — C(=O)S-; — SC(=O)-; —NR'—; — NR'C(=O) — ; — C(=O)NR'— ; — NR'C(=O)NR'— ; — OC(=O)NR'— ; — NR'C(=O)OR’— ; — NR'S(O)XNR'— ; — NR'S(O)XR’ — ; and — S(O)XNR' — , wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0022] As used herein, the term “helper lipid” means a compound selected from: a sterol such as cholesterol or a derivative thereof; a diacylglycerol or a derivative thereof, such as a glycerophospholipid, including phosphatidic acid (phosphatidate) (PA), phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and the like; and a sphingolipid, such as a ceramide, a sphingomyelin, a cerebroside, a ganglioside, or reduced analogues thereof, that lack a double bond in the sphingosine unit. An example of a diacylglycerol derivative is a glycerophospholipid-cholesterol conjugate in which one of the acyl chains is substituted with a moiety comprising cholesterol. The term encompasses lipids that are either naturally occurring or synthetic.

[0023] As used herein, the term “delivery vehicle” includes any preparation in which the lipid described herein is capable of being formulated and includes but is not limited to delivery vehicles comprising helper lipids.

[0024] As used herein, the term “nanoparticle” is any suitable particle in which the lipid can be formulated and that may comprise one or more helper lipid components. The one or more lipid components may include an ionizable lipid prepared by the method described herein and / or may include additional lipid components, such as the one or more helper lipid components. The term includes, but is not limited to, vesicles with one or more bilayers, including multilamellar vesicles, unilamellar vesicles and vesicles with anelectron-dense core. The term also includes polymer-lipid hybrids, including particles in which the lipid is attached to a polymer.

[0025] As used herein, the term “encapsulated,” with reference to incorporating a cargo molecule (e.g., mRNA) within a delivery vehicle refers to any association of the cargo with any component or compartment of the delivery vehicle such as a nanoparticle.

[0026] The term “sterol” refers to a naturally-occurring or synthetic compound having a gonane skeleton and that has a hydroxyl moiety attached to one of its rings, typically the A-ring.

[0027] The term “pharmaceutically acceptable salt” with reference to a form of the lipid of the disclosure in a protonated form (i.e., charged) and / or as part of a pharmaceutical formulation in which an LNP is formulated refers to a salt prepared from pharmaceutically acceptable, non-toxic acids, including inorganic and organic acids.SUMMARY

[0028] The present disclosure is based, at least in part, on the unexpected discovery that LNP formulations of nucleic acids comprising certain ionizable lipids that incorporate at least one lipophilic chain substituted with a sulfur atom and an ester moiety are more potent than an MC3 benchmark analogue (referred to herein as “nor-MC3”) for in vivo delivery of therapeutic RNA. Non-limiting examples described herein demonstrate that such lipids may increase the delivery of mRNA to the liver and the spleen relative to the benchmark lipid, nor-MC3.

[0029] According to one aspect of the disclosure, there is provided an ionizable, cationic, amino lipid or a pharmaceutically acceptable salt thereof comprising: a protonatable amino head group; two lipophilic chains, wherein the protonatable amino head group has a central nitrogen atom to which each of the two lipophilic chains are bonded; at least one of the two lipophilic chains has a structure of Formula C:Formula C wherein the wavy line represents a bond to the central nitrogen atom; wherein m and n are independently 2 to 8;E is an ester group that is -(C=O)O- or -O(C=O)-;R1is a linear, branched, monocyclic or polycyclic, optionally substituted, C3 to C20 alkyl group, C4 to C20 alkyl group, C5 to C20 alkyl group or Ce to C20 alkyl group comprising 0-2 carbon-carbon double bonds;R2is a linear, branched, optionally substituted, Ci to C10 alkyl group, C2 to C10 alkyl group, C3 to C10 alkyl group or C4 to C10 alkyl group comprising 0-2 carbon-carbon double bonds, or R2is bound to R3to form a ring structure as indicated by the dashed curved line; R3is H, or a linear, branched, monocyclic or polycyclic, optionally substituted, Ci to C10 alkyl group, comprising 0-2 carbon-carbon double bonds, or R3is bound to R2to form a ring structure as indicated by the dashed curved line;R4and R5are, independently, H, or a linear or branched, optionally substituted, Ci to C10 alkyl group, comprising 0-2 carbon-carbon double bonds, or R4and R5are bound to each other to form a ring structure; each lipophilic chain has between 15 and 40 carbon atoms in total; and wherein the ionizable, cationic, amino lipid: (i) imparts an apparent pKa of between 6 and 7.5 to a lipid nanoparticle when formulated therein; and (ii) has a ClogP of at least 11.

[0030] In one embodiment, a second one of the two lipophilic chains bonded to the central nitrogen atom of the head group has a structure as defined by Formula D:Formula D wherein the wavy line represents a bond to the central nitrogen atom of the head group;R6and R7are, independently, H, or a linear or branched, optionally substituted, Ci to C10 alkyl group, comprising 0-2 carbon-carbon double bonds, or R6and R7are bonded to each other to form a ring structure,A is O, S or a carbonyl (C=O), andQif A is O, then R8is an acyl group, wherein the wavy line represents the bond to the A, and wherein the R’ is as defined above for R1; if A is the carbonyl (C=O), then R8is an, wherein the wavy line represents the bond to the A, and wherein the R’ is as defined above for R1; if A is S, then R8is a group of Formula E:Formula E wherein the E of Formula E is the ester group that is -(C=O)O- or -O(C=O)-; and wherein the wavy line represents the bond to A, and wherein R9equals R1, R10equals R2, and R11equals R3.

[0031] According to another embodiment of the foregoing aspect, the head group is— (CH2)p-[Z-(CH2)q]r-(CH2)t-G wherein the wavy line represents a bond to the central nitrogen atom;Z is O, S or CH2; when r is >1, Z in one or more of the Z-(CH2)qmoieties is independently the CH2, O or S; G is OH, or NRaRbwherein Raand Rbare optionally deuterated Ci to C4 alkyl; p is 2 to 4; q is 2 to 4; r is 0 to 6; and t is 0 to 4.

[0032] According to another embodiment, the Z is S or O.

[0033] According to another aspect of the disclosure, there is provided an ionizable, cationic, amino lipid or a pharmaceutically acceptable salt thereof having a structure of: Formula A:N-(CH2)p-[Z-(CH2)q]r-(CH2)t-GY -X -fCl la)Formula A wherein m and n are, independently, 4 to 8;R1is a linear, cyclic or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties;E is an ester group that is -(C=O)O- or -O(C=O)-;R2and R3are bonded to each other or are not bonded to each other, as indicated by the dashed line, whereinif R2and R3are bonded to each other, thenR2, R3, and a CH-CH moiety to which R2and R3are bound form a ring structure comprising from 4 to 12 C atoms, optionally comprising one or more heteroatoms, optionally comprising 1-2 C=C double bonds of E or Z configuration, and wherein the ring structure is optionally substituted with one or more Ci to Cs alkyl groups, and if R2and R3are not bonded to each other, then R2and R3are, each independently: a linear or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties;X is either O, a carbonyl (C=O) or S, wherein if X is O, then,Y is , wherein the wavy line of Y is bonded to O;R3if X is the carbonyl (C=O), then Y is R3-O— . wherein the wavy line of Y is bonded to the carbon atom of the carbonyl; wherein R3is the linear, cyclic or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties, if X is S, thenY is a group of Formula BFormula B wherein the wavy line represents the bond to the S;R4is a linear, cyclic or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties;R5and R6are bonded to each other or not bonded to each other, as indicated by the dashed line, and if R5and R6are bonded to each other, thenR5and R6form part of a ring structure comprising from 4 to 12 carbon atoms, optionally comprising one or more heteroatoms, optionally comprising 1 to 2 C=C double bonds of E or Z configuration, optionally substituted with one or more Ci to Cs alkyl groups, and if R5and R6are not bonded to each other, then R5and R6are, independently: a linear or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties, optionally comprising one or more heteroatoms;Z is O or S; when r is >1, Z in one or more of the Z-(CH2)qmoieties is independently the CH2, O or S; G is OH, or NRaRbwherein Raand Rbare each independently, optionally deuterated, Ci to C4 alkyl; p is 2 to 4; q is 2 to 4; r is 0 to 6; and t is 0 to 4.

[0034] According to another embodiment, the carbonyl (C=O) of E is bonded to the R1group.

[0035] In another embodiment, the ionizable lipid has a structure of any one of compounds T-01-T-08 as defined below or a pharmaceutically acceptable salt thereof:

[0036] In another embodiment, the ionizable, cationic, amino lipid is selected from one of T-01 to T-06 above. In another embodiment, the one or more cycloalkyl moieties of the ionizable, cationic, amino lipid or pharmaceutically acceptable salt thereof are selected from a cyclopentyl, cyclohexyl or a cycloheptyl.

[0037] In another embodiment, the one or more heteroatoms of the ionizable, cationic, amino lipid or pharmaceutically acceptable salt thereof are selected from O, N and S.

[0038] In another aspect of the disclosure, there is provided a lipid nanoparticle comprising the ionizable, cationic amino lipid of any one of the foregoing aspects or embodiments and a nucleic acid.

[0039] In one embodiment, the lipid nanoparticle comprises a helper lipid.

[0040] In another embodiment, the helper lipid is selected from a sterol, such as cholesterol, a diacylglycerol, a glycerophospholipid-cholesterol conjugate, a sphingolipid and mixtures thereof.

[0041] According to another aspect, there is provided a method for administering a nucleic acid to a subject in need thereof, the method comprising preparing or providing the lipid nanoparticle as described above comprising the nucleic acid and administering the lipid nanoparticle to the subject.

[0042] According to another aspect, there is provided a method for delivering nucleic acid to a cell, the method comprising contacting the lipid nanoparticle described above with the cell in vivo or in vitro.

[0043] According to another aspect, there is provided a use of the ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof of any one of the foregoing aspects or embodiments thereof or the lipid nanoparticle described above in the manufacture of a medicament to treat or prevent a disease, disorder or condition that is treatable and / or preventable by a nucleic acid.

[0044] According to another aspect, there is provided a use of the ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof of any one of the foregoing aspects or embodiments thereof or the lipid nanoparticle described above to deliver a nucleic acid to a subject to treat or prevent a disease, disorder or condition that is treatable or preventable by the nucleic acid.

[0045] Other objects, features, and advantages of the present disclosure will be apparent to those of skill in the art from the following detailed description and figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIGURE 1 is a bar graph showing entrapment (%), particle size and poly dispersity index (PDI) of mRNA-containing lipid nanoparticles (LNPs) comprising the ionizable lipids nor-MC3, T-01, T-02, T-03, T-04, T-05 and T-06. The LNPs are composed of 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG and the amine-to- phosphate ratio (N / P) was 6.

[0047] FIGURE 2A shows luminescence intensity / mg in the liver for the mRNA- containing LNPs comprising the ionizable lipids nor-MC3, T-01, T-02, T-03, T-04, T-05 and T-06 after 4 hours post-intravenous administration to CD-I mice. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG (N / P = 6).

[0048] FIGURE 2B shows luminescence intensity / mg in the spleen for the mRNA- containing LNPs comprising the ionizable lipids nor-MC3, T-01, T-02, T-03, T-04, T-05 and T-06 after 4 hours post-intravenous administration to CD-I mice. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG (N / P = 6).DETAILED DESCRIPTION

[0049] Various aspects and embodiments of the disclosure are directed to ionizable lipids having structures of Formula A and pharmaceutically acceptable salts thereof.Formulations comprising such lipids find use in the delivery of nucleic acid to any target site of interest. In some embodiments, such lipids have been found to be particularly efficacious for the delivery of mRNA when formulated in a suitable delivery vehicle. In further embodiments, such lipids can be easily synthesized and prepared by processes having improved economics relative to known methods for making ionizable lipids.

