OA21603A - Lipid nanoparticles for delivery of nucleic acids, and related methods of use. - Google Patents

Abstract

The present disclosure provides for improved compositions of ionizable lipid nanoparticles for the 10 delivery of therapeutic nucleic acids to cells. Cationic ionizable lipids are engineered with improved stability to oxidative degradation while in storage, while retaining high transfection activity or potency in cells. These lipids are designed to be biodegradable, thus improving the tolerability of nanoparticles formed with them in vivo. In addition, targeting of these nanoparticles in a highly specific manner to dendritic cells is provided for through inclusion of antibody conjugates directed against cell surface 15 receptors

Description

LIPID NANOPARTICLES FOR DELIVERY OF NUCLEIC ACIDS AND METHODS OF USE THEREOF

RELATED APPLICATIONS

This patent application daims the benefit of and priority to U.S. Provisional Patent Application No. 63/118,534, fded on November 25, 2020, which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

This spécification includes a sequence listing submitted herewith, which includes the file entitled 191016-010403_ST25.txt having the following size: 7,061 bytes which was created November 24, 2021, the contents of which are incorporated by reference herein.

FIELD

The présent disclosure relates to cationic ionizable lipids and lipid nanoparticles (LNP). In some embodiments, a LNP comprising one or more cationic ionizable lipid(s) is useful for delivery of a nucleic acid compound, for dendritic cell targeting or methods of using these LNP compositions as vaccines. In some embodiments, a LNP can comprise bioreducible ionizable cationic lipids or unconjugated polyolefinic ionizable cationic lipids.

BACKGROUND

Lipid nanoparticles (LNP) are used for the delivery of therapeutic nucleic acids to cells. For example, LNP pharmaceutical compositions are employed in vaccines to deliver mRNA therapeutics. LNP formulations typically include an ionizable cationic lipid (ICL). However, it is known in the art that certain ICL compounds are undesirably sensitive to oxidation during storage. Therefore, there is a need for improved ICL compounds with improved stability to oxidative dégradation while in storage, while also providing desired transfection activity or potency in cells when incorporated in a LNP with a therapeutic agent such as a nucleic acid.

SNALP compositions are useful for delivery of nucleic acid thérapies for various infectious diseases. Infectious diseases such as tuberculosis, HIV/AIDS, malaria, and COVID-19 represent significant challenges to human health. Mycobacteria, for example, is a genus of bacteria responsible for tuberculosis (TB). According to the World Health Organization, worldwide, TB is one of the top causes of death and the leading cause of death from a single infectious agent. Despite current best efforts, there hâve been significant challenges in the development of effective vaccines for the prévention of many infectious diseases. New efforts in the identification of individual or combinations of antigenic peptides has helped improved the efficiency of vaccines. Nonetheless, significant opportunities remain in the engineering of adjuvants to help efficiently deliver and présent these antigenic sequences to professional antigen presenting cells, like dendritic cells. mRNA coding for antigenic peptides or proteins combined with ionizable cationic lipid nanoparticles represent a particularly promising strategy in the development of a vaccine. There is a need for safe and effective thérapies comprising SNALP pharmaceutical compositions for delivery of mRNA for treatment and prévention of various diseases, including vaccine compositions.

SUMMARY

In some embodiments, ionizable cationic lipids (ICLs) are provided. Cationic lipids are engineered with improved stability to oxidative dégradation while in storage, while retaining high transfection activity or potency in cells. Aspects of the disclosure are based in part on the discovery that the undesired oxidation and/or dégradation of ionizable lipids with polyene chains can be improved by including two or more methylene groups between a pair of alkynyl double bonds. Lipids disclosed herein comprise at least two carbon-carbon double bonds (olefins) spaced with at least two methylene or substituted methylene groups, wherein the substituted methylene is -C(Ri)(R2)- wherein Ri and R? are independently H, alkyl, or halogen. Lipids disclosed herein include two symmetrical polyene hydrocarbon chains each having two carbon-carbon double bonds (olefins) on either side of two, three or four methylene groups. The olefins in the lipid tails separated by at least two methylene groups renders the compounds described herein considerably less susceptible to oxidation compared to compounds separated by one methylene group, for example DLin-MC3-DMA, considered the gold standard in ionizable cationic lipid design and which was reported to hâve stability issues. In some embodiments, the compounds provided herein hâve greater than 30 %, greater than 50 %, greater than 75 %, greater than 90 %, and greater than 95 % réduction in oxidation byproducts when compared to the control LNP. In some embodiments, the compounds provided herein hâve greater than 30 %, greater than 50 %, greater than 75 %, greater than 90 %, and greater than 95 % réduction in oxidation byproducts when compared to the control LNP containing the DLin-KC2-DMA lipid.

In some embodiments, ionizable cationic lipid compositions are provided. In some aspects, the ionizable cationic lipid can comprise two polyene hydrocarbon chains, each including one or two alkenyl double bond moieties. In some aspects, the ionizable cationic lipid can comprise two polyene hydrocarbon chains, each including two or more methylene groups between two alkenyl double bond moieties. In some aspects, the ionizable cationic lipid can contain two Ci6 or Cis polyene hydrocarbon chains.

In some embodiments, liposomal compositions are provided comprising an ionizable cationic lipid having a pair of linear polyene Ci6 or Ci8 hydrocarbon chains each comprising an unsaturated linear ethylene, n-propylene or n-butylene between two adjacent unsaturated alkynyl double bonds in each polyene hydrocarbon chain. In some embodiments, a liposomal composition can comprise an ionizable lipid having a Chemical structure consisting of a pair of 16 or 18 carbon linear polyunsaturated lipid tails covalently bound to a head group comprising a dialkyl amino group having a pKa of between 6 and 7; wherein the head group comprises a heterocyclyl or alkyl portion covalently bound to the dialkyl amino group and optionally further comprising a phosphate group; and wherein each polyunsaturated lipid tail is unsaturated except for at least two olefins separated by at least two methylene groups along the length of the lipid tail, and each lipid tail optionally comprises a single acyl group at the end covalently bound to the head group. In some embodiments, each lipid tail is identical, and each lipid tail has a total of two olefins separated only by an unsubstituted ethylene, npropyl, or n-butyl. In some embodiments, each lipid tail further comprises an acyl group joined to an oxygen of the headgroup to form an ester.

In some embodiments, the dialkyl amino portion of the head group of the ionizable cationic lipid has a dialkyl amino Chemical structure of Formula (IV-A)

Rio

Formula IV-A, wherein n is 2, 3 or 4 in Formula (IV-A); and Rio and R12 in Formula (IV-A) are each independently selected from an alkyl group selected from the group consisting of: methyl, ethyl, and n-propyl, wherein the alkyl in Rio and R12 is optionally substituted with one or more hydroxyl. In some embodiments, Rio and R12 in Formula (IV-A) are each independently methyl, ethyl, (CH2)(CH₂)OH, or -(CH2)₂(CH₂)OH.

In some embodiments, an ionizable cationic lipid comprises a Chemical structure selected from

the group consisting of: R²² O

wherein

R²² is the first end of the lipid tail and ’ indicates attachment of the head group to the dialkyl amino Chemical structure of the head group. In some embodiments, an ionizable cationic lipid comprising the Chemical sub-structure of Formula (IV-A) further comprises an additional Chemical structure selected from the group consisting of:

R²²

wherein R²² is the first end of the lipid tail and ? indicates attachment of the head group to the dialkyl amino Chemical structure of the head group.

In some embodiments, an ionizable cationic lipid further comprises a pair of lipid tails attached to the head group, wherein each lipid tail comprises a hydrocarbon chain having the Chemical structure of Formula A or Formula B:

Formula A wherein a in Formula A is l, 2, 3 or 4; b is 2, 3 or 4; and c in Formula A is 3, 4, 5, 6, or7; or

Formula B wherein a in Formula B is 5, 6 or 7; and c in Formula B is 3, 4, or 5. In some embodiments, b is 4 and the sum of a, b and c in Formula A is 10, 11, 12 or 13. In some embodiments, the ionizable cationic lipid comprises a lipid tail of Formula A or Formula B at R²² in the Chemical structure shown above. In some embodiments, the ionizable cationic lipid comprises a lipid tail of Formula A, wherein b in Formula A indicates the position of attachment at R²² in the Chemical structure shown above.

In some embodiments, the ionizable cationic lipid comprises a lipid tail of Formula B, wherein in Formula B indicates the position of attachment at R²² in the Chemical structure shown above.