[0050] Embodiments disclosed herein relate to an ionizable, cationic, amino lipid or a pharmaceutically acceptable salt thereof having a structure of Formula A:Formula A wherein m and n are, independently, 4 to 8;R1is a linear, cyclic or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties;E is an ester group that is — (C=O)O — or — O(C=O) — ;R2and R3are bonded to each other or are not bonded to each other, as indicated by the dashed line, wherein if R2and R3are bonded to each other, thenR2, R3, and a CH-CH moiety to which R2and R3are bound form a ring structure comprising from 4 to 12 C atoms, optionally comprising one or more heteroatoms, optionally comprising 1-2 C=C double bonds of E or Z configuration, and wherein the ring structure is optionally substituted with one or more Ci to Cs alkyl groups, and if R2and R3are not bonded to each other, then R2and R3are, independently: a linear or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties;X is either O, a carbonyl (C=O) or S, wherein if X is O, then,Y is O , wherein the wavy line of Y is bonded to an O;R3^ if X is the carbonyl (C=O), then Y is R3-O— , wherein the wavy line of Y is bonded to the carbon atom of the carbonyl; and wherein R3is a linear, cyclic or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties if X is S, thenY is a group of Formula BFormula B wherein the wavy line represents the bond to the S;R4is a linear, cyclic or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties;R5and R6are bonded to each other or not bonded to each other, as indicated by the dashed line, and if R5and R6are bonded to each other, thenR5and R6form part of a ring structure comprising from 4 to 12 carbon atoms, optionally comprising one or more heteroatoms, optionally comprising 1 to 2 C=C double bonds of E or Z configuration, optionally substituted with one or more Ci to Cs alkyl groups, and if R5and R6are not bonded to each other, thenR5and R6are each, independently a linear or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties, optionally comprising one or more heteroatoms;Z is O or S; when r is >1, Z in any of the Z-(CH2)qmoieties is independently the CH2, O or S;G is OH, or NRaRbwherein Raand Rbare each independently, optionally deuterated, Ci to C4 alkyl; p is 2 to 4; q is 2 to 4; r is 0 to 6; and t is 0 to 4.Methods to produce lipids of Formula A

[0051] Lipids of Formula A or pharmaceutically acceptable salts thereof can be prepared using any suitable method known to those of skill in the art. Especially advantageous methods for the synthesis of said lipids are outlined in Schemes 2-17 below. Without intending to be limiting, the methods of Schemes 2-17 are subsequently exemplified in Schemes 18-27 with the synthesis of compounds T1-T7. Those skilled in the art would appreciate that alternative starting materials could be employed in the same sequences, leading to analogues of compound Tl-7 as defined by Formula A. Therefore, the synthetic schemes set forth below are merely illustrative of select embodiments.

[0052] Lipids of Formula A can be prepared from synthetic building block 2.5 (Scheme 2), wherein P can be H or a protecting group such as an acyl group or a trialkylsilyl group. In turn, 2.5 can be made starting with the mono-JV-alky lation of a suitable primary amine, for example, benzylamine (Bn-NTE) with alcohol 2.1, wherein L is a leaving group such as a halide (Cl, Br, I) or sulfonate (tosylate, mesylate, and the like), to produce 2.2.Subsequent reaction of 2.2 with 2.3, wherein L is a leaving group such as a halide (Cl, Br, I) or sulfonate (tosylate, mesylate, and the like), and P is as defined above, gives 2.4.Furthermore, the order of the steps leading to 2.4 can be inverted; i.e., benzylamine can be made to react with 2.3 first, then with 2.1, also resulting in formation of 2.4. Compound2.4 is then converted into 2.5 by catalytic debenzylation and introduction of an V-Boc protecting group in the resulting secondary amine.P-O— (CH2)n-CH2-LBn-NH2HO-(CH2)m^ 2.3HO- (CH2)m-CH2-Lcondition 2.2 conditions effective2.1 s effectiveHO-(CH2)m^ 1. H2, Pd(C) HO-(CH2)m^ P = acyl group or N-Bn > N-Boc trialkylsilyl group 2. or H5Scheme 2

[0053] Certain aminoalcohols of general formula 3.1 (Scheme 3) may be commercially available. In such cases, it is advantageous to prepare compounds of the type 2.5 by monoalkylation of 3.1 with 2.3, wherein L is a leaving group such as a halide (Cl, Br, I) or sulfonate (tosylate, mesylate, and the like), and P is as defined above, to produce 3.2, which is then converted into 2.5 under conditions effective. The mono-alkylation of 3.1 with 2.3 can be achieved as described in co-owned and co-pending PCT applications WO 2024 / 065043 and WO 2023 / 173203, which are incorporated herein by reference.

[0054] Certain aminoalcohols of general formula 4.1 (Scheme 4) may be commercially available.P-O-(CH2)n-CH2-L2 3HO- (CH2)m-CH2-NH2-:- -3 conditions effectiveHO-(CH2)m\ p = acyl group orN-Boc trialkylsilyl group P-O-(CH2)n^ 2.5or HScheme 3

[0055] In such cases, it may be advantageous to prepare compounds of the type 2.4 by mono-alkylation of 4.1 with 2.3. Product 2.4 thus obtained can then converted into 2.5 under conditions effective. The mono-alkylation of 4.1 with 2.3 can be achieved as described in co-owned and co-pending PCT applications WO 2024 / 065043 and WO 2023 / 173203, which are incorporated herein by reference.P-O- (CH2)n-CH2-L2 3 HO-(CH2)my Scheme 2HO- (CH2)m-CH2-NH-Bn -:- - N-Bn4 1 conditions effective P-O-(CH2)n— 4HO-(CH2)m\ p = acyl group orN-Boc trialkylsilyl groupP-O-(CH2)n—f2.5or HScheme 4

[0056] Indices m and n in the compounds of Schemes 2-4 above may differ or be the same. In the latter case, it may be advantageous to prepare a compound of the type 2.5, wherein m is equal to n, that is, compound 5.3 of Scheme 5, starting with the bis-N- alkylation of benzylamine with 2.1 to produce 5.1, followed by transformation into 5.2 under suitable conditions. One of the OH groups in 5.2 can then be selectively acylated, silylated, or sulfonylated as described in co-owned and co-pending WO 2023 / 147657, WO 2024 / 065043, and WO 2024 / 065041 (incorporated herein by reference) to produce 5.3.Bn-NH2HO-(CH2)m\ Scheme 2HO- (CH2)m-CH2-L - ► N— Bn - >vHO-(CH2)m^ conditions HO-(CH2)m\ P = acyl group orN-Boc - * N-Boc trialkylsilyl groupHO-(CH2)m^5 2effective P-O-(CH2)m— 5 3 or sulfonyl groupScheme 5

[0057] In certain cases, the aminoalcohol of general structure 3.1 is commercially available. In such a case, it may be advantageous to synthesize a compound of structure 5.3 by / V-alkylation of 3.1 with 6.1, wherein L is a leaving group such as a halide (Cl, Br, I) or sulfonate (tosylate, mesylate, and the like), and P’ is an acyl group, or a trialkylsilyl group, or H, leading to 6.2 (Scheme 6). Introduction of an N-Boc moiety converts 6.2 into 5.2, if P’ is H, or into 5.3, if P’ is an acyl or trialkylsilyl group. Furthermore, 5.2 can be converted into 5.3, P’ = acyl or trialkylsilyl group, by selective protection of one of the OH groups, or into 5.3, P’ = sulfonyl group, by selective sulfonylation of one of the OH groups.HO-(CH2)m-(CH2)-NH23.1P'-O— (CH2)m-CH2-L6.1 conditionsP' = acyl group or effective6.2 trialkylsilyl group or HHO-(CH2)m-\ conditions- HO-(CH2)m\N-Boc N-BocHO-(CH2)m^ effective P'-O-(CH2)m^5.2 product if P' = H 5.3 product if P' = acyl or trialkylsilylBoc effective5.3 P' = sulfoyl groupScheme 6

[0058] A lipid of Formula A wherein m and n differ, X is O, and Y is an acyl group, that is, a lipid of general structure 7.7 (Scheme 7), can be prepared starting from 2.5, wherein P is a trialkylsilyl group, RaSi, or an acyl group such as R4-C(O). The OH group in 2.5 is converted into a leaving group L such as a halide (Cl, Br, I) or sulfonate (tosylate, mesylate, and the like) to produce 7.1. Leaving group L can then be displaced with appropriate sulfur nucleophiles.

[0059] In certain embodiments, it is advantageous to create the sulfur nucleophile by treatment of a thioacetate ester such as 7.2 with a reagent that selectively cleaves the acetyl group, thereby releasing a nucleophilic form of the sulfur atom. The ensuing displacement reaction produces 7.3, if P in 2.5 and 7.1 is a trialkylsilyl group, or 7.4, if P in 2.5 and 7.1 is an acyl group. Furthermore, 7.3 can be transformed into 7.4 by releasing the silyl group and esterifying the resulting free alcohol. The Boc group in 7.4 can now be released and the resulting secondary amine 7.5 can be A-alkylated with 7.6, wherein L is a leaving group such as a halide (Cl, Br or I) or sulfonate (tosylate, mesylate, and the like), leading to formation of 7.7.HO-(CH2)m^ conditions L-(CH2)m^N-BN-Boc ocP-O-(CH2)n^ effective P-O-(CH2)n^ conditions effective2.5 P = R3Si or R4-CO 7.1 P = R3Si or R4-CO L = leaving group Boc7.3 7.4O product if P in 2.5 is R3Si product if P in 2.5 is R4-COR4O-(CH2)n^II 7.7 oScheme 7

[0060] In certain other embodiments, it is advantageous to displace leaving group L in 7.1 with the anion of thioacetic acid, leading to formation of thioacetate 8.1 (Scheme 8). Treatment of 8.1 with a reagent that selectively cleaves the acetyl group releases a nucleophilic form of the sulfur atom, which can then open the oxirane ring in an epoxide such as 8.2. If P in 7.1 and 8.1 is an acyl group, this results in formation of product 8.3. Acylation of the OH group in the latter with acid R4-COOH under conditions effective produces 7.4, which can be transformed into lipid 7.7 as seen above in Scheme 7.e ec ve conditions7.1 P = trialkylsilyl or acyl 8.1 P = trialkylsilyl or acyl effectiveg8.3 product if P = acyl 7.4O R2R0Y'R3S-(CH2)m-\N— (CH2)p-[Z-(CH2)q]r-(CH2)t-GR4O-(CH2)n^II 7.7OScheme 8

[0061] If P in 7.1 and 8.1 is a trialkylsilyl group, the reaction with epoxide 8.2 as per Scheme 8 would produce 9.1 (Scheme 9). This compound can be transformed into lipid 7.7 by sequential acylation of the OH group with acid RJ-COOH, release of the silyl protecting group, and acylation of the resulting alcohol 9.3 with acid R4-COOH, leading to 7.4, which can then be converted into 7.7 as seen in Scheme 7 above.Scheme 9

[0062] A lipid of Formula A wherein m and n differ and X is S, that is, a lipid of general structure 10.5 (Scheme 10), can be prepared from 9.3. The OH group in the latter can be converted into a leaving group L (halide such as Cl, Br, I, or sulfonate such as tosylate, mesylate, and the like). The resulting 10.1 can be caused to react with thioacetate 10.2 in the presence of a reagent that selectively cleaves the acetyl group, thereby releasing a nucleophilic form of the S atom. The ensuing substitution reaction produces 10.3, which can be A-deblocked to give amine 10.4. The A-alkylation of 10.4 with 7.6 as per Scheme 7 gives 10.5.Scheme 10

[0063] In certain embodiments, it may be advantageous to displace leaving group L in 10.1 with the anion of thioacetic acid, leading to formation of thioacetate 11.1 (Scheme 11). Treatment of 11.1 with a reagent that selectively cleaves the acetyl group releases a nucleophilic form of the sulfur atom, which can then open the oxirane ring in an epoxide such as 11.2 and cause the formation of product 11.3. Acylation of the OH group in the latter with R4-COOH under conditions effective produces 10.3, which can be transformed into lipid 7.7 as seen above in Scheme 10.Scheme 11

[0064] The synthetic diagrams above illustrate synthetic methods for the preparation of lipids of Formula A, wherein groups R1and R4, R2and R5, R3and R6, and indices m and n differ. However, in certain embodiments one or more of R1and R4, R2and R5, R3and R6, and indices m and n may be the same. The above synthetic methods are equally applicable in such cases. In certain cases, however, the above synthetic methods can be simplified. For example, certain embodiments contemplated herein are such that indices m and n are equal. A lipid of Formula A wherein X is O, Y is an acyl group, and m=n, that is, a lipid of general structure 12.4, can be prepared from a compound of the type 5.3 wherein P is a sulfonyl group, SO2-R’, with R’ = Me, Ph, 4-tolyl, etc., and no silyl protection is present; i.e., compound 12.1 (Scheme 12). As seen earlier in Scheme 7, an appropriate sulfur nucleophile can be generated by treatment of a thioacetate ester such as 7.2 with a reagent that selectively cleaves the acetyl group. Reaction of the sulfur nucleophile thus generated with 12.1 results in displacement of the sulfonate and formation of 12.2. Esterification of the free OH group in the latter with R4-COOH produces 12.3, which can be transformed into 12.4 by the method of Scheme 7. Thesynthetic sequence of Scheme 12 thus avoids OH protection as a silyl group and a later deprotection step.12.1 (= 5.3 with P = R'-SO2) 12.2 effectiveO R2O R2.R 0' / yR3Scheme 7RO>Y'R3S-(CH2)m^ S-(CH2)m— \ N— (CH2)p-[Z-(CH2)q]r-(CH2)t-GN-Boc R< O-(CH2)n^R4O-(CH2)m^ | 12.3 II 12.4OOScheme 12

[0065] A lipid of Formula A wherein X is S and m = n, that is, a lipid of general structure 13.3 (Scheme 13), can be prepared from compound 12.2 starting with conversion of the free OH group into a leaving group L (L = halogen such as Cl, Br, I or sulfonate such as mesylate, tosylate, triflate and the like). Displacement of L in the resulting 13.1 with an appropriate sulfur nucleophile generated by treatment of a thioacetate ester such as 10.2 with a reagent that selectively cleaves the acetyl group produces 13.2, which can be transformed into 13.3 by the method of Scheme 10.conditions S-(CH2)m\ Boc - »- N-Boceffective L-(CH2)m— conditions effective12.2 13.1 L = halogen or sulfonate

[0066] In certain embodiments contemplated herein, the lipid with the general structure of Formula A is such that m = n, R2= R5and R3= R6; i.e., the embodiments in question can be represented as structure 14.6 (Scheme 14). As an alternative to the synthetic routes shown above, compounds 14.6 can be prepared starting with the conversion of the OH groups in compound 2.5 wherein P = H into leaving groups L (L = halogen such as Cl, Br, I or sulfonate such as mesylate, tosylate, triflate and the like). Displacement of L in the resulting 14.1 with the anion of thioacetic acid produces bis-thioacetate 14.2. Treatment of the latter with a reagent that selectively cleaves the acetyl groups, thereby liberating a nucleophilic form of the sulfur atom, and consequent reaction with epoxide 8.2, leading to the formation of 14.3. Selectively mono-esterification of 14.3 with acid R^COOH in the presence of a suitable coupling agent, as described in co-owned and co-pending application PCT / CA2023 / 051727 fded on December 21, 2023, gives 14.4, which can be further esterified with acid R4-COOH in the presence of a suitable coupling agent to produce 14.5. The latter can be converted into lipid 10.6 by the method of Scheme 10.HO-(CH2)m^ conditions L-(CH2)m-\ AcSH N-Boc N-BocHO-(CH2)meffective L-(CH2)m^14 1conditions effective2.5 P = H R2O R3HOS-(CH2)m-\ - S-(CH2)m^N-Boc N-BocScheme 14

[0067] Further contemplated herein are certain embodiments, wherein a lipid with the general structure of Formula A is such that m = n, R1= R4, R2= R5and R3= R6; i.e., the embodiments in question can be represented as structure 15.2 (Scheme 15). Such a compound can be prepared by doubly esterifying 14.3 with acid R^COOH in the presence of a suitable coupling agent and then transforming diester 15.1 into lipid 15.2 by the method of Scheme 10.Scheme 15

[0068] Without intending to be limiting, examples of lipids of Formula A are T-01-T-08 below.

[0069] Building blocks of the type 2.5 that can be used for the synthesis of T-01-T-08 are provided in Scheme 16 as compoundsl6.1-16.4, A method for their preparation is also shown.BocScheme 2 16.2 precursor of T-03, T-04Scheme 16

[0070] The synthesis of lipids T-01 and T-02 can be carried out starting with the conversion of 16.1 into bis-tosylate 17.1, reaction of which with thioacetic acid in the presence of EtsN, leads to the formation of 17.2 (Scheme 17). The latter can be treated with MeONa in the presence of 1 -octene oxide, whereupon product 17.3 is obtained. The OH groups are esterified with 3-cyclohexylpropanoic acid in the presence of EDCI and the Boc group is released from the resulting 17.4 to produce amine 17.5 or its TFA salt.TsCI AcSHBoc17.4 Z = Boc — 1T1_TFAA17.5 Z = H -1Scheme 17

[0071] Amine 17.5 can be transformed into T-01 by reductive amination with commercial aldehyde 18.1, followed by release of the silyl group in 18.2 with a source of fluoride ion, for example, HF-pyridine complex. Furthermore, 17.5 can be converted into T-02 by N- alkylation with 2-(2-bromoethoxy)ethan-l-ol (Scheme 18). The same method can be used to convert 16.2, into lipids T-03 and T-04, and 16.3 into T-05 and T-06.Scheme 18

[0072] The synthesis of lipids T-07 and T-08 requires additional building blocks 19.3 and19.8, which can be made as shown in Scheme 19.

[0073] The synthesis of lipids T-07 and T-08 can be achieved starting with the conversion of compound 14.4 into tosylate 20.1 (Scheme 20). Reaction of 20.1 with 19.3 in the presence of NaOMe produces 20.2. The silyl group in the latter can be released by treatment with a source of fluoride ion, for example, HF-pyridine complex, and the resulting alcohol 20.3 can then be converted into tosylate 20.4. Reaction of 20.4 with 19.8 in the presence of NaOMe produces 20.5, which upon treatment with an appropriateprotonic acid, for example, trifluoroacetic acid, is transformed into amine 20.6 or its trifluoroacetate salt. Amine 20.6 or its TFA salt can be converted into lipids T-07 and T- 08 (Scheme 21) by the method outlined in Scheme 18 above.O ^

[0074] Those skilled in the art will appreciate that the use of alternative building blocks in the schemes above can produce lipids of Formula A other than T-01-T-08, such as the representative embodiments T-09-T-20 below. Methods that could be employed to prepare T-09-T-20 are provided in co-owned and co-pending U.S. provisional patent application No. 63 / 664,792 fded on June 27, 2024, which is incorporated herein by reference.

[0075] Moreover, the final ^-alkylation step can be used to introduce moi eties other than those provided in the schemes above, leading to additional structural diversity. For example, the reaction of an amine such as 17.5 with the known epoxide 22.1 (Matile, S., et al., Angew. Chem. Int. Ed. 2020, 59, 6273-6277), in a solvent such as DMF and optionally in the presence of a base such as K2CO3, is anticipated to produce T-20 (Scheme 22).Formulation of the above lipids in a delivery vehicle

[0076] The lipids of the disclosure may be formulated in a variety of drug delivery vehicles (also referred to herein as a “delivery vehicle”) known to those of ordinary skill in the art. An example of a delivery vehicle is a lipid nanoparticle, which includes liposomes, lipoplexes, polymer nanoparticles comprising lipids, polymer-based nanoparticles, emulsions, and micelles.

[0077] In one embodiment, a lipid having the structure of Formula A of the disclosure is formulated in a delivery vehicle by mixing them with additional lipids, including helper lipids, such as vesicle forming lipids and optionally an aggregation inhibiting lipid, such as a hydrophilic polymer-lipid conjugate (e.g., PEG-lipid).

[0078] As set forth previously, a helper lipid includes a sterol, a diacylglycerol, a ceramide or derivatives thereof.

[0079] Examples of sterols include cholesterol, or a cholesterol derivative, such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, beta-sitosterol, fucosterol, and the like.

[0080] Examples of diacylglycerols include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), and mixtures thereof. In certain embodiments, the phospholipid is DPPC, DSPC, a DSPC- cholesterol conjugate or mixtures thereof. These lipids may be synthesized or obtained from natural sources, such as from egg. The DSPC-cholesterol conjugate is a lipid in which one of the acyl chains is substituted with a cholesterol moiety link to the head group by a succinate linker.

[0081] A suitable ceramide derivative is egg sphingomyelin or dihydrosphingomyelin.

[0082] Delivery vehicles incorporating the lipids of the disclosure can be prepared using a wide variety of well described formulation methodologies known to those of skill in the art, including but not limited to extrusion, ethanol injection and in-line mixing. In one embodiment, the preparation method is an in-line mixing technique in which aqueous andorganic solutions are mixed using a rapid-mixing device as described in Kulkami et al., 2018, ACS Nano, 12:4787 and Kulkami et al., 2017, Nanoscale, 36:133347, each of which is incorporated herein by reference in its entirety.

[0083] The delivery vehicle can also be a nanoparticle that is a lipoplex that comprises a lipid core stabilized by a surfactant. Vesicle-forming lipids may be utilized as stabilizers. The lipid nanoparticle in another embodiment is a polymer-lipid hybrid system that comprises a polymer nanoparticle core surrounded by stabilizing lipid. Nanoparticles comprising lipids of the disclosure may alternatively be prepared from polymers without lipids. Such nanoparticles may comprise a concentrated core of a therapeutic agent that is surrounded by a polymeric shell or may have a solid or a liquid dispersed throughout a polymer matrix.

[0084] Lipids described herein can also be incorporated into emulsions, which are drug delivery vehicles that contain oil droplets or an oil core. An emulsion can be lipid- stabilized. For example, an emulsion may comprise an oil filled core stabilized by an emulsifying component such as a monolayer or bilayer of lipids.

[0085] Lipids described herein may be incorporated into a micelle. Micelles are selfassembling particles composed of amphipathic lipids or polymeric components that are utilized for the delivery of agents present in the hydrophobic core.Delivery of nucleic acid, genetic material, proteins, peptides or other charged agents

[0086] Lipids disclosed herein may facilitate the incorporation of a compound or molecule (referred to herein also as “cargo” or “cargo molecule”) bearing a net negative or positive charge into the delivery vehicle and subsequent delivery to a target cell in vitro or in vivo.

[0087] In one embodiment, the cargo molecule is genetic material, such as a nucleic acid. The nucleic acid includes, without limitation, RNA, including small interfering RNA (siRNA), small nuclear RNA (snRNA), micro RNA (miRNA), messenger RNA (mRNA) or DNA such as vector DNA or linear DNA. The nucleic acid length can vary and can include nucleic acid of 5-50,000 nucleotides in length. The nucleic acid can be in any form, including single stranded DNA or RNA, double stranded DNA or RNA, or hybrids thereof. Single stranded nucleic acid includes antisense oligonucleotides.

[0088] In one embodiment, the cargo is an mRNA, which includes a polynucleotide that encodes at least one peptide, polypeptide or protein. The mRNA includes, but is not limited to, small activating RNA (saRNA) and trans-amplifying RNA (taRNA), as described in WO 2022 / 251953A1, which is incorporated herein by reference.

[0089] The mRNA as used herein encompasses both modified and unmodified mRNA. In one embodiment, the mRNA comprises one or more coding and non-coding regions. The mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, or may be chemically synthesized.

[0090] In those embodiments in which an mRNA is a chemically synthesized molecule, the mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and / or backbone modifications. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C 5 -bromouridine, C5 -fluorouridine, C5 -iodouridine, C5-propynyl- uridine, C5-propynyl-cytidine, C5 -methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8 -oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0091] The mRNAs of the disclosure may be synthesized according to any of a variety of known methods. For example, mRNAs in certain embodiments may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.

[0092] In some embodiments, in vitro synthesized mRNA may be purified before encapsulation to remove undesirable impurities including various enzymes and other reagents used during mRNA synthesis.

[0093] The present disclosure may be used to encapsulate mRNAs of a variety of lengths. In some embodiments, the present disclosure may be used to encapsulate in vitro synthesized mRNA ranging from about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb in length.

[0094] Typically, mRNA synthesis includes the addition of a “cap” on the 5' end, and a “tail” on the 3' end. The presence of the cap is important in providing resistance tonucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

[0095] In some embodiments, mRNAs include a 5' and / or 3' untranslated region. In some embodiments, a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 50 and 500 nucleotides in length.

[0096] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between 50 and 500 nucleotides in length or longer.

[0097] While mRNA provided from in vitro transcription reactions may be desirable in certain embodiments, other sources of mRNA are contemplated, such as mRNA produced from bacteria, fungi, plants, and / or animals.