In some embodiments, the ionizable cationic lipid has a Chemical structure of Formula (I-A)

a is l, 2, 3, 4, 5 or 6; b is 2, 3 or 4; c is 3, 4, 5, 6, or 7; and the sum of a, b and c is 10 or 12; q is l, 2, 3 or 4; each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with one or more hydroxyl; and

, wherein v is 0 or l; q is l, 2 or 3 and q2 is 1 or

2. In some embodiments, q is 1, 2 or 3 when v is 0, and q is 1, 2, 3 or 4 when v is 1 in Formula I-A.

In some embodiments, v in Formula I-A is 0. In some embodiments, v in Formula I-A is 0 and q is 1,2 or 3. In some embodiments, v in Formula I-A is 0 and q is 1 or 2. In some embodiments, the ionizable lipid is a cationic lipid selected from the group consisting of Compounds 17-19, and 2325:

In some embodiments, the ionizable lipid is a cationic lipid selected from the group consisting of: AKG-UO-1, AKG-UO-2, AKG-UO-4, and AKG-UO-5. In some embodiments, the ionizable lipid is AKG-UO-1:

AKG-UO-1 O

In some embodiments, the ionizable lipid is AKG-UO-1 A:

In some embodiments, the ionizable lipid is AKG-UO-1B:

O

AKG-UO-18

In some embodiments, the ionizable lipid is AKG-UO-2

In some embodiments, the ionizable lipid is AKG-UO-4:

In some embodiments, the ionizable lipid is AKG-UO-4A:

In some embodiments, the ionizable lipid is AKG-UO-5:

O

AKG-UO-5

In some embodiments, the ionizable lipid is AKG-UO-6, AKG-UO-7, AKG-UO-7, AKG-UO5 8, AKG-UO-9, or AKG-UO-10:

O

O

AKG-UO-6

AKG-UO-7

AKG-UO-8

AKG-UO-9

AKG-UO-10

In some embodiments, the ionizable lipid comprises a head group that includes a methylated phosphate moiety. In some embodiments, the ionizable lipid has a Chemical structure of Formula I-A 5 wherein v is 1. In some embodiments, the ionizable lipid has a Chemical structure of Formula I-A wherein v is 1 and q is 3 or 4. In some embodiments, the ionizable lipid is selected from the group consisting of:

In some embodiments, the ionizable lipid is AKG-UO-3:

In some embodiments, the ionizable lipid has a Chemical structure of Formula II-A:

wherein a is 1, 2, 3, 4, 5 or 6; b is 2, 3 or 4; c is 4, 5, 6, 7 or 8;

Rio

q is l or 2; and each of Rio and R|₂ is independently (Ci-C₄)alkyl optionally substituted with one or more hydroxyL

In some embodiments, the ionizable lipid is selected from the group consisting of Compound 5 l-3 and Compounds 5-8:

In some embodiments, the ionizable lipid is selected from the group consisting of Compound

H

In some embodiments, the ionizable lipid has a Chemical structure of Formula II-A:

wherein a is l, 2, 3, 4, 5 or 6; b is 2, 3 or 4; c is 4, 5, 6, 7 or 8;

q’ is l or 2; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with one or more hydroxyL

In some embodiments, the ionizable lipid is a compound selected from the group consisting of Compounds 9-19:

In sonie embodiments, the ionizable lipid has a Chemical structure of Formula II-A:

wherein a is 1, 2, 3, 4, 5 or 6; b is 2, 3 or 4; c is 4, 5, 6, 7 or 8;

or 2; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with one or more hydroxyl. In some embodiments, q is l, 2 or 3 when v is 0 or q is 3 or 4 when v is l, in Formula IIA.

In some embodiments, the lipids are designed to be biodégradable, thus improving the tolerability of nanoparticles formed with them in vivo.

In some embodiments, the ionizable lipid has a Chemical structure of Formula II-B:

wherein a is 5, 6 or 7; and c is 3, 4, or 5;

q and q’ are each independently l or 2; and

Rio and R12 are each (Ci-C4)alkyl optionally substituted with hydroxyl.

In some embodiments, the ionizable cationic lipid is a compound selected from the group consisting of Compounds 29-34:

In some embodiments, the ionizable lipid is a bioreducible cationic lipid. In some embodiments, the ionizable lipid is a bioreducible cationic lipid comprising a sterol Chemical structure. In some embodiments, the ionizable lipid has a Chemical structure of Formula (VI-A):

(VI-A) or a pharmaceutically acceptable sait thereof, wherein q is 3 or 4 and R3 is

In some embodiments, the ionizable cationic lipid is selected from the group consisting of Compounds 35-38:

In some embodiments, a lipidic nanoparticle composition comprises lipids and nucleic acids, the lipidic nanoparticles comprising an ionizable lipid of Formula I, II, III, IV or combinations thereof or pharmaceutically acceptable salts thereof. In some embodiments, a lipidic nanoparticle composition comprises lipids and nucleic acids, the lipidic nanoparticles comprising an ionizable lipid of Formula I-A, II-A, II-B, IV-A, or VI-A or combinations thereof or pharmaceutically acceptable salts thereof.

In some embodiments, a lipidic nanoparticle composition comprises lipids and nucleic acids, the lipidic nanoparticles comprising an ionizable lipid comprising a polyene hydrocarbon chain of Formula A, Formula A', Formula A, or Formula B or combinations thereof or pharmaceutically acceptable salts thereof.

In some embodiments, the présent disclosure also provides for compositions of lipid nanoparticles (LNP) for the delivery of therapeutic nucleic acids to cells. Aspects of the disclosure are based in part on the discovery that LNP compositions combining various ionizable cationic lipids with certain low amounts of a phosphatidyl-L-serine below 20 mol% of the total lipid in the composition (e.g., 2.5-10 mol % of the total lipid in the composition) surprisingly demonstrated significantly enhanced targeting of encapsulated nucleic acids.

In some embodiments, the LNP compositions comprise: (a) a nucleic acid, (b) an ionizable cationic lipid, (c) a sterol (e.g., cholestérol or a cholestérol dérivative, or a phytosterol such as betasitosterol), (d) a phospholipid comprising phosphatidylserine (e.g., a mixture of phosphatidylserine and DSPC), and (e) a conjugated lipid (e.g., PEG-DMG). In one aspect, the LNP compositions comprise: (a) a nucleic acid, (b) an ionizable cationic lipid, (c) a sterol (e.g., cholestérol or a cholestérol dérivative, or a phytosterol such as beta-sitosterol); (d) phospholipids comprising a phosphatidylserine lipid in a total amount of 1-10 mol% (e.g., 2.5-10 mol%, 3-9 mol%, 5.0-7.5 mol%) of the total lipid in the composition, and additional phospholipids (e.g., DSPC), and (e) a conjugated lipid (e.g., PEGDMG). In one aspect, the LNP compositions comprise: (a) a nucleic acid, (b) an ionizable cationic lipid, (c) a sterol (e.g., cholestérol or a cholestérol dérivative, or a phytosterol such as beta-sitosterol); (d) phospholipids comprising a phosphatidylserine lipid in a total amount of l-10 mol% (e.g., 2.5-10 mol%, 3-9 mol%, 5.0-7.5 mol%) of the total lipid in the composition, and additional phospholipids (e.g., DSPC), and (e) a conjugated lipid (e.g., PEG-DMG) in a total amount of 0.5-4.5 mol% (e.g., 0.5-2.5 mol%, 1.5 mol%) of the total lipid in the composition. In one aspect, the LNP compositions comprise: (a) a nucleic acid, (b) an ionizable cationic lipid in a total amount of 40-65 mol% (e.g., 50 mol %) of the total lipid in the composition, (c) a sterol (e.g., cholestérol or a cholestérol dérivative, or a phytosterol such as beta-sitosterol) in a total amount of 25-40 mol% (e.g., 38.5 mol%) of the total lipid in the composition; (d) phospholipids comprising a phosphatidylserine lipid in a total amount of l-ΊΟ mol% (e.g., 2.5-10 mol%, 3-9 mol%, 5.0-7.5 mol%) of the total lipid in the composition, and additional phospholipids (e.g., DSPC), and (e) a conjugated lipid (e.g., PEG-DMG) in a total amount of 0.5-4.5 mol% (e.g., 0.5-2.5 mol%, 1.5 mol%) of the total lipid in the composition. In one aspect, the LNP compositions comprise: (a) a nucleic acid, (b) an ionizable cationic lipid in a total amount of 40-65 mol% (e.g., 50 mol %) of the total lipid in the composition, (c) a sterol (e.g., cholestérol or a cholestérol dérivative, or a phytosterol such as beta-sitosterol) in a total amount of 25-40 mol% (e.g., 38.5 mol%) of the total lipid in the composition; (d) phospholipids in a total amount of 5-25 mol% of the total lipid in the composition, wherein the phospholipids comprise a phosphatidylserine lipid in a total amount of l-ΊΟ mol% (e.g., 2.5-10 mol%, 3-9 mol%, 5.0-7.5 mol%) of the total lipid in the composition, and additional phospholipids (e.g., DSPC) (e.g., 10 mol% of the total lipid in the composition), and (e) a conjugated lipid (e.g., PEG-DMG) in a total amount of 0.5-4.5 mol% (e.g., 0.5-2.5 mol%, 1.5 mol%) of the total lipid in the composition.

In one aspect, the LNP compositions comprise: (a) a nucleic acid, (b) a ionizable cationic lipid having a pair of linear polyene Ci6 or Cis hydrocarbon chains each comprising an unsaturated linear ethylene, n-propylene or n-butylene between two adjacent unsaturated alkynyl double bonds in each polyene hydrocarbon chain, the ionizable cationic lipid présent in the composition in a total amount of 40-65 mol% of the total lipid in the composition, (c) a sterol (e.g., cholestérol) in a total amount of 25-40 mol% of the total lipid in the composition; (d) phospholipids in a total amount of 5-25 mol% of the total lipid in the composition, the phospholipids including a phosphatidylserine lipid (e.g., phosphatidyl-L-serine lipid) in a total amount of l -10 mol% of the total lipid in the composition, and additional phospholipids (e.g., DSPC in a total amount of 10 mol% of the total lipid in the composition), and (e) a conjugated lipid (e.g., PEG-DMG) in a total amount of 0.5-2.5 mol% of the total lipid in the composition. In one aspect, the LNP compositions comprise: (a) a mRNA nucleic acid, (b) an ionizable cationic of Formula (I-A), Formula (II-A), or Formula (II-B) when v is 0 in a total amount of 40-65 mol% of the total lipid in the composition, (c) cholestérol in a total amount of 25-40 mol% of the total lipid in the composition; (d) a L-serine phosphatidylserine lipid (e.g., DPPS or DSPS) in a total amount of l-10 mol% (e.g., 2.5-10 mol%, 3-9 mol%, 5.0-7.5 mol%) of the total lipid in the composition, and DSPC in a total amount of 5-25 mol% of the total lipid in the composition, and (e) a conjugated lipid (e.g., PEG-DMG) in a total amount of 0.5-2.5 mol% of the total lipid in the composition. In one aspect, the LNP compositions comprise: (a) a mRNA nucleic acid, (b) an ionizable cationic of Formula (I-A) when v is 0 in a total amount of 40-65 mol% of the total lipid in the composition, (c) cholestérol in a total amount of 25-40 mol% of the total lipid in the composition; (d) a L-serine phosphatidylserine lipid (e.g., DPPS or DSPS) in a total amount of 3-9 mol% of the total lipid in the composition, and DSPC in a total amount of 5-25 mol% of the total lipid in the composition, and (e) a conjugated lipid (e.g., PEG-DMG) in a total amount of 0.5-2.5 mol% of the total lipid in the composition.