[0098] The mRNA sequence may comprise a reporter gene sequence, although the inclusion of a reporter gene sequence in pharmaceutical formulations for administration is optional. Such sequences may be incorporated into mRNA for in vitro studies or for in vivo studies in animal models to assess biodistribution.

[0099] In another embodiment, the cargo is an siRNA. An siRNA becomes incorporated into endogenous cellular machineries to result in mRNA breakdown, thereby preventing transcription. Since RNA is easily degraded, its incorporation into a delivery vehicle can reduce or prevent such degradation, thereby facilitating delivery to a target site.

[0100] The siRNA encompassed by embodiments of the disclosure may be used to specifically inhibit expression of a wide variety of target polynucleotides. The siRNA molecules targeting specific polynucleotides may be readily prepared according to procedures known in the art. An siRNA target site may be selected and corresponding siRNAs may be chemically synthesized, created by in vitro transcription, or expressed from a vector or PCR product. A wide variety of different siRNA molecules may be used to target a specific gene or transcript. The siRNA may be double-stranded RNA, or a hybrid molecule comprising both RNA and DNA, e.g., one RNA strand and one DNA strand. The siRNA may be of a variety of lengths, such as 15 to 30 nucleotides in length or 20 to 25 nucleotides in length. In certain embodiments, the siRNA is double-strandedand has 3' overhangs or 5' overhangs. In certain embodiments, the overhangs are UU or dTdT 3'. In particular embodiments, the siRNA comprises a stem loop structure.

[0101] In a further embodiment, the cargo molecule is a microRNA or small nuclear RNA. Micro RNAs (miRNAs) are short, noncoding RNA molecules that are transcribed from genomic DNA, but are not translated into protein. These RNA molecules are believed to play a role in regulation of gene expression by binding to regions of target mRNA. Binding of miRNA to target mRNA may downregulate gene expression, such as by inducing translational repression, deadenylation or degradation of target mRNA. Small nuclear RNA (snRNA) are typically longer noncoding RNA molecules that are involved in gene splicing. The snRNA molecules may have therapeutic importance in diseases that are an outcome of splicing defects.

[0102] In another embodiment, the cargo is a DNA vector as described in co-owned and co-pending WO 2022 / 251959, which is incorporated herein by reference. The DNA vectors may be administered to a subject for the purpose of repairing, enhancing or blocking or reducing the expression of a cellular protein or peptide. Accordingly, the nucleotide polymers can be nucleotide sequences including genomic DNA, cDNA, or RNA.

[0103] As will be appreciated by those of skill in the art, the vectors may encode promoter regions, operator regions or structural regions. The DNA vectors may contain double-stranded DNA or may be composed of a DNA-RNA hybrid. Non-limiting examples of double-stranded DNA include structural genes, genes including operator control and termination regions, and self-replicating systems such as vector DNA.

[0104] Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes and triplex-forming oligonucleotides. In order to have prolonged activity, the single-stranded nucleic acids will preferably have some or all of the nucleotide linkages substituted with stable, non-phosphodi ester linkages, including, for example, phosphorothioate, phosphorodithioate, phophoroselenate, or O-alkyl phosphotriester linkages.

[0105] The DNA vectors may include nucleic acids in which modifications have been made in one or more sugar moi eties and / or in one or more of the pyrimidine or purine bases. Such sugar modifications may include replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, azido groups or functionalized as ethers or esters. In another embodiment, the entire sugar may be replaced with sterically and electronicallysimilar structures, including aza-sugars and carbocyclic sugar analogs. Modifications in the purine or pyrimidine base moiety include, for example, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substitutes known to those of skill in the art.

[0106] The DNA vector may be modified in certain embodiments with a modifier molecule such as a peptide, protein, steroid or sugar moiety. Modification of a DNA vector with such molecule may facilitate delivery to a target site of interest. In some embodiments, such modification translocates the DNA vector across a nucleus of a target cell. By way of example, a modifier may be able to bind to a specific part of the DNA vector (typically not encoding of the gene-of-interest), but also has a peptide or other modifier that has nucleus-homing effects, such as a nuclear localization signal. A nonlimiting example of a modifier is a steroid-peptide nucleic acid conjugate as described by Rebuffat et al., 2002, Faseb J. 16(11): 1426-8, which is incorporated herein by reference. The DNA vector may contain sequences encoding different proteins or peptides. Promoter, enhancer, stress or chemically -regulated promoters, antibiotic-sensitive or nutrientsensitive regions, as well as therapeutic protein encoding sequences, may be included as required. Non-encoding sequences may be present as well in the DNA vector.

[0107] The nucleic acids used in the present method can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries or prepared by synthetic methods. Synthetic nucleic acids can be prepared by a variety of solution or solid phase methods. Generally, solid phase synthesis is preferred. Detailed descriptions of the procedures for solid phase synthesis of nucleic acids by phosphite-triester, phosphotriester, and H-phosphonate chemistries are widely available.

[0108] In one embodiment, the DNA vector is double stranded DNA and comprises more than 700 base pairs, more than 800 base pairs or more than 900 base pairs or more than 1000 base pairs.

[0109] In another embodiment, the DNA vector is a nanoplasmid or a minicircle.

[0110] Gene editing systems can also be incorporated into delivery vehicles comprising the charged lipid. This includes a Cas9-CRISPR, TALEN and zinc finger nuclease gene editing system. In the case of Cas9-CRISPR, a guide RNA (gRNA), together with a plasmid or mRNA encoding the Cas9 protein may be incorporated into a delivery vehicle comprising the lipids described herein. Optionally, a ribonucleoprotein complex may be incorporated into a delivery vehicle comprising the lipid described herein. Likewise, thedisclosure includes embodiments in which genetic material encoding DNA binding and cleavage domains of a zinc finger nuclease or TALEN system are incorporated into a delivery vehicle together with the lipids of the disclosure.

[0111] While a variety of nucleic acid cargo molecules are described above, it will be understood that the above examples are non-limiting and the disclosure is not to be considered limiting with respect to the particular cargo molecule encapsulated in the delivery vehicle.

[0112] For example, the lipids described herein may also facilitate the incorporation of proteins and peptides into a delivery vehicle, which includes ribonucleoproteins. This includes both linear and non-linear peptides, proteins or ribonucleoproteins.

[0113] While pharmaceutical compositions are described above, the lipids described herein can be a component of any nutritional, cosmetic, cleaning or foodstuff product.Pharmaceutical formulations

[0114] The ionizable lipids of the disclosure may be present in a salt form. The salt is typically a pharmaceutically acceptable salt. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, and zinc. In one embodiment, the base is selected from ammonium, calcium, magnesium, potassium and sodium. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and the like.

[0115] In some embodiments, the delivery vehicle comprising the cargo molecule is part of a pharmaceutical composition and is administered to treat and / or prevent a disease condition. The treatment may provide a prophylactic (preventive), ameliorative or a therapeutic benefit. The pharmaceutical composition will be administered at any suitable dosage.

[0116] In one embodiment, the pharmaceutical compositions is administered parentally, i.e., intra-arterially, intravenously, subcutaneously or intramuscularly. In yet a further embodiment, the pharmaceutical compositions are for intra- tumoral or in-utero administration. In another embodiment, the pharmaceutical compositions are administered intranasally, intravitreally, subretinally, intrathecally or via other local routes.

[0117] The pharmaceutical composition comprises pharmaceutically acceptable salts and / or excipients.

[0118] The compositions described herein may be administered to a patient. The term patient as used herein includes a human or a non-human subject.

[0119] The following examples are given for the purpose of illustration only and not by way of limitation on the scope of the invention.

[0120] The article “a” or “an” as used herein is meant to include both singular and plural, unless otherwise indicated.EXAMPLESMaterials

[0121] The lipid l,2-distearoyl-5«-glycero-3-phosphorylcholine (DSPC) was purchased from Lipoid Inc. (Newark, NJ). l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene gly col-2000 (PEG-DMG) was purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol and lOx Phosphate Buffered Saline (pH 7.4) were purchased from Sigma Aldrich (St Louis, MO). The ionizable amino-lipid was synthesized as previously described in WO 2022 / 246555, which is incorporated herein by reference.

[0122] An mRNA encoding firefly luciferase synthesized by NanoVation Therapeutics (Vancouver, BC) was used to analyse luciferase activity.MethodsPreparation of lipid nanoparticles (LNP) containing mRNA

[0123] Ionizable amino-lipid described herein, DSPC, cholesterol, and PEG-DMG, were dissolved in ethanol at the appropriate ratios to a final concentration of 10 or 20 mM total lipid. Nucleic acid (mRNA) was dissolved in an appropriate buffer such as 25 mM sodium acetate pH 4 or sodium citrate pH 4 to a concentration necessary to achieve the appropriate amine-to-phosphate ratios. The aqueous and organic solutions were mixed using a rapidmixing device as described in Kulkami et al., 2018, ACS Nano, 12:4787 and Kulkami et al., 2017, Nanoscale, 36: 133347 (each incorporated herein by reference) at a flow rate ratio of 3: 1 (v / v; respectively) and a total flow rate of 20 mL / min. The resultant mixture was dialyzed directly against 1000-fold volume of PBS pH 7.4. All formulations were concentrated using an Amicon™ centrifugal filter unit and analysed using the methods described below.Analysis of LNP

[0124] Particle size analysis of LNPs in PBS was carried out using backscatter measurements of dynamic light scattering with a Malvern Zetasizer™ (Worcestershire, UK). The reported particle sizes correspond to the number-weighted average diameters(nm). Total lipid concentrations were determined by extrapolation from the cholesterol content, which was measured using the Cholesterol E-Total Cholesterol Assay (Wako Diagnostics, Richmond, VA) as per the manufacturer’s recommendations. Encapsulation efficiency of the formulations was determined using the Quant-iT RiboGreen™ Assay kit (Invitrogen, Waltham, MA). Briefly, the total siRNA or mRNA content in solution was measured by lysing lipid nanoparticles in a solution of TE containing 2% Triton Tx-100, and free DNA vector in solution (external to LNP) was measured based on the RiboGreen™ fluorescence in a TE solution without Triton. Total siRNA or mRNA content in the formulation was determined using a modified Bligh-Dyer extraction procedure. Briefly, LNP formulations containing siRNA or mRNA were dissolved in a mixture of chloroform, methanol, and PBS that results in a single phase and the absorbance at 260 nm measured using a spectrophotometer.In vivo analysis in CD-I mice

[0125] LNP -mRNA encoding firefly luciferase were injected intravenously (tail-vein) into 6-8 weeks old CD-I mice. Four hours following injection, the animals were euthanized and the liver and spleen and isolated. Tissue was homogenized in Gio Lysis buffer and a luciferase assay performed using the Steady Gio Luciferase assay kit (as per manufacturers recommendations).Organic synthesis of lipids T1-T7.

[0126] Unless otherwise specified, all reagents and solvents were commercial products and were used without further purification. All reactions were performed under an argon atmosphere. Reaction mixture from aqueous workups were dried by passing over a plug of anhydrous Na2SO4 held in a filter tube and concentrated under reduced pressure on a rotary evaporator. Thin-layer chromatography was performed on silica gel plates coated with silica gel (Merck 60 F254 plates). Visualization of developed chromatograms was performed by staining with L or potassium permanganate solution. Medium-pressure column chromatography was performed on a Teledyne ISCO system.JH and13C nuclear magnetic resonance (NMR) spectra were recorded at room temperature in CDCh solutions.JH NMR spectra were referenced to residual CHCh (7.26 ppm) and13C NMR spectra were referenced to the central line of the CDCh triplet (77.00 ppm). Chemical shifts are reported in parts per million (ppm) on the 5 scale. Multiplicities are reported as “s” (singlet), “d” (doublet), “f ’ (triplet), “q” (quartet), “m” (multiplet), and furtherqualified as “app” (apparent) and “br” (broad). Low-resolution mass spectra (m / z) were obtained in the electrospray (ESI) mode.Nor-MC3 control

[0127] The ionizable lipid, nor-MC3, was used in Example 2 as a control. This ionizable lipid has the structure:nor-MC3

[0128] WO 2022 / 246571 describes the synthesis of the foregoing ionizable lipid and is incorporated herein by reference.Example 1: Methods for chemically synthesizing ionizable lipids of the disclosureSynthesis of building blocksProcedure for \,\-dialkylation of benzylamine: 5,5'-(benzylazanediyl)bis(pentan-l- ol).