In some embodiments, a composition comprises : (a) a polyunsaturated ionizable cationic lipid; and (b) a charged phospholipid phosphatidylserine lipid

In some embodiments, a composition comprises : (a) an ionizable cationic lipid of Formula IV-A; and (b) an anionic phospholipid targeting moiety selected from the group consisting of: DSPS (L-isomer), DPPS (L-isomer), DMPS (L-isomer), DOPS (L-isomer), DSPS (D-isomer), DSPG, DPPG, N-Glu-DSPE, and N-Suc-DSPE. In some embodiments, a composition comprises: (a) an ionizable cationic lipid of Formula IV-A; and (b) an anionic phospholipid targeting moiety of Formula V-A. In some embodiments, a composition comprises : (a) an ionizable cationic lipid of Formula IVA; and (b) an anionic phospholipid targeting moiety selected from the group consisting of: DSPS (Lisomer), and DPPS (L-isomer).

In some embodiments, a composition comprises: (a) an ionizable cationic lipid of Formula IV; and (b) an anionic phospholipid targeting moiety of Formula V-A. In some embodiments, a composition comprises: (a) an ionizable cationic lipid of Formula IV; and (b) an anionic phospholipid targeting moiety selected from the group consisting of: DSPS (L-isomer), DPPS (L-isomer), DMPS (L-isomer), DOPS (L-isomer), DSPS (D-isomer), DSPG, DPPG, N-Glu-DSPE, and N-Suc-DSPE. In some embodiments, a composition comprises: (a) an ionizable cationic lipid of Formula IV; and (b) an anionic phospholipid targeting moiety of Formula V-A. In some embodiments, a composition comprises: (a) an ionizable cationic lipid of Formula IV-A; and (b) an anionic phospholipid targeting moiety selected from the group consisting of: DSPS (L-isomer), and DPPS (L-isomer).

In one aspect, the LNP compositions comprise: (a) an mRNA nucleic acid, (b) an ionizable cationic lipid selected from the group consisting of AKG-KC2-OA, AKG-KC3-OA, Dlin-KC2-DMA and Dlin-KC3-DMA in a total amount of 40-65 mol% of the total lipid in the composition, (c) cholestérol (or a dérivative thereof) in a total amount of 25-40 mol% of the total lipid in the composition; (d) a mixture of two or more phospholipids in a total amount of 5-25 mol% of the total lipid in the composition, the phospholipids comprising L-serine phosphatidylserine lipid (e.g., DPPS or DSPS) in a total amount of 3-9 mol% (e.g., 5.0-7.5 mol%) of the total lipid in the composition, and (e) a conjugated lipid (e.g., PEG-DMG) in a total amount of 0.5-2.5 mol% of the total lipid in the composition.

AKG-KC2-0A

AKG-KC3-0A

Dlin-KC2-DMA

Dlin-KC3-DMA

In one aspect, the nucleic acid lipid nanoparticle (LNP) composition comprises: a nucleic acid, the ionizable cationic lipid AKG-UO-1, and a (L-Serine) PS lipid in a total amount of 2.5-10 mol% 5 of the total lipid content of the LNP composition. In some embodiments, the nucleic acid is mRNA, the PS lipid is (L-Serine) DSPS, (L-Serine) DPPS, or a mixture thereof, and the LNP composition further comprises cholestérol and a second phospholipid selected from the group consisting of: DSPC, DPPC and DOPC. The the LNP composition further comprises 0.5-1.5 mol% PEG-DMG or PEGDSG, based on the total lipid content in the LNP composition.

In one aspect, the nucleic acid lipid nanoparticle (LNP) composition comprises: a nucleic acid, an ionizable cationic lipid selected from KC2OA, KC2, KC2-01, ALC-0315, and SM 102; and a (LSerine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition. In some embodiments, the LNP composition has a N/P ratio 3 to 8 (e.g. ratio of 5-7 or 5).

In one aspect, the nucleic acid lipid nanoparticle (LNP) composition comprises: a nucleic acid, 15 an ionizable cationic lipid selected from AKG-UO-6 and AKG-UO-7; and a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition. In some embodiments, the N/P ratio 3 to 8 (e.g. ratio of 5-7 or 5 or 7).

In one aspect, the nucleic acid lipid nanoparticle (LNP) vaccine composition comprises: a mRNA nucleic acid with a N/P ratio of 3 to 8; an ALC-0315 ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition; cholestérol in a total amount of 2540 mol% of the total lipid content of the LNP composition; a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition; DSPC phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition; and PEG-DMG in a total amount of 0-2.5 mol% of the total lipid content of the LNP composition.

In one aspect, the nucleic acid lipid nanoparticle (LNP) vaccine composition comprises: a mRNA nucleic acid with a N/P ratio of 3 to 8; a Dlin-KC2-DMA ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition; cholestérol in a total amount of 25-40 mol% of the total lipid content of the LNP composition; a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition; DSPC phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition; and PEG-DMG in a total amount of 0-2.5 mol% of the total lipid content of the LNP composition.

In one aspect, the lipid nanoparticle (LNP) vaccine composition comprises: a mRNA nucleic acid with a N/P ratio of 3 to 8; a KC3-OA ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition; cholestérol in a total amount of 25-40 mol% of the total lipid content of the LNP composition; a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition; DSPC phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition; and PEG-DMG in a total amount of 0-2.5 mol% of the total lipid content of the LNP composition.

In one aspect, the nucleic acid lipid nanoparticle (LNP) vaccine composition comprises: a mRNA nucleic acid with a N/P ratio of 3 to 8; an ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition; cholestérol in a total amount of 25-40 mol% of the total lipid content of the LNP composition; a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition; and DSPC phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition; and PEG-DMG in a total amount of 02.5 mol% of the total lipid content of the LNP composition.

One aspect of the disclosure relates to the use of a (L-Serine) PS lipid in a LNP in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition for targeting of the LNP to dendritic cells. In some embodiments, the LNP comprises mRNA. In some embodiments, the LNP further comprises cholestérol. In some embodiments, the LNP further comprises ICL. In some embodiments, the LNP further comprises one or more additional phospholipids including DSPC. In some embodiments, the LNP further comprises a conjugated lipid. In some embodiments, the LNP comprises a mRNA nucleic acid with a N/P ratio of 3 to 8; an ionizable cationic lipid (ICL) in a total amount of 40-65 mol% of the total lipid content of the LNP composition; cholestérol in a total amount of 25-40 mol% of the total lipid content of the LNP composition; a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition; DSPC phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition; and a conjugated lipid in a total amount of 0-2.5 mol% of the total lipid content of the LNP composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of the oxidative dégradation mechanisms of lipid esters of linoleic acid containing conjugated multiple unsaturations that are particularly sensitive to oxidation.

FIG. 2 shows the reaction of the reduced c-terminal cysteine of a Fab’ antibody fragment with a maleimide terminated-poly(ethylene glycol) 2000 derivatized distearoylphosphatidylethanolamine. RI and R2 are stearic acid. The final antibody lipopolymer conjugale is an intermediate that is subsequently inserted into the outer lipid layer of lipidic nanoparticle to make it actively targeted.

FIG. 3A. Impact of DSPS inclusion from 0-2.5 mol % on transfection efficiency of dendritic cells (MutuDC1940) using mCherry mRNA LNPs formulated with DLin-KC2-DMA as the ionizable cationic lipid. ICL was kept at 50 mol%, cholestérol at 38.5 mol%, PEG-DMG at 1.5 mol% and the DSPS content varied. Inclusion of DSPS was made by reducing the DSPC content by the same mol% of DSPS that was added. Cells were incubated with each formulation at a concentration of 1 ug mRNA/mL for 24 h. UT sample corresponds to cells where no LNPs were added. Lipofect refers to Lipofectamine treated sample.

FIG. 3B. Impact of DSPS inclusion from 0-7.5 mol % on transfection efficiency of dendritic cells (MutuDC1940) using mCherry mRNA LNPs formulated with DLin-KC2-DMA as the ionizable cationic lipid. ICL was kept at 50 mol%, cholestérol at 38.5 mol%, PEG-DMG at 1.5 mol% and the DSPS content varied. Inclusion of DSPS was made by reducing the DSPC content by the same mol% of DSPS that was added. Cells were incubated with each formulation at a concentration of 1 ug mRNA/mL for 24 h. UT sample corresponds to cells where no LNPs were added. Lipofect refers to Lipofectamine treated sample.

FIG. 3C. Impact of DSPS inclusion from 0-7.5 mol % on transfection efficiency of dendritic cells (MutuDC 1940) using mCherry mRNA LNPs formulated with DLin-KC2-DMA as the ionizable cationic lipid. ICL was kept at 50 mol%, cholestérol at 38.5 mol%, PEG-DMG at 1.5 mol% and the 23

DSPS content varied. Inclusion of DSPS was made by reducing the DSPC content by the same mol% of DSPS that was added. Cells were incubated with each formulation at a concentration of 0.3 ug mRNA/mL for 24 h. UT sample corresponds to where no LNPs were added.