[0129] Anhydrous K2CO3 (6.4 g, 46.7 mmol, 2.5 equiv) was added to a solution of 5- bromopentan-l-ol (6.2 g, 37.0 mmol) and benzylamine (2 g, 18.7 mmol) in dry acetonitrile (50 mL). The reaction mixture was heated to 75 °C under argon atmosphere and stirred for 16 hours, diluted with water and extracted with EtOAc (3 x 50 mL). The combined extracts were dried (ISfeSCL) and concentrated. The residue was purified by silica chromatography (0-10% MeOH in DCM) to yield the desired product (3.8 g, 73%).1H NMR (400 MHz, CDCh) 8 7.38-7.21 (m, 5H), 3.63 (t, J= 6.5 Hz, 4H), 3.57 (s, 2H), 2.48- 2.40 (m, 4H), 1.61-1.47 (m, 8H), 1.43-1.31 (m, 4H).

[0130] The following compounds were prepared by the same method: 6,6'-(benzylazanediyl)bis(hexan-l-ol).

[0131] From 6-bromo-l -hexanol and benzylamine: 'H NMR 57.37-7.20 (m, 5H), 3.69- 3.60 (m, 4H), 3.55 (s, 2H), 2.45-2.37 (m, 4H), 1.63-1.43 (m, 10H), 1.33 (m, 6H).7,7'-(benzylazanediyl)bis(heptan-l-ol).

[0132] From 7-bromo-l -heptanol and benzylamine.XH NMR (400 MHz, CDCh) 8 7.30- 7.13 (m, 5H), 3.57 (t, J= 6.58, 4H), 3.48 (s, 2H), 3.34 (br, 2H), 2.33 (t, J =6.53 Hz, 4H), 1.88 - 0.88 (m, 20H).Procedure for TBS protection: ((7-bromoheptyl)oxy)(tert-butyl)dimethylsilane.

[0133] Imidazole (5 g, 74.1 mmol) was added over 20 min to 7-bromoheptan-l-01 (4.8 g, 24.7 mmol) and tert-Butyldimethylsilyl chloride (4.47 g, 29.64 mmol) in dimethylformamide (30 ml) at 0 °C. The reaction was warmed to room temperature and stirred for 6 hours, diluted with water and extracted with ether (3 x 20.0 mL). The combined extracts were dried (Na2SO4) and concentrated gave a pale-yellow oil (7.2 g). Distillation via kugelrohr at 140 °C, 0.02-0.04 mm Hg afforded the desired product (6.2 g, 82%) as a colorless oil.XH NMR (400 MHz, CDCh) 8 3.56-3.53 (t, J = 6.54 Hz, 2H), 3.50-3.46 (t, J= 6.54 Hz, 1H), 3.37-3.36 (t, J = 6.87 Hz, 1H), 1.82-1.70 (m, 2H), 1.48- 1.26 (m, 8H), 0.84 (s, 9H), 0.001 (s, 6 H).Procedure for the / V-alkylation of an / V-alkyl benzylamine: 6-(benzyl(7-((tert- butyldimethylsilyl)oxy)heptyl)amino)hexan-l-ol.

[0134] A solution of 6-(benzylamino)hexan-l-ol (3 g , 14.5 mmol) and ((7- bromoheptyl)oxy)(tert-butyl)dimethylsilane (4.5 g, 14.5 mmol) in dry acetonitrile (10 mL) containing suspended K2CO3 (3 g, 21.7 mmol, 1.5 equiv) was heated to 75 °C under argon and stirred for 16 hours, diluted with water and extracted with EtOAc (3 x 50 mL). The combined extracts were dried (Na2SO4) and concentrated. The residue was purified by silica chromatography (0-50% EtOAc in hexanes) to yield the desired product (4.2 g, 67%).XH NMR (400 MHz, CDCh) 8 7.28-7.15 (m, 5H,), 3.57-3.52 (m, 4H,), 3.48 (s, 2H), 2.35-2.31 (m, 4H,), 1.52-1.40 (m, 9H), 1.26 -1.21 (m, 10H), 0.84 (s, 9H), 0.001 (s, 6 H).Procedure for tandem catalytic \-debenzylation - Boc protection: tert-butyl bis(5- hydroxypentyl)carbamate (16.1).Boc16.1

[0135] Palladium hydroxide (320 mg, 2.4 mmol) was added to a solution of 5,5'- (benzylazanediyl)bis(pentan-l-ol) (2.2g, 7.9 mmol) and di-tert-butyl dicarbonate (1.72 g, 7.7 mmol) in methanol (30 mL) and the mixture was stirred under hydrogen (balloon) at rt for 12 hours. The solution was fdtered through celite, and the fdtrate was concentrated under reduced pressure. The residue was purified by silica chromatography (0-30% EtOAc in hexanes) to yield 16.1 (2.1 g, 95%). 'H NMR (400 MHz, CDCh) 8 3.65 (t, J= 6.5 Hz, 4H), 3.18 (s, 4H), 1.66 -1.49 (m, 8H), 1.46 (s, 9H), 1.41-1.29 (m, 4H).

[0136] The following compounds were prepared by the same method: te / 7-Butyl bis(6-hydroxyhexyl)carbamate (16.2).But-3-en-l-ylcyclohexane (19.5).

[0140] Commercial (4- bromo-butyl)cyclohexane (116 mmol, 25.4 g) was added to a RBF under inert atmosphere containing 1.0 M tBuOK in THF (139 mmol, 139 mL). The reaction was then stirred at RT for 30 min. The reaction was diluted with hexanes (200 ml) and fdtered through celite, then evaporated to yield a dark crude oil. The crude oil was fdtered through a silica plug eluding with hexanes (200 mL), then evaporated, yielding the desired alkene (>95% yield). This was carried forward without further purification. 'H NMR (400 MHz, CDCh) 8 5.81 (ddt, J= 16.9, 10.2, 6.7 Hz, 1H), 5.03-4.96 (m, 1H), 4.95- 4.89 (m, 1H), 2.09-2.01 (m, 2H), 1.78-1.00 (m, 11H), 0.94-0.81 (m, 2H).Procedure for epoxidation: 2-(2-cyclohexylethyl)oxirane (19.6).

[0141] Crude but-3-en-l- ylcyclohexane (20.3 g, 147 mmol) was added to a RBF under inert atmosphere, then diluted with DCM (440 mL). Formic acid (440 mmol, 16.6 mL) was then added. Aqueous hydrogen peroxide (30 wt%) (294 mmol, 30.0 mL) was then added at room temperature to the vigorously stirred mixture via syringe pump over 5 h. After completion of the addition, the reaction was stirred at RT overnight, then washed with water (500 mL). The layers were separated, and the organic phase was collected. The aqueous phase was back extracted with DCM (2x300 mL). The combined organic phases were dried (NazSCh), filtered, and evaporated to yield the crude epoxide. This material was filtered through a silica plug (-100 g) eluding with 10% EtOAc in hexanes (500 mL), then evaporated, yielding the desired oxirane (10.3 g, 67 mmol, 46% yield), which was used without further purification. 'H NMR (400 MHz, CDCh) 82.92 - 2.85 (m, 1H), 2.74 (dd, J= 5.0, 3.9 Hz, 1H), 2.46 (dd, J= 5.0, 2.7 Hz, 1H), 1.76 - 1.06 (m, 13H), 0.95 - 0.79 (m, 2H).Procedure for epoxide opening: N-(2-hydroxyoctyl) ethanethioate (19.2).

[0142] To a solution of 2-hexyloxirane (10 g, 78 mmol, 1 eq.) and AcSH (6.7 mL, 93.6 mmol, 1.2 eq) in DMF at 0 °C, was added NaHCCh (0.66 g, 7.8 mmol, 0.1 eq). The resulting mixture was allowed to warm to RT and stirred for 3 hrs, then poured into water (100 mL). The aq. phase was extracted with n-hexanes (3 x 50 mL), the combined organic layers were dried over Na2SO4 and concentrated in vacuo. The resulting residue was purified by column chromatography eluting with EtOAc:hexane (0-10%) to afford the title compound (12.1 g, 59.2 mmol, 76%) as yellow oil. 'H NMR (400 MHz, CDCh) 5 3.73 (tq, J= 6.1, 4.2 Hz, 1H), 3.15 (dd, J= 14.0, 3.7 Hz, 1H), 2.88 (dd, J= 14.0, 7.5 Hz, 1H), 2.37 (s, 3H), 1.99 (td, J = 4.8, 2.3 Hz, 1H), 1.63 - 1.18 (m, 10H), 0.93 - 0.82 (m, 3H).

[0143] The following compound was prepared by the same method: N-(4-cyclohexyl-2-hydroxybutyl) ethanethioate (19.7).

[0144] XH NMR (400 MHz, CDCh) 83.72 (tdd, J= 7.4, 5.4, 3.7 Hz, 1H), 3.17 (dd, J = 14.0, 3.8 Hz, 1H), 2.90 (dd, J= 14.0, 7.5 Hz, 1H), 2.39 (s, 3H), 2.00 (s, 1H), 1.77 - 1.08 (m, 13H), 0.91 (qt, J= 12.6, 6.1 Hz, 2H). l-(Acetylthio)octan-2-yl hexanoate (19.3).19.3

[0145] To a solution of EDC.HC1 (13.6 g, 70.8 mmol, 1.2 eq) and 4-DMAP (7.93 g, 64.9 mmol, 1.1 eq) in CH2CI2 (50 mL), was added hexanoic acid (11.1 mL, 88.5 mmol, 1.5 eq) and stirred for 10 min, then followed by slow addition a solution of thioacetate alcohol (12.1 g, 59 mmol, 1 eq) in CH2CI2 (10 mL). The resulting mixture was stirred at room temp for 18 hr, diluted with CH2CI2 (50 mL) and poured into water (100 mL). The organic layer was separated, and the aq. phase was further extracted with CH2CI2 (2 x 30 mL). The combined organic layers were dried (Na2SO4), filtered, and concentrated in vacuo. The resulting residue was purified by automated flash column chromatography eluting with EtOAc:«-hexanes (0-10-%) to afford the title compound (14.3 g, 47.3 mmol, 80%) as a colorless oil.XH NMR (400 MHz, CDCh) 84.97 (tt, J= 7.0, 4.6 Hz, 1H), 3.24 (dd, J =14.0, 4.4 Hz, 1H), 2.97 (dd, J= 14.0, 6.9 Hz, 1H), 2.33 (s, 3H), 2.27 (t, J= 7.5 Hz, 2H), 1.60 (dp, J= 12.2, 6.8 Hz, 4H), 1.41 - 1.10 (m, 12H), 0.88 (dt, J= 9.9, 6.7 Hz, 6H).

[0146] The following compound was prepared by the same method: l-(acetylthio)-4-cyclohexylbutan-2-yl 3-cyclohexylpropanoate (19.8).84.96 (qd, J= 6.7, 4.3 Hz, 1H), 3.26 (dd, J= 14.0, 4.4 Hz, 1H), 3.00 (dd, J= 14.0, 6.9 Hz, 1H), 2.36 (s, 3H), 2.31 (t, 2H), 1.77 - 1.49 (m, 14H), 1.22 (tdd, J= 14.6, 11.2, 6.0 Hz, 10H), 0.98 - 0.81 (m, 2H).Procedure for tosylation: ((ter / -butoxycarbonyl)azanediyl)bis(pentane-5,l-diyl) bis(4- methylbenzenesulfonate) (17.1).Boc17.1

[0148] Solid TsCl (2 g, 10.5 mmol, 1.5 equiv) was added to a solution of 16.1 (1 g, 3.5 mmol, 1 equiv), TEA (2 mL, 14 mmol, 2 equiv) and DMAP (213 mg, 1.75 mmol) in DCM (20.0 mL) at 0 °C under nitrogen. The reaction was warmed to room temperature and stirred for 2 hours, diluted with water and extracted with DCM (3 x 20.0 mL). The combined extracts were dried (Na2SO4) and concentrated. The residue was purified by silica chromatography (0-20% EtOAc in hexanes) to yield 17.1 (1.2 g, 60%). 'H NMR (400 MHz, CDCh) 8 7.84-7.73 (m, 4H), 7.39-7.32 (m, 4H), 4.02 (t, J= 6.4 Hz, 4H), 3.09 (s, 4H), 2.46 (s, 6H), 1.74-1.62 (m, 4H), 1.43 (s, 14H), 1.37-1.23 (m, 6H).

[0149] The following compounds were prepared by the same method:((teT -ButoxycarbonyI)azanediyl)bis(heptane-7,l-diyl) bis(4-methylbenzenesulfonate).Boc6-((tert-Butoxycarbonyl)(7-((tert-butyldimethylsilyl)oxy )heptyl)amino)hexyl 4- methyl-benzenesulfonate (20.1).Boc i20.1

[0151] From 16.4 with 1.5 equiv of TsCl and 2 equiv of EtsN. 'H NMR (400 MHz, CDCh) 8 7.81-7.78 (d, J= 8.32 Hz, 2H), 7.37-7.35 (d, J= 8.32 Hz, 2H), 4.03-4.00 (t, J = 6.53 Hz, 2H), 3.62-3.59 (t, J= 6.53 Hz, 2H), 3.12 (brs, 4H,), 2.46 (s, 3H), 1.70-1.61 (m, 2H), 1.50-1.43 (m, 15H), 1.34-1.20 (m, 10H), 0.90 (s, 9H), 0.05 (s, 6 H).Procedure for mesylation: ((tert-butoxycarbonyl)azanediyl)bis(hexane-6,l-diyl) dimethanesulfonate.Boc i

[0152] To a solution of tert-butyl A,A-bis(6-hydroxyhexyl)-carbamate (2.5 g, 5.3 mmol) and triethylamine (3.6 mL, 2.6 g, 26 mmol) in CH2CI2 (15 mL) in a RBF under inert atmosphere, was added methanesulfonyl chloride (21 mmol, 1.6 mL) dropwise via syringe at 0°C. The mixture was stirred for 15 min at 0°C then quenched with water. The biphasic mixture was separated, and the organic layer was collected. The aqueous phase was back- extracted with DCM (2x50 mL). The combined organic phases were dried (Na2SO4), filtered through a silica plug, eluting with 5% MeOH in DCM (50 mL), and evaporated to yield the crude product as a yellow oil (>95% yield). 'H NMR (400 MHz, CDCI3) 84.21 (t, J= 6.5 Hz, 4H), 3.16-3.12 (m, 4H), 3.00 (s, 6H), 1.80-1.69 (m, 4H), 1.51 (m, J = 7.5 Hz, 4H), 1.44 (s, 9H), 1.47-1.38 (m, 4H), 1.35-1.25 (m, 4H).Procedure for thioacetate formation: 5'„S''-((( rt-butoxycarbonyl)azanediyl)- bis(pentane-5,l-diyl)) diethanethioate (17.2).Boc17.2

[0153] To a solution of 17.1 (1.1 g, 1.8 mmol) and TEA (1.1 mL, 7.3 mmol) in DMF (10 mL) was added thioacetic acid (410 mg, 5.4 mmol). The mixture was stirred at rt for 16 hours, diluted with water (50.0 mL) and extracted with EtOAc (3 x 40.0 mL). The residue was purified by silica chromatography (0-20% EtOAc in Hexanes) to yield 17.2 (649 mg,87%).XH NMR (400 MHz, CDCI3) 8 3.15 (brs, 4H), 2.88 (t, J= 7.3 Hz, 4H), 2.34 (s, 6H), 1.56 (m, 8H), 1.46 (s, 9H), 1.39-1.30 (m, 4H).

[0154] The following compounds were prepared by the same method:5'„S"-((( / c / -Biitoxycarbonyl)azanediyl)bis(hexane-6.1-diyl)) diethanethioate.Boc i

[0155]

[0156] From ((terLbutoxycarbonyl)azanediyl)bis(hexane-6,l-diyl) dimethanesulfonate.XH NMR (400 MHz, CDCh) 83.20-3.06 (m, 4H), 2.89-2.80 (m, 4H), 2.31 (s, 6H), 1.44 (s, 9H), 1.60-1.20 (m, 16H).5'„S"-((( / c / 7-Biitoxycarbonyl)azanediyl)bis(heptane-7.1-diyl)) diethanethioate.Boc

[0157] From ((tert-butoxycarbonyl)azanediyl)bis(heptane-7, 1 -diyl) bis(4- methylbenzenesulfonate).XH NMR (400 MHz, CDCh) 8 3.19 - 2.98 (m, 4H), 2.84 (t, J = 7.34 Hz, 4H), 2.30 (s, 6H), 1.62 - 1.11 (m, 29H).Synthesis of lipids T-01-T-08Procedure for epoxide opening with a thiolate: tert-butyl bis(5-((2-hydroxyoctyl)- thio)pentyl)carbamate (17.3).OH Boc OH17.3

[0158] To a solution of 17.2 (600 mg, 1.48 mmol) and 2-hexyloxirane (417 mg, 3.3 mmol) in EtOH (5 mL) was added NaOH (177 mg, 4.44 mmol) at rt under nitrogen. The reaction was stirred at reflux for 4 hours, cooled to rt, diluted with water (40.0 mL) and extracted with DCM (3 x 40.0 mL). The combined organics were dried (Na2SO4) and concentrated. The residue was purified by silica chromatography (0-50% EtOAc in Hexanes) to yield 17.3 (809 mg, 94%).XH NMR (400 MHz, CDCh) 8 3.65 (m, 2H), 3.16 (s, 4H), 2.75 (dd, J= 13.6, 3.4 Hz, 2H), 2.54 (t, J= 7.4 Hz, 4H), 2.45 (dd, J= 13.6, 9.0 Hz, 2H), 1.67-1.42 (m, 22H), 1.42-1.23 (m, 19H), 0.94-0.86 (m, 6H).

[0159] The following compounds were prepared by the same method: / c / 7-Butyl bis(6-((2-hydroxyoctyl)thio)hexyl)carbamate.Boc i

[0161] Obtained in 60% yield from ^^-(((tert-butoxy-carbonyhazanediyhbis^eptane- 7,1 -diyl)) di ethanethioate after silica gel column chromatography (0-50% EtOAc in hexanes).XH NMR (400 MHz, CDCh) 8 3.61 - 3.46 (m, 2H), 3.28 - 2.95 (m, 4H), 2.75- 2.53 (m, 4H), 2.50 - 2.32 (m, 4H), 1.67 - 1.07 (m, 45H), 0.88 - 0.73 (m, 10H).Procedure for esterification: (((( / er / -butoxycarbonyl)azanediyl)bis(pentane-5,l-diyl))- bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (17.4).

[0162] A solution of 17.3 (700 mg, 1.2 mmol), 3-cyclohexylpropanoic acid (374 mg, 2.4 mmol), EDCI-HC1 (687 mg, 3.6 mmol) and DMAP (146 mg, 1.2 mmol) in CH2CI2 (5 mL) was stirred at rt under nitrogen for 18 hours then concentrated. The residue was purified by silica chromatography (0-10% EtOAc in Hexanes) to yield 17.4 (910 mg, 88%). 'H NMR (400 MHz, CDCh) 84.96 (dtd, J= 8.0, 6.1, 4.4 Hz, 2H), 3.16 (s, 4H), 2.72-2.60 (m, 4H), 2.56 (t, J= 7.2 Hz, 4H), 2.41-2.27 (m, 4H), 1.77-1.49 (m, 22H), 1.47 (s, 9H), 1.44- 1.09 (m, 32H), 0.98-0.84 (m, 10H).

[0163] The following compounds were prepared by the same method:(((( / er / -ButoxycarbonyI)azanediyl)bis(hexane-6,l-diyl))bis(sulfanediyl))bis(octane- 1,2-diyl) bis(3-cyclohexylpropanoate).

[0164] Obtained in 90% yield from tert-butyl bis(6-((2-hydroxyoctyl)thio)- hexyl)carbamate after chromatography (0-10% EtOAc in hexanes). 'H NMR (400 MHz, CDCh) 54.96 (dtd, J= 8.0, 6.1, 4.4 Hz, 2H), 3.16 (s, 4H), 2.72-2.60 (m, 4H), 2.56 (t, J = 7.2 Hz, 4H), 2.41-2.27 (m, 4H), 1.77-1.49 (m, 22H), 1.47 (s, 9H), 1.45-1.07 (m, 34H), 0.98-0.84 (m, 10H).((((tert-ButoxycarbonyI)azanediyl)bis(heptane-7,l-diyl))bis(sulfanediyl))bis(octane- 1,2-diyl) bis(3-cyclohexylpropanoate).

[0165] Obtained in 63% yield from tert-butyl bis(7-((2-hydroxyoctyl)thio)- heptyl)carbamate after silica gel column chromatography (0-7% EtOAc in hexane). 'H NMR (400 MHz, CDCh) 8 5.02 - 4.82 (m, 2H), 3.20 - 2.99 (m, 4H), 2.77 - 2.44 (m, 8H), 2.30 (t, J= 7.13 Hz, 4H), 1.77 - 1.05 (m, 73H), 0.92 - 0.78 (m, 10H).12-(tert-ButoxycarbonyI)-2,2,3,3-tetramethyI-4-oxa- 19-thia- 12-aza-3-silaheptacosan- 21-yl hexanoate (20.2).Boc

[0166] NaOMe 25 wt% in MeOH (1.67 mL, 7.31 mmol, 1.3 equiv) was added dropwise to a cold (-20 °C) solution of thioester 19.3 (2.04 g, 6.74 mmol, 1.2 equiv) and tosylate 20.1 (3.37 g, 5.62 mmol, 1.0 equiv), in degassed DMF (6 mL). The mixture was stirred for 45 min, then quenched with sat. NH4CI (10 mL) and extracted with hexanes (3 x 10 mL). The combined extracts were washed with brine, dried (NfeSCL), and concentrated in vacuo. The residue was purified by silica gel column chromatography (5 % EtOAc in Hexane) to afford 20.2 (2.25 g, 3.27 mmol, 58%) as a colorless oil. 'H NMR (400 MHz, CDCh) 8 5.02 - 4.83 (m, 1H), 3.58 (t, J= 6.56 Hz, 2H), 3.12 (t, J= 7.41 Hz, 4H), 2.66 - 2.47 (m, 4H), 2.29 (t, J= 7.52 Hz, 2H), 1.77 - 1.19 (m, 43H), 0.96 - 0.78 (m, 15H), 0.03(s, 6H).12-l-((6-((tert-Butoxycarbonyl)(7-hydroxyheptyl)amino)hexyl)thio)octan-2-yl hexanoate (20.3).

[0167]

[0168] HF - pyridine (0.45 mL, 3.49 mmol, 1.2 equiv) was added dropwise to a solution of 20.2 (2.0 g, 2.91 mmol, 1.0 equiv) in CH2CI2 (5 mL) in an ice bath. The mixture was stirred for 10 min, then quenched with sat. NaHCCL (5 mL) and extracted with CH2CI2 (3 x 10 mL). The combined extracts were washed with water, dried (Na2SO4), and concentrated in vacuo. The residue was purified by silica gel column chromatography (15% EtOAc in Hexane) to afford 20.3 (1.54 g, 2.68 mmol, 92%) as a colorless oil. 'H NMR (400 MHz, CDCh) 8 5.07 - 4.78 (m, 1H), 3.64 (t, J= 6.49 Hz, 2H), 3.32 - 3.03 (m, 4H), 2.76 - 2.41 (m, 4H), 2.30 (t, J= 7.45 Hz, 2H), 1.86 - 1.04 (m, 43H), 0.88 (q, J= 7.02 Hz, 6H). l-((6-((ter / -ButoxycarbonyI)(7-(tosyloxy )heptyl)amino)hexyI)thio)octan-2-yI hexanoate (20.4).B i oc

[0169] p-Toluenesulfonyl chloride (0.75 g, 3.92 mmol, 1.5 equiv) in CH2CI2 (2.0 mL) was added to a solution of 20.3 (1.5 g, 2.61 mmol, 1.0 equiv), triethylamine (0.47 mL, 3.40 mmol, 1.3 equiv) and A A^-di meth l aminopyridine (0.032 g, 0.26 mmol, 0.1 equiv ) in CH2CI2 (3.0 mL) cooled in an ice bath. The mixture was stirred at room temperature for 4 h. The solution was diluted with more CH2CI2 (5 mL) and sequentially washed with aqueous saturated NaHCCL (2 5 mL) and water (5 mL), then dried (Na2SO4) and evaporated. The residue was purified by silica gel column chromatography (9 % EtOAc in Hexane) to afford 20.4 (1.43 g, 1.98 mmol, 75%) as a pale yellowish oil. 'H NMR (400 MHz, CDCh) 8 7.79 (d, J= 8.11 Hz, 2H), 7.34 (d, J= 7.97 Hz, 2H), 4.95 (m, 1H), 4.01 (t, J= 6.46 Hz, 2H), 3.25 - 2.90 (m, 4H), 2.71- 2.46 (m, 4H), 2.45 (s, 3H), 2.30 (t, J= 7.47 Hz, 2H), 1.93 - 1.07 (m, 43H), 0.88 (t, J= 7.07 Hz, 6H).15-(te / 7-Butoxy carbonyl)- l-cyclohexyl-5-(2-cyclohexylethyl)-3-oxo-4-oxa-7,22-dithia- 15-azatriacontan-24-yl hexanoate (20.5).