FIG. 3D. Impact of DSPS inclusion from 0-7.5 mol % on transfection efficiency of dendritic cells (MutuDCl940) using mCherry mRNA LNPs formulated with DLin-KC2-DMA as the ionizable cationic lipid. ICL was kept at 50 mol%, cholestérol at 38.5 mol%, PEG-DMG at 1.5 mol% and the DSPS content varied. Inclusion of DSPS was made by reducing the DSPC content by the same mol% of DSPS that was added Cells were incubated with each formulation at a concentration of 0.1 ug mRNA/mL for 24 h. UT sample corresponds to cells where no LNPs were added.

FIG. 4. Transfection of murine dendritic cells (MutuDCl940) using LNPs containing various ICLs (KC2, KC2-OA, KC3-OA, and SM-102) and 5 mol % DSPS, and comparison to LNPs using GIu-DSPE or Suc-DSPE rather than DSPS. UT sample corresponds to cells where no LNPs were added.

FIG. 5. DSPS or DPPS increase mCherry LNP transfection with KC2, KC2-01, KC2-PA, KC3-01, and KC3-OA comprising ICLs. UT sample corresponds to cells where no LNPs were added.

FIG. 6A. Comparison of various Chemical forms of phosphatidylserine with AKG-UO-l containing LNPs in transfecting murine dendritic cells. UT sample corresponds to cells where no LNPs were added. Lipo refers to Lipofectamine MessengerMax (ThermoFisher) used according to manufacturées instructions at the same dosage level as the LNPs.

FIG. 6B. Comparison of DSPS to other negatively charged phospholipids in transfecting murine dendritic cells using AKG-UOl containing LNPs. UT sample corresponds to cells where no LNPs were added. Lipo refers to Lipofectamine MessengerMax (ThermoFisher) used according to manufacturer’s instructions at the same dosage level as the LNPs.

FIG. 7. Impact of DSPS concentration in AUG-UO-1 containing LNPs on transfection of dendritic cells. UT sample corresponds to cells where no LNPs were added.

FIG. 8. Impact of PEG-DMG concentration in AUG-UO-1 containing LNPs with and without 5 mol % DSPS on transfection of dendritic cells. The Y-axis shows the % PEG used in the composition followed by the concentration of mRNA added to the cells (0.11, 0.33, or l pg/mL). UT sample corresponds to cells where no LNPs were added.

FIG. 9A. Oxidative dégradation of lipid suspensions of ICLs with a single methylene between two olefins (KC2, KC3, and O-l 1769) and those with four methylenes between the two olefins (KC201, KC3-01, and UO-l).

FIG. 9B. Oxidative dégradation of liposomes containing O-H 769, an ICL with a single methylene between two olefins liposomes containing UO-1, an ICL with four methylenes between the two olefins.

FIG. 10A. Effect of N/P on mCherry expression of KC2-01 containing LNPs at l pg/ml in murine dendritic cells. LIT sample corresponds to cells where no LNPs were added.

FIG. 10B. Effect ofN/P on mCherry expression of KC2-01 containing LNPs at 0.33 pg/ml in murine dendritic cells. UT sample corresponds to cells where no LNPs were added.

FIG. 11. Transfection efficiency of LNPs with and without DSPS (7.5 mol %) and containing different ionizable cationic lipids. UT sample corresponds to cells where no LNPs were added.

FIG. 12. Transfection efficiency of LNP formulations containing various concentrations of DOPS (0, 10, and 25 mol % as % of total lipid) and mCherry mRNA in murine dendritic cells.

FIG. 13A. mRNA sequence of VRN-029, a SARS-COV2 spike protein generating sequence.

FIG. 13B. The effect of PEG-DMG (C14) concentration (mol %) on LNP vaccine immunogenicity. Total anti-spike antibody titers and CD4 responses from mice immunized with mRNA-LNPs using 7.5% DSPS and the ionizable lipid UO1 with increasing mol% of PEG-DMG. The middle graph shows day 34 endpoint antibody titers. The right graph shows the corresponding CD4 T cell responses.

FIG. 13C. The effect of PEG-DPPE (Cl6) concentration (mol %) on LNP vaccine immunogenicity. Total anti-spike antibody titers from mice immunized with mRNA-LNPs using 7.5% DSPS and the ionizable lipid UO1 with increasing mol% of PEG-DPPE. The middle graph shows day 34 endpoint antibody titers. The mol% of PEG-DPPE inversely impacted antibody levels. The right graphs shows the corresponding CD4 T cell responses.

FIG. 13D. Total anti-spike antibody titers and CD4 responses from mice immunized with mRNA-LNPs using 7.5% DSPS and the ionizable lipid KC2OA with either 1.5 mol% PEG-DMG (14C) or PEG-DSG ( 18C). The left graph shows day 34 endpoint antibody titers. The right graph shows the corresponding CD4 T cell responses.

FIG. 13E. Total anti-spike antibody titers and CD4 responses from mice immunized with mRNA-LNPs using 7.5% DSPS and the ionizable lipid UO1 with either 1.5 mol% PEG-DMG (14C) or PEG-DSG ( 18C). The left graph shows day 34 endpoint antibody titers. The right graph shows the corresponding CD4 T cell responses.

FIG. 13F. Effect of phosphatidylserine incorporation in mRNA-LNP immunogenicity. Total anti-spike antibody titers (A) and spike-specific CD4 T cell responses from mice immunized with 25 mRNA-LNPs using various ionizable lipids and PEG-lipids plus/minus 7.5 mol% DSPS Antibody data were were log-transformed and analyzed using two-way ANOVA with a Sidak’s multiple comparison test. CD4 T cell data were analyzed using a REML mixed-effects model with a Sidak’s multiple comparison test.

FIG. 13G. Effect of phosphatidylserine lipid tail (DPPS vs DSPS) composition on mRNALNP priming of B (Panel A) and T cell (Panel B) responses. Antibody data were log-transformed prior to analysis. Data were analyzed using one-way ANOVA with a Tukey’s multiple comparison test.

FIG. 14A. Comparison of the mCherry expression of KC2-01 LNPs, 7.5 mol% DSPS (D isomer) and DSPS (L isomer) at l pg/mL mRNA for 24h.

FIG. 14B. Comparison of the mCherry expression of KC2-01 LNPs, 7.5 mol% DSPS (D isomer) and DSPS (L isomer) at 0.33 pg/mL mRNA for 24h.

FIG. 15. Comparison of the mCherry expression of KC2 LNPs, with 5 and 7.5 mol% DSPS (L-isomer) to LNPs prepared with SM-102 or ALC-0315 at 1 pg/mL mRNA for 24h. The Y-axis is mean fluorescence intensity (MFI). UT sample corresponds to cells where no LNPs were added.

FIG. 16. Comparison of the mCherry expression of UO1, UO6 and UO7 formulations alone, or with added 7.5mol% D-isomer of DSPS, at 1 pg/mL mRNA for 24 h. UT sample corresponds to cells where no LNPs were added.

FIG. 17. Comparison of the mCherry expression of UO1, SM 102, ALC-0315 formulations alone, or with added DSPS, at 1 pg/mL mRNA for 24h. Lipo refers to Lipofectamine MessengerMax (ThermoFisher) used according to manufacturer’s instructions at the same dosage level as the LNPs. UT sample corresponds to cells where no LNPs were added.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the compositions and methods of the présent disclosure.

Stabilized Nucleic Acid Lipid Particles (SNALP) are used as a vehicle for the systemic delivery of mRNA or other nucleic acid therapeutics. SNALP compositions include cationic lipids such as MC3 or KC2, comprising a protonatable tertiary amine head group joined to a pair of linear 18 carbon aliphatic chains containing a pair of carbon-carbon double bonds separated by a single methylene group (e.g., linoleic acid). However, while the structure of these hydrocarbon chains, each containing a pair of double bonds separated by a single methylene group, imparts désirable biological properties to the SNALP compositions, this Chemical sub-structure also results in the undesired problem of increased sensitivity of the compound to oxidative dégradation. For example, FIG. 1 is a depiction of the oxidative dégradation mechanisms of lipid esters of linoleic acid containing conjugated multiple unsaturations that are particularly sensitive to oxidation. What is needed are novel cationic lipids suitable for use in a SNALP composition, but having enhanced résistance to oxidative dégradation.

Disclosed herein are compounds, compositions and methods related to the treatment of bacterial infections. As used herein, the term “compound”, “drug” and “active agent” are used interchangeably. Some aspects of the disclosure relate to novel ionizable lipids or bioreducible ionizable lipids. These lipids are cationic (i.e. positively charged) at acidic pH, such as encountered intracellularly following endocytosis or phagocytosis by a cell. The same lipids, and compositions containing them, are near neutral in charge when présent at pH 7.4. These lipids may also hâve multiple olefins that are separated by at least two methylene groups présent in their alkyl or acyl groups.

Some aspects of the disclosure relate to the process for the synthesis of the novel ionizable lipids.

Other aspects relate to compositions comprising lipidic nanoparticles comprising ionizable cationic lipid, the lipidic nanoparticles containing nucleic acids. In some embodiments, nucleic acids are encapsulated into the lipidic nanoparticles.

Other aspects of the disclosure relate to the use of these ionizable lipids or lipidic nanoparticles compositions comprising ionizable lipids in vaccines for the prévention of infectious diseases or cancer. In some embodiments, the infectious disease can be a bacterial or a viral infection. In some embodiments, the compositions described herein can be used to prevent infections related to tuberculosis, HIV/AIDS, malaria, or coronavirus-related infections such as COVID-19. In other embodiments, the infection is influenza, hepatitis B, hepatitis C, Dengue, human papillomavirus (HPV), norovirus, mumps, measles, Meningococcal disease, pneuomococcal disease, polio, rotovirus, respiratory syncytial virus (RSV), rubella, shingles/herpes zoster virus, tetanus, or whooping cough.

In some embodiments, the compounds and compositions described herein may promote efficient uptake and transfection of target cells, including tissue macrophages and dendritic cells. The efficient delivery nucleic acids coding for antigen spécifie for infectious viruses or bacteria, and subséquent présentation of that antigen to elicit the desired immune response to protect against corresponding infections is a resuit. In some embodiments, the nucleic acid can be a synthetic nucleic 27 acid (e.g., engineered codon optimized mRNA) encoding an epitope of coronavirus such as SARSCoV, MERS-CoV or SARS-CoV-2. In some embodiments the nucleic acid can be a synthetic nucleic acid (e.g. engineered codon optimized mRNA) encoding the S-protein (spike protein) or a fragment thereof of coronavirus such as SARS-CoV, MERS-CoV or SARS-CoV-2.

Définitions

For convenience, certain terms employed in the spécification, examples, and appended daims are collected here. Unless defined otherwise, ail technical and scientific tenus used herein hâve the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

As used herein, the following terms and phrases are intended to hâve the following meanings: The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

As used herein the tenu comprising or comprises is used in reference to compositions, methods, and respective component(s) thereof, that are présent in a given embodiment, yet open to the inclusion of unspecified éléments.

As used herein the tenu consisting essentially of ' refers to those éléments required for a given embodiment. The tenu permits the presence of additional éléments that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the disclosure.

The term consisting of ' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

The term “comprising” when used in the spécification includes “consisting of ’ and consisting essentially of.

If it is referred to “as mentioned above” or “mentioned above”, “supra” within the description it is referred to any of the disclosures made within the spécification in any of the preceding pages.

If it is referred to “as mentioned herein”, “described herein”, “provided herein,” or “as mentioned in the présent text,” or “stated herein” within the description it is referred to any of the disclosures made within the spécification in any of the preceding or subséquent pages.

As used herein, the term “about” means acceptable variations within 20%, within 10% and within 5% of the stated value. In certain embodiments, about can mean a variation of +/-1%, 2%,

3%, 4%, 5%, 10% or 20%.

The term effective amount as used herein with respect to a compound or the composition means the amount of active compound (also referred herein as active agent or drug) sufficient to cause a bactericidal or bacteriostatic effect. In one embodiment, the effective amount is a therapeutically effective amount meaning the amount of active compound that is sufficient alleviate the symptoms of the bacterial infection being treated.

The term subject (or, altematively, patient) as used herein refers to an animal, preferably a mammal, most preferably a human that receives either prophylactic or therapeutic treatment.

The term “administration” or “administering” as used herein includes ail means of introducing the compounds or the pharmaceutical compositions to the subject in need thereof, including but not limited to, oral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal and the like. Administration of the compound or the composition is suitably parentéral. For example, the compounds or the composition can be preferentially administered intravenously, but can also be administered intraperitoneally or via inhalation like is currently used in the clinic for liposomal amikacin in the treatment of mycobacterium avium (see Shirley et al., Amikacin Liposome Inhalation Suspension: A Review in Mycobacterium avium Complex Lung Disease. Drugs. 2019 Apr; 79(5):555-562)

The terms “treat,” “treating,” and “treatment,” as used herein, refer to therapeutic or preventative measures such as those described herein.

The term “pharmaceutically acceptable sait refers to a relatively non-toxic, inorganic or organic acid addition sait of a compound of the présent disclosure which sait possesses the desired pharmacological activity.

The term alkyl means saturated carbon chains having from one to twenty carbon atoms which may be linear or branched or combinations thereof, unless the carbon chain is defined otherwise. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tertbutyl, pentyl, hexyl, heptyl, octyl, and the like. Unless stated otherwise specifically in the spécification, an alkyl group is optionally substituted.

The term “phosphatidylserine”, with any of it’s acyl chain compositions, refers to the Z,-isomer of serine in the headgroup unless specified in a particular example.

The term “lipid conjugate” refers to a conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, polysarcosine (see e.g. WO2021191265A1 which is herein incorporated by reference in its entirety for ail purposes), 29 polyamide oligomers (e.g., ATTA-lipid conjugales), PEG-lipid conjugales, such as PEG coupled to dialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled to cholestérol, PEG coupled to phosphatidylethanolamines, PEG conjugated to ceramides (see, e.g., U.S. Pat. No. 5,885,613, the disclosure of which is herein incorporated by reference in its entirety for ail purposes), cationic PEG lipids, and mixtures thereof. PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties. In preferred embodiments, non-ester containing linker moieties are used.

The abbreviations for the ionizable cationic lipids may be truncated in the Examples from that used in the Tables, For example, AKG-UO-1 or AKG-K.C2-01 may be referred to as UO1 or KC201.

The abbreviation UT used in various studies refers to untreated samples.

The term ”lipidic nanoparticle”, or “LNP”, refers to particles having a diameter of from about 5 to 500 nm. In some embodiments, the lipid nanoparticle comprises one or more active agents. In some embodiments, the lipid nanoparticle comprises a nucleic acid. In some embodiments, the nucleic acid is condensed in the interior of the nanoparticle with a cationic lipid, polymer, or polyvalent small molécule and an extemal lipid coat that interacts with the biological milieu. Due to the répulsive forces between phosphate groups, nucleic acids are naturally stiff polymers and prefer elongated configurations. In the cell, to cope with volume constraints DNA can pack itself in the appropriate solution conditions with the help of ions and other molécules. Usually, DNA condensation is defined as the collapse of extended DNA chains into compact, orderly particles containing only one or a few molécules. By binding to phosphate groups, cationic lipidic can condense DNA by neutralizing the phosphate charges and allow close packing.

In some embodiments, the active agent is encapsulated into the LNP. In some embodiments, the active agent can be an anionic compounds, for example, but not limited to DNA, RNA, natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA and small interfering RNA), nucleoprotein, peptide, nucleic acid, ribozyme, DNA-containing nucleoprotein, such as an intact or partially deproteinated viral particles (virions), oligomeric and polymeric anionic compounds other than DNA (for example, acid polysaccharides and glycoproteins)). In some embodiments, the active agent can be intermixed with an adjuvant.

In a LNP vaccine product, the active agent is generally contained in the interior of the LNP. In some embodiments, the active agent comprises a nucleic acid. Typically, water soluble nucleic acids are condensed with cationic lipids or polycationic polymers in the interior of the particle and the surface of the particle is enriched in neutral lipids or PEG-lipid dérivatives. Additional ionizable cationic lipid may also be at the surface and respond to acidification in the environment by becoming positively charged, facilitating endosomal escape.

Ionizable lipids can hâve different properties or functions with respect to LNPs. Due to the pKa of the amino group, the lipid molécules can become positively charged in acidic conditions. Under these conditions, lipid molécules can electrostatically bind to the phosphate groups of the nucleic acid which allows the formation of LNPs and the entrapment of the nucleic acid. In some embodiments, the pKa can be low enough that it renders the LNP substantially neutral in surface charge in biological fluids, such as blood, which are at physiological pH values. High LNP surface charge is associated with toxicity, rapid clearance from the circulation by the fixed and free macrophages, hemolytic toxicities, including immune activation (Filion et al Biochim Biophys Acta. 1997 Oct 23; 1329(2):345-56).

In some embodiments, pKa can be high enough that the ionizable cationic lipid can adopt a positively charged form at acidic endosomal pH values. This way, the cationic lipids can combine with endogenous endosomal anionic lipids to promote membrane lytic nonbilayer structures such as the hexagonal HI1 phase, resulting in more efficient intracellular delivery. In some embodiments, the pKa ranges between 6.2-6.5. For example, the pKa can be about 6.2, about 6.3, about 6.4, about 6.5. Unsaturated tails also contribute to the lipids’ ability to adopt nonbilayer structures. (Jayaraman et al., Angew Chem Int Ed Engl. 2012 Aug 20;51(34):8529-33).

Release of nucleic acids from LNP formulations, among other characteristics such as liposomal clearance and circulation half-life, can be modified by the presence of polyethylene glycol and/or sterols (e.g. cholestérol) or other potential additives in the LNP, as well as the overall Chemical structure, including pKa of any ionizable cationic lipid included as part of the formulation.

The term “bioreducible” refers to compounds that undergo accelerated dégradation due to the cleavage of disulfide linkages in a reductive environment. Unlike other nucleic acid therapeutics such as siRNA, the success of mRNA-based thérapies dépends on the availability of a safe and efficient delivery vehicle that encapsulâtes the mRNA. mRNA is fragile and needs a protective coating for it to remain active until it reaches its target site. mRNA containing LNPs are a promising vaccine option for Covd-19 immunity (Jackson et aL, Preliminary Report. N Engl J Med. 2020 Nov 12;383(20): 19201931). The efficiency and tolerability of LNPs has been attributed to the amino lipid and unlike many biomaterial applications that may hâve a required service lifetime of weeks or months, functional LNP mediated delivery of mRNA occurs within hours obviating the need for persistent lipids. Indeed in applications where chronic dosing is required this will be especially important. It has been demonstrated that LNPs enter cells via endocytosis and accumulate in endolysosomal compartments. The ionizable cationic lipid (ICL) is able to effectively deliver mRNA to the cytosol after endocytosis while being susceptible to enzymatic hydrolysis in late endosomes/Iysosomes by lipases or hydrolysis triggered by the reductive environment of the lysosome allowing complété biodégradation. The extracellular space is a relatively oxidative environment, while the intracellular space is a reductive one, allowing a disulfide linked molécule to remain intact in the extracellular space but be rapidly reduced once intemalized (Huang et aL, Mol Ther. 2005 Mar; 11(3):409-17, 2005). Some embodiments, provide bioreducible disulfide linked ICL molécules (see compounds 29-36, Table 2) that are stable in LNP formulation and while in circulation but undergo cleavage in the reductive environment of the lysosome. Such compounds and compositions can facilitate rapid biological destruction of the lipids and can prevent potentially toxic accumulation of ICL lipids (as observed in rats with DLin-MC3-DMA (Sabins et al., Mol Ther. 2018 Jun 6;26(6): 1509-1519).