[0170] NaOMe, 25 wt% in MeOH (0.61 mL, 2.68 mmol, 1.3 equiv) was added dropwise to a cold (-20 °C) solution of thioester 19.8 (0.91 g, 2.47 mmol, 1.2 equiv) and tosylate 13.6 (1.5 g, 2.06 mmol, 1.0 equiv), in degassed DMF (5 mL). The mixture was stirred for 45 min, then quenched with sat. NH4CI (10 mL) and extracted with hexanes (3 x 10 mL). The combined extracts were washed with brine, dried (NfeSCL), and concentrated in vacuo. The residue was purified by silica gel column chromatography (4% EtOAc in Hexane) to afford 20.5 (1.2 g, 1.36 mmol, 66%) as a colorless oil. 'H NMR (400 MHz, CDCh) 8 5.04 - 4.82 (m, 2H), 3.22 - 3.00 (m, 4H), 2.75 - 2.44 (m, 8H), 2.38 - 2.22 (m, 4H), 1.81 - 1.04 (m, 67H), 0.99 - 0.77 (m, 10H). LRMS m / z 882.6 [M+H]+.Procedure for Boc group release: ((azanediylbis(pentane-5,l-diyl))bis(sulfanediyl))- bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (17.5).17.5

[0171] A solution of 17.4 (0.29 g, 0.34 mmol, 1.0 equiv) and trifluoroacetic acid (TFA) (0.13 mL, 1.7 mmol, 5.0 equiv) in CH2CI2 (3 mL) was stirred at rt for 5 hours then concentrated. The mixture was diluted with CH2CI2 (5 mL), neutralized with sat. NaHCCL and the organic phase was collected. The aqueous phase was back extracted with CH2CI2 (3 5 mL). The combined extracts were washed with water, dried (NfeSCL), and concentrated in vacuo. The residue was purified by silica gel column chromatography (5% MeOH in DCM) to afford 17.5 (0.16 g, 0.21 mmol, 62%) as a pale yellowish oil. 'H NMR (400 MHz, CDCh) 8 5.00 - 4.84 (m, 2H), 2.95 - 2.85 (m, 4H), 2.72 - 2.40 (m, 7H), 2.31 (t, J= 7.35 Hz, 4H), 1.77 - 1.09 (m, 56H), 0.98 - 0.71 (m, 10H).

[0172] The following compounds were prepared by the same method:((Azanediylbis(hexane-6,l-diyl))bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexyl- propanoate).

[0173] Obtained in 60% yield as a pale yellow oil from ((((tert-butoxycarbonyl)- azanediyl)bis(hexane-6,l-diyl))bis(sulfane-diyl))bis(octane-l,2-diyl) bis(3-cyclohexyl- propanoate) after silica gel column chromatography (5% MeOH in DCM). 'H NMR (400 MHz, CDCh) 5 5.05 - 4.77 (m, 2H), 2.98 - 2.74 (m, 5H), 2.74 - 2.39 (m, 8H), 2.31 (t, J = 6.33 Hz, 4H), 1.82 - 1.03 (m, 58H), 0.97 - 0.75 (m, 10H).((Azanediylbis(heptane-7,l-diyl))bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclo- hexylpropanoate).

[0174] Obtained in 62% yield from ((((fer / -butoxycarbonyl)azanediyl)bis-(heptane-7,l- diyl))bis-(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) as a pale yellow oil after silica gel column chromatography (0-5% MeOH in DCM). 'H NMR 8 5.02 - 4.81 (m, 2H), 3.12 (s, 1H), 2.77 - 2.39 (m, 12H), 2.35 - 2.23 (t, J= 6.23 Hz, 4H), 1.88 - 1.03 (m, 62H), 0.94 - 0.72 (m, 10H). l-Cyclohexyl-5-(2-cyclohexylethyl)-3-oxo-4-oxa-7,22-dithia-15-azatriacontan-24-yl hexanoate (20.6).

[0175] Obtained from 20.5 in 47% yield as a pale yellow oil after silica gel column chromatography (5% MeOH in DCM).XH NMR (400 MHz, CDCh) 85.04 - 4.77 (m, 2H), 2.76 - 2.36 (m, 12H), 2.36 - 2.22 (m, 4H), 2.16 (br, 1H), 1.85 - 1.00 (m, 58H), 0.99 - 0.68 (m, 10H).Procedure for reductive \-alkylation: 13-(4-((ter / -butyldimethylsilyl)oxy)butyl)-l- cyclohexyl-5-hexyl-3-oxo-4-oxa-7,19-dithia-13-azaheptacosan-21-yl 3-cyclohexyl- propanoate (18.2).

[0176] Sodium triacetoxyborohydride (0.13 g, 0.60 mmol, 1.5 equiv.) was added portion wise to a solution of 17.5 (0.3 g, 0.40 mmol, 1.0 equiv) and 4-[tert- butyl(dimethyl)silyl] oxy butanal (0.13 g, 0.60 mmol, 1.5 equiv), in DCE (3.00 mL). The mixture was stirred at room temperature under nitrogen for 18 hours. The reaction was quenched with sat. NaHCCti (5 mL) and extracted with CH2CI2 (3 x 5 mL). The combined extracts were washed with water, dried (Na2SO4), and concentrated in vacuo. The residue was purified by silica gel column chromatography (5% MeOH in DCM) to afford 18.2 (0.23 g, 0.24 mmol, 62%) as a colorless oil.XH NMR (400 MHz, CDCh) 8 5.00 - 4.83 (m, 2H), 3.64 (t, J= 6.12 Hz, 2H), 2.97 - 2.36 (m, 14H), 2.33 - 2.26 (m, 4H), 1.77 - 1.04 (m, 58H), 0.99 - 0.73 (m, 19H), 0.05 (s, 6H).

[0177] The following compounds were prepared by the same method: 14-(4-((ter / -Butyldimethylsilyl)oxy)butyl)-l-cyclohexyl-5-hexyl-3-oxo-4-oxa-7,21- dithia-14-azanonacosan-23-yI 3-cyclohexylpropanoate.

[0178] Obtained in 59% yield as a pale yellow oil from ((azanediylbis(hexane-6,l- diyl))bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexyl-propanoate) after silica gel column chromatography (0-7% MeOH in DCM).1H NMR (400 MHz, CDCh) 8 5.01 - 4.82 (m, 2H), 3.63 (t, J= 6.21 Hz, 2H), 2.69 - 2.44 (m, 14H), 2.40 - 2.18 (m, 4H), 1.91 - 1.06 (m, 62H), 1.00 - 0.69 (m, 19H), 0.04 (s, 6H).15-(4-((ter / -Butyldimethylsilyl)oxy)butyl)-l-cyclohexyl-5-hexyl-3-oxo-4-oxa-7,23- dithia-15-azahentriacontan-25-yI 3-cyclohexylpropanoate.

[0179] Obtained in 65% yield as a pale yellow oil from ((azanediylbis(heptane-7,l- diyl))bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclo-hexylpropanoate) after silica gel column chromatography (0-5% MeOH in DCM). 'H NMR (400 MHz, CDCh) 5 5.07 - 4.85 (m, 2H), 3.61 (t, J= 5.95 Hz, 2H), 2.78 - 2.23 (m, 18H), 1.89 - 1.04 (m, 66H), 1.02 - 0.71 (m, 19H), 0.04 (s, 6H).15-(4-((ter / -ButyldimethyIsilyl)oxy)butyl)-l-cyclohexyI-5-(2-cyclohexyIethyI)-3-oxo-4- oxa-7,22-dithia-15-azatriacontan-24-yl hexanoate.

[0180] Obtained as a colorless oil in 62% yield from 20.6 after purification by silica gel column chromatography (5% MeOH in DCM). 'H NMR (400 MHz, CDCh) 5 5.04 - 4.78 (m, 2H), 3.61 (t, J = 5.85 Hz, 2H), 2.80 - 2.38 (m, 14H), 2.36 - 2.25 (m, 4H), 1.89 - 1.04 (m, 64H), 1.02 - 0.71 (m, 17H), 0.04 (s, 6H).Procedure for silyl group release: ((((4-hydroxybutyl)azanediyl)bis-(pentane-5,l- diyl))bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-01).

[0181] To a solution of 18.2 (0.15 g, 0.16 mmol, 1.0 equiv.) in CH2CI2 (0.5 mL) was added HF-pyridine (0.03 mL, 0.24 mmol, 1.5 equiv.) at 0°C under nitrogen. The mixture was stirred at 0°C until completion after 15 min. The reaction mixture was quenched by sat. NaHCCL (3.0 mL) and CH2CI2 (3.0 mL). The layers were separated, and the organic phase was collected. The aqueous phase was back extracted with CH2CI2 (2 x 5 mL). The combined organic phases were dried (Na2SO4) and concentrated in vacuo. The residue was purified by silica gel column chromatography (7% MeOH in DCM) to afford T-01 (80 mg, 0.09 mmol, 60%) as a colorless oil. 'H NMR (400 MHz, CDCh) 84.99 - 4.84 (m, 2H), 3.71 (t, J= 5.32 Hz, 2H), 3.10 - 2.92 (m, 6H), 2.68 - 2.45 (m, 8H), 2.32 (t, J= 6.71 Hz, 4H), 1.92 - 1.00 (m, 58H), 0.94 - 0.67 (m, 10H). LRMS m / z 826.6 [M+H]+.

[0182] The following compounds were prepared by the same method:((((4-Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl))bis(sulfanediyl))bis(octane-l,2- diyl) bis(3-cyclo-hexylpropanoate) (T-03).

[0183] Obtained in 53% yield as a colorless oil from 14-(4-((tert- butyldimethylsilyl)oxy)butyl)-l-cyclohexyl-5-hexyl-3-oxo-4-oxa-7,21-dithia-14-azanona- cosan-23-yl 3-cyclohexylpropanoate after silica gel column chromatography (0-8% MeOH in DCM).XH NMR (400 MHz, CDCh) 5 5.00 - 4.83 (m, 2H), 3.69 (t, J= 5.22 Hz, 2H), 3.08 - 2.79 (m, 6H), 2.72 - 2.45 (m, 8H), 2.44 - 2.22 (m, 4H), 1.98 - 1.03 (m, 62H), 0.97 - 0.69 (m, 10H). LRMS m / z 854.6 [M+H]+.((((4-Hydroxybutyl)azanediyl)bis(heptane-7,l-diyl))bis(sulfanediyl))bis(octane-l,2- diyl) bis(3-cyclohexylpropanoate) (T-05).

[0184] Obtained in 60% yield as a colorless oil from 15-(4-((tert- butyldimethylsilyl)oxy)butyl)-l-cyclohexyl-5-(2-cyclohexylethyl)-3-oxo-4-oxa-7,22- dithia-15-azatriacontan-24-yl hexanoate after silica gel column chromatography (7% MeOH in DCM).XH NMR (400 MHz, CDCh) 8 5.02 - 4.82 (m, 2H), 3.71 (t, J= 5.34 Hz, 2H), 3.08 - 2.90 (m, 6H), 2.70 - 2.48 (m, 8H), 2.31 (t, J= 7.15 Hz, 4H), 1.91 - 1.80 (m, 2H), 1.79 - 1.02 (m, 64H), 1.01 - 0.70 (m, 10H). LRMS m / z 882.6 [M+H]+. l-Cyclohexyl-5-(2-cyclohexylethyl)-15-(4-hydroxybutyl)-3-oxo-4-oxa-7,22-dithia-15- azatriacontan-24-yl hexanoate (T-07).