The terms “encapsulation” and “entrapped,” as used herein, refer to the incorporation or association of the mRNA, DNA, siRNA or other nucleic acid pharmaceutical agent in or with a lipidic nanoparticle. As used herein, the term “encapsulated” refers to complété encapsulation or partial encapsulation. A siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA. A siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest.

The term “mol% with regard to cholestérol refers to the molar amount of cholestérol relative to the sum of the molar amounts of cholestérol and non-PEGylated phospholipid expressed in percentage points. For example, “55 moL% cholestérol” in a liposome containing cholestérol and HSPC refers to the composition of 55 mol. parts of cholestérol per 45 mol. parts of HSPC.

The term “mol% with regard to PEG-lipid refers to the ratio of the molar amount of PEGlipid and non-PEGylated phospholipid expressed in percentage points. For example, “5 mol.% PEGDSPE” in a LNP containing HSPC and PEG-DSPE refers to the composition having 5 mol. parts of PEG-DSPE per 100 mol. parts of HSPC.

As used herein, the term “pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, 32 dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonie agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animais.

Various aspects and embodiments are described in further detail in the following subsections.

Compounds

Provided herein are compounds, compositions and methods for the treatment or prévention of infectious diseases, including tuberculosis. According to aspects of the disclosure, the cationic lipids comprise the compounds having Formula I, II, III or IV or pharmaceutically acceptable salts thereof. According to aspects of the disclosure, the ionizable cationic lipids comprise the compounds having one or more Chemical sub-structures selected from the group consisting of: Formula IV, Formula IVA, Formula A, Formula A’, Formula A”, Formula A’”, and/or Formula B. In some aspects of the disclosure, the ionizable cationic lipids can comprise compounds having Formula I, Formula I-A, Formula LA’, Formula 1-A”, Formula II, Formula II-A, Formula II-A’, Formula II-B, Formula II-B’, Formula III, or Formula IILA or pharmaceutically acceptable salts thereof. In some aspects of the disclosure, the LNP can comprise compounds having Formula V or Formula V-A or pharmaceutically acceptable salts thereof. In some aspects of the disclosure, the LNP can comprise compounds having Formula VI or Formula VI-A or pharmaceutically acceptable salts thereof. In some aspects of the disclosure, the LNP can comprise compounds having Formula VII or pharmaceutically acceptable salts thereof. In some aspects of the disclosure, the LNP can comprise compounds having Formula VIII or pharmaceutically acceptable salts thereof. According to aspects of the disclosure, the cationic lipids comprise the compounds having (a) an ionizable cationic lipid selected from a compound of Formula I, Formula I-A, Formula I-A’, Formula I-A”, Formula II, Formula II-A, Formula ILA’, Formula II-B, Formula II-B’, Formula III, or Formula IILA, or a sterol lipid of Formula VLA, or a branched lipid of Formula VIII and (b) a sterol of Formula VI, and optionally further comprising (c) a alkylene glycol lipid of Formula VIL In some embodiments, the LNP further comprises a phospholipid of Formula V or Formula V-A. In some embodiments, the LNP further comprises an anionic phospholipid targeting moiety from Table 3.

Also provided herein are compounds, compositions and methods for the treatment or prévention of infectious diseases, including tuberculosis. According to aspects of the disclosure, the cationic lipids comprise the compounds having Formula A or pharmaceutically acceptable salts thereof. In some embodiments, the cationic lipids two fatty acyl groups as in Formula II, II, III or IV.

Disclosed herein are compounds of Formula I, Formula II, Formula III, Formula IV or pharmaceutically acceptable salts thereofthat are useful in the préparation of vaccines. Also disclosed herein are compositions comprising the cationic lipids of Formula I, Formula II, Formula III, Formula IV or pharmaceutically acceptable salts thereof. In some embodiments, the vaccine is used for the prévention mycobacterium infections. In some embodiments, the vaccine can be used for the prévention of tuberculosis, nontuberculous mycobacteria (NTM), nontuberculosis lung disease, leprosy, mycobacterium avium-intracellulare, mycobacterium kansasii, mycobacterium marinum, mycobacterium ulcerans, mycobacterium chelonae, mycobacterium fortuitum, mycobacterium abscessusand other infectious diseases such as coronaviruses (COVID-19, SARS CoV2, SARS-CoV, MERS-CoV), diphtheria, ebola, flu (Influenza), hepatitis, Hib disease, HIV/AIDS, HPV (Human Papillomavirus), malaria, measles, meningococcal disease, mumps, norovirus, plague, pneumococcal disease, polio, respiratory syncytial virus (RSV), rotavirus, rubella (German Measles), shingles (Herpes Zoster), tetanus (Lockjaw), whooping cough (Pertussis) and zika.

Provided herein are compounds, compositions and methods for the treatment or prévention of infectious diseases, including tuberculosis. According to aspects of the disclosure, the cationic lipids comprise the compounds having Formula I, II, III or IV or pharmaceutically acceptable salts thereof. In some embodiments, the cationic lipids two fatty acyl groups as in Formula II, II, III or IV.

One aspect of the disclosure provides a lipid comprising one or more polyunsaturated polyene hydrocarbon chains of Formula A:

Formula A.

wherein a is l, 2, 3 or 4; b is 2, 3 or 4; and c is 3, 4, 5, 6, or 7. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains where b is 4. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A, where the sum of a, b and c is 10, 11, 12 or 13. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A, where a is 4; b is 4; and c is 4 or 5. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A, where a is l, 2 or 3; b is 4; and c is 3, 4, 5, 6, or 7. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A, where a is 5 or 6; b is 2, 3 or 4; and c is 3, 4, 5, 6, or 7. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A, where the sum of a, b and c is 10, 11, 12 or 13. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A, where the sum of a, b and c is 12. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A, where b is 2 and the sum of a, b and c is 12. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A, where b is 3 and the sum of a, b and c is 12. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A, where b is 4 and the sum of a, b and c is 12. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A.

One aspect of the disclosure provides a lipid comprising one or more polyunsaturated polyene hydrocarbon chains of Formula A:

Formula A'.

wherein a is 1, 2 or 3; b is 2, 3 or 4; and c is 3, 4, 5, 6, or 7. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains where b is 4. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A', where the sum of a, b and c is 10, 11, 12 or 13.

One aspect of the disclosure provides a lipid comprising one or more polyunsaturated polyene hydrocarbon chains of Formula A:

Formula A”.

wherein a is 4; b is 4; and c is 4 or 5. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A” where the sub of a, b and c is 12.

One aspect of the disclosure provides a lipid comprising one or more polyunsaturated polyene hydrocarbon chains of Formula A':

Formula A'”.

wherein a is 5 or 6; b is 2, 3 or 4; and c is 3, 4, 5, 6, or 7. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A', where the sum of a, b and c is 10, 11, 12 or 13. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A'”, where the sum of a, b and c is 12. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A'”, where b is 2 and the sum of a, b and c is 12. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A', where b is 3 and the sum of a, b and c is 12. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A', where b is 4 and the sum of a, b and c is 12. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A”'.

One aspect of the disclosure provides a lipid comprising one or more polyunsaturated polyene hydrocarbon chains of Formula B:

Formula B wherein a is 5, 6 or 7; and c is 3, 4, or 5. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains where b is 4. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula B, where the sum of a and c is 9, 10 or 11.

In some embodiments, an ionizable lipid comprises the Chemical structure of Formula (IV-A): Rio ' A (IV-A), or a pharmaceutically acceptable sait thereof, wherein

O U R²² o

V-TV ^ry° _R2p

Yis R²² O , R²² O , R²² , or n is an integer 2, 3 or 4;

R²² is a polyene hydrocarbon chain of Formula A, Formula A', Formula A, or Formula A' or Formula B ; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with hydroxyl.

In some aspects, R²² in Formula (IV-A) is a polyene hydrorcarbon chain of Formula A. In some aspects, R²² in Formula (IV-A) is a polyene hydrocarbon chain of Formula A'. In some aspects, R²² in Formula (IV-A) is a polyene hydrocarbon chain of Formula A. In some aspects, R²² in Formula (IV-A) is a polyene hydrocarbon chain of Formula A”'. In some aspects, R²² in Formula (IV-A) is a polyene hydrocarbon chain of Formula B.

In some aspects, Rio and R12 are each independently selected from methyl, ethyl, propyl, (CH2)(CH2)OH, and -(CH2)2(CH2)OH in Formula (IV-A). In some aspects, Rio and R12 are each independently methyl in Formula (IV-A). In some aspects, Rio and R12 are each independently ethyl in Formula (IV-A). In some aspects, at least one of Rio and R12 is n-propyl optionally substituted with hydroxyl in Formula (IV-A). In some aspects, Rio is methyl and R12 is selected from methyl, ethyl, (CH2)(CH2)OH, and -(CH2)2(CH2)OH in Formula (IV-A). In some aspects, Rio is methyl and R12 is selected from -(CH2)(CH2)OH, and -(CH2)2(CH2)OH in Formula (IV-A). In some aspects, Rio is methyl and R12 is selected from -(CH2)(CH₂)OH, and -(CH₂)2(CH₂)OH in a compound comprising the Chemical structure of Formula (IV-A). In some aspects, Rio and R12 are independently selected from methyl or ethyl, optionally substituted with one or more hydroxyl in Formula (IV-A). In some aspects, one or both of Rio and R12 in Formula (IV-A) are -(CH2)(CH2)OH, or -(CH2)2(CH2)OH in Formula (IV-A). In some aspects, Rio is methyl and R12 is methyl or ethyl substituted with hydroxyl in Formula (IV-A). In some aspects, one or both of Rio in Formula (IV-A) is methyl and R12 is (CH2)(CH2)OH in Formula (IV-A). In some aspects, one or both of Rio in Formula (IV-A) is methyl and R12 is -(CFbHCFDOH in Formula (IV-A).

In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalently bound to the Y portion of Formula (IV-A) wherein Y is R O ; n is an integer 2, 3 or 4; R²² is a polyene hydrocarbon chain of Formula A, Formula A', Formula A, Formula A' or Formula B; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with hydroxyl. In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalently bound to the Y portion of Formula (IV-A) ^RZ°z wherein Y is R²² O ; n is an integer 2, 3 or 4; R²² is a polyene hydrocarbon chain of Formula A, Formula A', Formula A, or Formula A'''; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with hydroxyl. In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalentiy bound to the Y portion of Formula (IVR²² .0

A) wherein Y is R²² O ; n is an integer 2, 3 or 4; R²² is a polyene hydrocarcarbon chain of Formula B; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with hydroxyl.

In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene r²²i ΖΤ Zo hydrocarbon chains covalentiy bound to the Y portion of Formula (IV-A) wherein Y is, R ; n is an integer 2, 3 or 4; R²² is a polyene hydrocarbon chain of Formula A, Formula A', Formula A'', Formula A”' or Formula B; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with hydroxyl. In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalentiy bound to the Y portion of Formula (IV-A) r²²J Zr Zo wherein Y is, R²² ; n is an integer 2, 3 or 4; R²² is a polyene hydrocarbon chain of Formula A,

Formula A', Formula A, or Formula A”'; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with hydroxyl. In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalentiy bound to the Y portion of Formula (IVr²²i Zr Zo

A) wherein Y is, R²² ; n is an integer 2, 3 or 4; R²² is a polyene hydrocarbon chain of Formula

B; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with hydroxyl.

In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene

O

R^{22 0} T

R²² /0 hydrocarbon chains covalentiy bound to the Y portion of Formula (IV-A) wherein Y is ; n is an integer 2, 3 or 4; R²² is a polyene hydrocarbon chain of Formula A, Formula A', Formula A, Formula A'” or Formula B; and each of Rio and Ri₂ is independently (Ci-C4)alkyl optionally substituted with hydroxyl. In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains of Formula (IV-A):

or a pharmaceutically acceptable sait thereof, wherein Y is O ; n is an integer 2, 3 or 4; R²² is a polyene hydrocarbon chain of Formula A, Formula A', Formula A”, or Formula A'; and each of Rio and Ri₂ is independently (Ci-C4)alkyl optionally substituted with hydroxyl.

In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene O hydrocarbon chains covalently bound to the Y portion of Formula (IV-A) wherein Y is R²² ; n is an integer 2, 3 or 4; R²² is a polyene hydrocarbon chain of Formula A, Formula A', Formula A, Formula A”' or Formula B; and each of Rio and Ri₂ is independently (Ci-C4)alkyl optionally substituted with hydroxyl.

In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene R²² hydrocarbon chains covalently bound to the Y portion of Formula (IV-A) wherein Y is n is an integer 2, 3 or 4; R²² is a polyene hydrocarbon chain of Formula A, Formula A', Formula A'', Formula A' or Formula B; and each of Rio and Ri₂ is independently (Ci-C4)alkyl optionally substituted with hydroxyl.

In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalently bound to the Y portion of Formula (IV):

pharmaceutically acceptable sait thereof, wherein Y is

or

R²²

R²² A? „ n is an integer 2, 3 or 4; and R is a polyene hydrocarbon chain of Formula A, Formula A', Formula A, Formula A' or Formula B. In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalently bound to the Y portion of Formula (IV):

yÜx V' • (IV), or a pharmaceutically acceptable sait thereof, wherein Y is O ; n is an integer 2, 3 or 4; and R²² is a polyene hydrocarbon chain of Formula A. In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains covalently bound to the Y portion of Formula (IV):

' (IV), or a pharmaceutically acceptable sait thereof, wherein Y is O ; n is an integer 2; and R²² is a polyene hydrocarbon chain of Formula A'.

One aspect of the disclosure provides a compound of Formula I or pharmaceutically acceptable salts thereof:

Formula I wherein Y is independently a methyl or ethyl group, wherein the two fatty acyl groups hâve between 16-18 carbons and contain two unconjugated olefins.

Another aspect of the disclosure provides a composition comprising ionizable lipids, the lipidic nanoparticles comprising an ionizable lipid of Formula I or pharmaceutically acceptable salts thereof

Formula I wherein Y is independently a methyl or ethyl group, wherein the two fatty acyl groups hâve a total of between 16-18 carbons and contain two olefins separated by two to four methylene groups.

In some embodiments, the two fatty acyl groups hâve 16 carbons. In some embodiments, the two fatty acyl groups hâve 17 carbons. In some embodiments, the two fatty acyl groups hâve 18 carbons.

In some embodiments, an ionizable lipid of Formula I-A is provided, or pharmaceutically acceptable salts thereof:

Formula I-A wherein a is l, 2, 3, 4, 5 or 6; b is 2, 3 or 4; c is 3, 4, 5, 6, or 7; the sum of a, b and c is 10 or 12; L is

each of Rio and R12 is independently (C,-C4)alkyl optionally substituted with hydroxyl; v is 0 or l; q is l, 2, 3 or 4; and q2 is l or 2. In some aspects, v is 0, q is l, 2 or 3 and the sum of a, b and c is 12 in an ionizable lipid of Formula I-A. In some aspects, v is l, q 5 is 3 or 4 and the sum of a, b and c is 12 in an ionizable lipid of Formula I-A. In some aspects, Rio and

R12 are independently selected from methyl, ethyl, and propyl each optionally substituted with a single hydroxyl in an ionizable lipid of Formula I-A'. In some aspects, the sum of a, b and c is 12, and Rio and R12 are independently selected from methyl, ethyl, -(CH2)(CH2)OH, and -(CH2)2(CH2)OH.

In some embodiments, an ionizable lipid of Formula I-A' is provided, or pharmaceutically

IO acceptable salts thereof:

or ethyl; v is 0 or l ; q is 2, 3 or 4; and q2 is l or 2. In some aspects, v is 0 , q is l, 2 or 3; and the sum of a and c is 6 or 8 in an ionizable lipid of Formula I-A'. In some aspects, v is l, q is 3 or 4; and the sum of a and c is 6 or 8 in an ionizable lipid of Formula I-A'.

In some embodiments, an ionizable lipid of Formula I-A is provided, or pharmaceutically acceptable salts thereof:

Formula I-A wherein a is 4, 5 or 6; b is 2, 3 or 4; c is 3, 4, 5, 6, or 7;

L is

; each of Rio and Ru is independently (Ci-C4)alkyl optionally substituted with hydroxyl; v is 0 or l; q is l, 2, 3 or 4; and q2 is l or 2. In some aspects, v is 0, q is l, 2 or 3 and the sum of a, b and c is 12 in an ionizable lipid of Formula I-A. In some aspects, v is l, q is 3 or 4 and the sum of a, b and c is 12 in an ionizable lipid of Formula I-A. In some aspects, Rio and R12 are independently selected from methyl, ethyl, and propyl each optionally substituted with a single hydroxyl in an ionizable lipid of Formula I-A. In some aspects, the sum of a, b and c is 12, and Rio and R12 are independently selected from methyl, ethyl, -(CH2)(CH2)OH, and -(CH₂)2(CH₂)OH.

Another aspect of the disclosure provides for a compound of Formula II or pharmaceutically acceptable salts thereof:

Formula II wherein R. is a substituent comprising a dialkylamino group of one of the structures shown above, wherein the two fatty acyl groups are between 16-18 carbons and contain two olefins that are separated by at least two methylene groups.

In some embodiments, the two fatty acyl groups hâve 16 carbons. In some embodiments, the two fatty acyl groups hâve 17 carbons. In some embodiments, the two fatty acyl groups hâve 18 10 carbons.

Another aspect of the disclosure provides for a compound of Formula II-A or pharmaceutically acceptable salts thereof:

wherein a is l, 2, 3, 4, 5 or 6; b is 2, 3 or 4;

c is 4, 5, 6, 7 or 8; R2 is

q and q’ are each independently l or 2; and Rio and R12 are each methyl or ethyl. In some aspects, the sum of a, b and c is 11 or 13 in an ionizable lipid of Formula I-A. In some aspects, an ionizable lipid of Formula I-A is characterized by one or more of the following: a is 1, 2 or 3; q is 2; q’ is 1; and at least one of Rio 5 or R12 is ethyl. In some aspects, b is 4 in an ionizable lipid of Formula II-A. In some aspects, a is 4, b is 4 and c is 4 in an ionizable lipid of Formula II-A. In some aspects, a is 1, b is 4 and c is 8 in an ionizable lipid of Formula II-A. In some aspects, a is 2, b is 4 and c is 5 in an ionizable lipid of Formula II-A.

Another aspect of the disclosure provides for a compound of Formula II-A' or 10 pharmaceutically acceptable salts thereof:

Rio

or O ; q and q’ are each independently 1 or 2; and Rio and R12 are each methyl or ethyl. In some aspects, the sum of a, b and c is 11 or 13 in an ionizable lipid of

Formula I-A'. In some aspects, an ionizable lipid of Formula I-A is characterized by one or more of the following: q is 2; q’ is 1; and at least one of Rio or R₎₂ is ethyl. In some aspects, b is 4 in an ionizable lipid of Formula II-A'. In some aspects, a is 4, b is 4 and c is 4 in an ionizable lipid of Formula II-A'. In some aspects, a is 1, b is 4 and c is 8 in an ionizable lipid of Formula II-A'. In some aspects, a is 2, b is 4 and c is 5 in an ionizable lipid of Formula II-A'.