[0185] Obtained as a colorless oil in 56% yield from 20.6 after purification by silica gel column chromatography (7% MeOH in DCM).XH NMR (400 MHz, CDCh) 8 5.02 - 4.82(m, 2H), 3.70 (t, J= 5.34 Hz, 2H), 3.08 - 2.90 (m, 6H), 2.70 - 2.48 (m, 8H), 2.38-2.25 (m, 4H), 1.91- 1.02 (m, 62H), 1.01 - 0.70 (m, 10H). LRMS m / z 854.6 [M+H]+.Procedure for nucleophilic N- alkylation: ((((2-(2-hydroxyethoxy)ethyl)azanediyl)- bis(pentane-5,l-diyl))bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropan- oate) (T-02).

[0186] To a solution of crude 17.5 (270 mg) and 2-(2-bromoethoxy)ethan-l-ol (57 mg, 0.34 mmol) in dry acetonitrile (5 mL) was added anhydrous K2CO3 (60 mg, 0.44 mmol) at rt under nitrogen. The reaction was stirred at reflux for 16 hours, cooled to rt, diluted with water (10.0 mL) and extracted with EtOAc (3 x 20.0 mL). The combined organics were dried (NfeSCL) and concentrated. The residue was purified by silica chromatography (0- 7% MeOH in DCM) to yield T-02 (110 mg, 38%).XH NMR (400 MHz, CDCh) 84.96 (m, 2H), 3.76-3.66 (m, 2H), 3.62 (t, J= 4.6 Hz, 4H), 2.75-2.40 (m, 14H), 2.39-2.27 (m, 4H), 1.77-1.44 (m, 23H), 1.42-1.05 (m, 32H), 0.90-0.86 (m, 10H).

[0187] The following compounds were prepared by the same method:((((2-(2-Hydroxyethoxy)ethyl)azanediyl)bis(hexane-6,l-diyl))bis(sulfanediyl))bis- (octane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-04).

[0188] Obtained as a colorless oil in 36% yield from ((azanediylbis(hexane-6,l- diyl))bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexyl-propanoate) after silica gel column chromatography (0-4% MeOH in DCM). 'H NMR (400 MHz, CDCh) 8 5.06 - 4.78 (m, 2H), 3.71 - 3.49 (m, 6H), 2.76 - 2.38 (m, 12H), 2.30 (t, J= 6.66 Hz, 4H), 1.88 - 0.97 (m, 57H), 0.98 - 0.60 (m, 13H). LRMS m / z 870.6 [M+H]+.((((2-(2-Hydroxyethoxy)ethyl)azanediyl)bis(heptane-7,l-diyl))bis(sulfanediyl))bis- (octane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-06).

[0189] Obtained in 47% yield as a colorless oil from ((azane-diylbis(heptane-7, 1 - diyl))bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclo-hexylpropanoate) after silica gel column chromatography 'H NMR (400 MHz, CDC13) 55.00 - 4.82 (m, 2H), 3.70 - 3.53 (m, 6H), 2.74 - 2.38 (m, 13H), 2.28 (t, J= 7.67 Hz, 4H), 1.85 - 1.02 (m, 64H), 0.99 - 0.75 (m, 10H). LRMS m / z 898.6 [M+H]+. l-Cyclohexyl-5-(2-cyclohexylethyl)-15-(2-(2-hydroxyethoxy)ethyl)-3-oxo-4-oxa-7,22- dithia-15-azatriacontan-24-yl hexanoate (T-08).

[0190] Obtained as a pale yellow oil in 30% yield from 20.6 after purification by silica gel column chromatography (6% MeOH in DCM). 'H NMR (400 MHz, CDCI3) 55.01 - 4.77 (m, 2H), 3.74 (t, J= 6.21 Hz, 2H), 3.54- 3.39 (m, 4H), 2.89 - 2.18 (m, 14H), 1.91 - 1.07 (m, 62H), 1.00 - 0.65 (m, 10H).Example 2: mRNA-containing LNPs comprising ionizable lipids of the disclosure exhibit in vivo delivery of mRNA to the liver and spleen that is superior to the nor- MC3 benchmark

[0191] LNP formulations containing 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG with a nitrogen-to-phosphorous ratio (N / P) of 6 and mRNA encoding luciferase were prepared as described in the Materials and Methods. The poly dispersity index (PDI), encapsulation efficiency and size of the LNP formulations are shown in Figure 1. The ionizable lipid of each formulation is as indicated in the figures.

[0192] The LNP formulations were subsequently tested for in vivo transfection efficiency in the liver and spleen after injection to CD-I mice. The mRNA dose was 1 mg / kg. Luminescence intensity in the liver and spleen was measured at 4 hours postinjection.

[0193] The results in Figure 2A show luminescence intensity per mg liver relative to the nor-MC3 benchmark. Results for luminescence intensity per mg spleen relative to the nor-MC3 benchmark are shown in Figure 2B.

Claims

WE CLAIM:

1. An ionizable, cationic, amino lipid or a pharmaceutically acceptable salt thereof comprising: a protonatable amino head group; two lipophilic chains, wherein the protonatable amino head group has a central nitrogen atom to which each of the two lipophilic chains are bonded; at least one of the two lipophilic chains has a structure of Formula C:Formula C wherein the wavy line represents a bond to the central nitrogen atom; wherein m and n are independently 2 to 8;E is an ester group that is -(C=O)O- or -O(C=O)-;R1is a linear, branched, monocyclic or polycyclic, optionally substituted, C3 to C20 alkyl group, C4 to C20 alkyl group, C5 to C20 alkyl group or Ce to C20 alkyl group comprising 0-2 carbon-carbon double bonds;R2is a linear, branched, optionally substituted, Ci to C10 alkyl group, C2 to C10 alkyl group, C3 to C10 alkyl group or C4 to C10 alkyl group comprising 0-2 carbon-carbon double bonds, or R2is bound to R3to form a ring structure as indicated by the dashed curved line; R3is H, or a linear, branched, monocyclic or polycyclic, optionally substituted, Ci to C10 alkyl group, comprising 0-2 carbon-carbon double bonds, or R3is bound to R2to form a ring structure as indicated by the dashed curved line;R4and R5are, independently, H, or a linear or branched, optionally substituted, Ci to C10 alkyl group, comprising 0-2 carbon-carbon double bonds, or R4and R5are bound to each other to form a ring structure; each lipophilic chain has between 15 and 40 carbon atoms in total; and wherein the ionizable, cationic, amino lipid: (i) imparts an apparent pKa of between 6 and 7.5 to a lipid nanoparticle when formulated therein; and (ii) has a ClogP of at least 11.

2. The ionizable, cationic, amino lipid or the pharmaceutically acceptable salt thereof of claim 1, wherein a second one of the two lipophilic chains bonded to the central nitrogen atom of the head group has a structure as defined by Formula D:Formula D wherein the wavy line represents a bond to the central nitrogen atom of the head group; R6and R7are, independently, H, or a linear or branched, optionally substituted, Ci to Cio alkyl group, comprising 0-2 carbon-carbon double bonds, or R6and R7are bonded to each other to form a ring structure,A is O, S or a carbonyl (C=O), andQif A is O, then R8is an acyl group, wherein the wavy line represents the bond to the A, and wherein the R’ is as defined above for R1; if A is the carbonyl (C=O), then R8is an R'— O— , wherein the wavy line represents the bond to the A, and wherein the R’ is as defined above for R1; if A is S, then R8is a group of Formula E:Formula E wherein the E of Formula E is the ester group that is -(C=O)O- or -O(C=O)-; and wherein the wavy line represents the bond to A, and wherein R9equals R1, R10equals R2, and R11equals R3.

3. The ionizable, cationic, amino lipid or the pharmaceutically acceptable salt thereof of claim 1 or 2, wherein the head group is— (CH2)p-[Z-(CH2)q]r-(CH2)t-G wherein the wavy line represents a bond to the central nitrogen atom;Z is O, S or CH2; when r is >1, Z in one or more of the Z-(CH2)qmoi eties is independently the CH2, O or S; G is OH, or NRaRbwherein Raand Rbare optionally deuterated Ci to C4 alkyl; p is 2 to 4; q is 2 to 4; r is 0 to 6; and t is 0 to 4.

4. The ionizable, cationic, amino lipid or the pharmaceutically acceptable salt thereof of claim 3, wherein the Z is S or O.

5. An ionizable, cationic, amino lipid or the pharmaceutically acceptable salt thereof having a structure of Formula A:N-(CH2Jp-[Z-(CH2Jq]r-(CH2)t-GY -X - (Cl l2) / Formula A wherein m and n are, independently, 4 to 8;R1is a linear, cyclic or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties;E is an ester group that is -(C=O)O- or -O(C=O)-;R2and R3are bonded to each other or are not bonded to each other, as indicated by the dashed line, wherein if R2and R3are bonded to each other, thenR2, R3, and a CH-CH moiety to which R2and R3are bound form a ring structure comprising from 4 to 12 C atoms, optionally comprising one or more heteroatoms, optionally comprising 1-2 C=C double bonds of E or Z configuration, and wherein the ring structure is optionally substituted with one or more Ci to C5 alkyl groups, and if R2and R3are not bonded to each other, then R2and R3are, each independently: a linear or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties;X is either O, a carbonyl (C=O) or S, wherein if X is O, then,Y is 9 , wherein the wavy line of Y is bonded to an O; andif X is the carbonyl (C=O), then Y is R3-O^, wherein the wavy line of Y isbonded to the carbon atom of the carbonyl, and wherein R3is the linear, cyclic or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties; if X is S, thenY is a group of Formula BFormula B wherein the wavy line represents the bond to the S;R4is a linear, cyclic or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties;R5and R6are bonded to each other or not bonded to each other, as indicated by the dashed line, and if R5and R6are bonded to each other, thenR5and R6form part of a ring structure comprising from 4 to 12 carbon atoms, optionally comprising one or more heteroatoms, optionally comprising 1 to 2 C=C double bonds of E or Z configuration, optionally substituted with one or more Ci to C5 alkyl groups, and if R5and R6are not bonded to each other, thenR5and R6are each, independently, a linear or branched, optionally substituted, C3 to C20 alkyl group, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising one or more cycloalkyl moieties, optionally comprising one or more heteroatoms,Z is O or S; when r is >1, Z in one or more of the Z-(CH2)qmoieties is independently the CH2, O or S;G is OH, or NRaRbwherein Raand Rbare each independently, optionally deuterated, Ci to C4 alkyl;p is 2 to 4; q is 2 to 4; r is 0 to 6; and t is 0 to 4.

6. The ionizable, cationic, amino lipid of claim 5, wherein the carbonyl (C=O) of E is bonded to the R1group.

7. The ionizable, cationic, amino lipid of any one of claims 1 to 6, having a structure of any one of compounds T-01-T-08 as defined below or a pharmaceutically acceptable salt thereof:

8. The ionizable, cationic, amino lipid or the pharmaceutically acceptable salt thereof of claim 6, wherein the one or more cycloalkyl moieties are selected from at least one cyclopentyl, cyclohexyl or cycloheptyl.

9. The ionizable, cationic, amino lipid or the pharmaceutically acceptable salt thereof of claim 6 or 8, wherein the one or more heteroatoms are selected from O, N and S.

10. A lipid nanoparticle comprising the ionizable, cationic amino lipid of any one of claims 1 to 9 and a nucleic acid.

11. The lipid nanoparticle of claim 10, comprising a helper lipid.

12. The lipid nanoparticle of claim 11, wherein the helper lipid is selected from cholesterol, a diacylglycerol, a glycerophospholipid-cholesterol conjugate, a sphingolipid and mixtures thereof.

13. A method for administering a nucleic acid to a subject in need thereof, the method comprising preparing or providing the lipid nanoparticle of claim 10, 11 or 12 comprising the nucleic acid and administering the lipid nanoparticle to the subject.

14. A method for delivering nucleic acid to a cell, the method comprising contacting the lipid nanoparticle of claim 10, 11 or 12 with the cell in vivo or in vitro.

15. Use of the ionizable, cationic, amino lipid or the pharmaceutically acceptable salt thereof of any one of claims 1 to 7 or the lipid nanoparticle of claim 10, 11 or 12 in the manufacture of a medicament to treat or prevent a disease, disorder or condition that is treatable and / or preventable by a nucleic acid.

16. Use of the ionizable, cationic, amino lipid or the pharmaceutically acceptable salt thereof of any one of claims 1 to 9 or the lipid nanoparticle of claim 10, 11 or 12 to delivera nucleic acid to a subject to treat or prevent a disease, disorder or condition that is treatable or preventable by the nucleic acid.

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