Another aspect of the disclosure provides for acompound of Formula II-B or pharmaceutically

wherein a is 5, 6 or 7; and c is 3, 4, or 5; R2 is

Rio

F^io

O ; q and q’ are each independently 1 or 2; and Rio and R12 are each methyl or ethyl. In some aspects, the sum of a and c is 9 or 11 in an ionizable lipid of Formula I-B. In some aspects, an ionizable lipid of Formula I-B is characterized by one or more of the following: q is 2; q’ is 1; and at least one of Rio or R12 is ethyl. In some aspects, an ionizable lipid of Formula I-B is characterized by one or more of the following: q is 1; q’ is 2; and Rio and R12 are each methyl. In some aspects, c is 4 in an ionizable lipid of Formula II-B. In some aspects, a is 5 or 7 and c is 4 in an ionizable lipid of Formula II-B. In some aspects, a is 5 and c is 4 in an ionizable lipid of Formula II-B. In some aspects, a is 7 and c is 4 in an ionizable lipid of Formula II-B.

Another aspect of the disclosure provides for a compound of Formula II-B' or pharmaceutically acceptable salts thereof:

Formula (Π-Β') ^10

Ο ; q and q’ are each independently 1 or 2; and Rio and R12 are each methyl. In some aspects, the sum of a and c is 9 or 11 in an ionizable lipid of Formula I-B'. In some aspects, c is 4 in an ionizable lipid of Formula II-B'. In some aspects, a is 5 or 7 and c is 4 in an ionizable lipid of Formula II-B'. In some aspects, a is 5 and c is 4 in an ionizable lipid of Formula II-B. In some aspects, a is 7 and c is 4 in an ionizable lipid of Formula II-B'. In some aspects, a is 5 and c is 3 in an ionizable lipid of Formula II-B. In some aspects, a is 7 and c is 3 in an ionizable lipid of Formula II-B'.

Another aspect of the disclosure provides for a compound of Formula III or pharmaceutically acceptable salts thereof:

Formula III wherein Y is a methyl or ethyl group, wherein the two fatty acyl groups are disulfide fatty acyl groups having between 16-l 8 carbons and containing a single olefin.

In some embodiments, the two fatty acyl groups hâve 16 carbons. In some embodiments, the two fatty acyl groups hâve 17 carbons. In some embodiments, the two fatty acyl groups hâve 18 carbons.

Another aspect of the disclosure provides for a compound of Formula III-A or pharmaceutically acceptable salts thereof:

Formula III-A wherein a is 5, 6 or 7; c is 3, 4, or 5; q is 2 or 3 and Rio and R12 are methyl or ethyl. In some aspects, an ionizable lipid can comprise a compound of Formula III-A wherein a is 5 or 7. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula A, where the sum of a and c is 8, 9 or IO.

Another aspect of the disclosure provides for a compound of Formula III-A' or pharmaceutically acceptable salts thereof:

Formula III-A' wherein a is 5 or 7; c is 3, 4, or 5; q is 2 or 3 and Rio and R12 are methyl or ethyl. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains where c is 3. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula III-A', where the sum of a and c is 8 or 10. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula III-A', where q is 2, and the sum of a and c is 8 or 10. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula III-A', where q is 2, Rio and Ru are each methyl and the sum of a and c is 8 or 10. In some aspects, an ionizable lipid can comprise two polyunsaturated polyene hydrocarbon chains of Formula III-A', where q is 2, Rio and Ru are each methyl and c is 3.

In some embodiments, the compound in Formula I-III has a pKa between 6 and 7.

In some embodiments, a lipidic nanoparticle composition comprises lipids and nucleic acids, the lipidic nanoparticles comprising a compound of Formula I, II, III, combinations thereof or pharmaceutically acceptable salts thereof.

In some embodiments, an LNP comprises an ionizable lipid having a structure of Formula (IV):

vüf । (IV), or a pharmaceutically acceptable sait thereof,

independently alkyl, alkenyl, alkynyl, or heteroalkyl, each of which is optionally substituted with R^B; each R^b is independently alkyl, halo, hydroxy, amino, cycloalkyl, or heterocyclyl; n is an integer between l and 10 (inclusive); and dénotés the attachaient point.

In some embodiments, Y is R²² 6

In some embodiments, the compound in Formula IV has a pKa between 6 and 7.

In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains of Formula (IV-A):

Rio

N

Rl2 (IV-A), or a pharmaceutically acceptable sait thereof, wherein Y is

R²² /0

R²² O ; n is an integer 2, 3 or 4; R²² is a polyene hydrocarcarbon chain of Formula A, Formula A', Formula A”, or Formula A'; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with hydroxyl. In some aspects, Rio and R12 in Formula (IV-A) are independently selected from methyl, ethyl, -(CH2)(CH₂)OH, and -(CH₂)2(CH₂)OH.

In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains of Formula (IV-A):

Rio

^N\

Rl2 (IV-A), or a pharmaceutically acceptable sait thereof, wherein Y is,

ΙΟ r²²j ztO

R²² ; n is an integer 2, 3 or 4; R²² is a polyene hydrocarcarbon chain of Formula A, Formula

A', Formula A'', or Formula A', or Formula B; and each of Rio and R12 is independently (CiC4)alkyl optionally substituted with hydroxyl. In some aspects, Rio and R12 in Formula (IV-A) are independently selected from methyl, ethyl, -(CH2)(CH2)OH, and -(CH2)2(CH2)OH.

In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains of Formula (IV-A):

R10

^N\

Rl2 (IV-A), or a pharmaceutically acceptable sait thereof, wherein Y is

O ; n is an integer 2, 3 or 4; R²² is a polyene hydrocarcarbon chain of Formula A, Formula A', Formula A'', or Formula A”', or Formula B; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with hydroxyl. In some aspects, Rio and R12 in Formula (IV-A) are independently selected from methyl, ethyl, -(CFbXCFyOH, and -(CH2)2(CH2)OH.

In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains of Formula (IV-A):

Rio

Ο

R12 ₂₂ (IV-A), or a pharmaceutically acceptable sait thereof, wherein Y is R ; n is an integer 2, 3 or 4; R²² is a polyene hydrocarcarbon chain of Formula A, Formula A', Formula A, or Formula A', or Formula B; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with hydroxyl. In some aspects, Rio and R12 in Formula (IV-A) are independently selected from methyl, ethyl, -(CH2)(CH2)OH, and -(CH2)2(CH2)OH.

In some embodiments, an ionizable lipid comprises one or more polyunsaturated polyene hydrocarbon chains of Formula (IV-A):

Rw

R^{22 R}12 R22 L (IV-A), or a pharmaceutically acceptable sait thereof, wherein Y is is an integer 2, 3 or 4; R²² is a polyene hydrocarcarbon chain of Formula A, Formula A', Formula A, or Formula A', or Formula B; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with hydroxyl. In some aspects, Rio and R12 in Formula (IV-A) are independently selected from methyl, ethyl, -(CH2)(CH2)OH, and -(CH2)2(CH2)OH.

In some embodiments, the compounds hâve the structure of the compounds listed in Table 1 or Table 2.

Table 1A shows examples of cationic lipids. Table 2 shows examples of bioreducible cationic lipids.

Table IA. Exemplary cationic lipids

Table 1 A. Exemplary cationic lipids (continued)

Table IA. Exemplary cationic lipids (continued)

Table IA. Exemplary cationic lipids (continued)

Table IB Additional Exemplary cationic lipids

AKG-UO-6

AKG-UO-7

AKG-UO-8

AKG-UO-9

AKG-U0-1O

Table 2. Exemplary bioreducible cationic lipids

Table 2. Exemplary bioreducible cationic lipids (continued)

In some embodiments, the ionizable lipid encapsulate the nucleic acid. In some embodiments, the ionizable lipid encapsulate the nucleic acid in a LNP formulation. In some embodiments, the nucleic acid is a siRNA molécule. In some embodiments, the nucleic acid is a mRNA molécule. In some embodiments, the nucleic acid is a DNA molécule.

In some embodiments, compositions further comprising ligands, such as antibody conjugates, 10 directed against cell surface receptors to target lipid nanoparticles in a highly spécifie manner to dendritic cells are provided. In some embodiments, the composition further comprises a targeting ligand, wherein the targeting ligand is oriented to the outside of the nanoparticle. In some embodiments, the targeting ligand is an antibody.

In some embodiments, the lipidic nanoparticles are in an aqueous medium.

In some embodiments, the nucleic acid is entrapped in the lipidic nanoparticle with a compound disclosed herein, including compounds of Formula I, II, III, IV or combinations thereof, wherein the nucleic acid is either RNA or DNA. In some embodiments, the nucleic acid is entrapped in the lipidic nanoparticle with a compound disclosed herein, including compounds of Formula I, ΙΑ, II, II-A, II-B, III, III-A, IV, IV-A, IV-B, V, V-A, VLA, VII, VIII or combinations thereof, wherein the nucleic acid is either RNA or DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid is DNA.

In some embodiments, the lipidic nanoparticle comprises a membrane comprising phosphatidylcholine and a sterol. In some embodiments, the sterol is cholestérol. In some embodiments, the lipidic nanoparticle comprises a membrane comprising phosphatidylcholine, ionizable cationic lipid (ICL). In some embodiments, the ICL hâve a structure of Formula I, II, III or IV, and cholestérol, wherein the membrane séparâtes the inside of the lipidic nanoparticles from the aqueous medium. In some embodiment, the ICL hâve a structure as shown in Table l A and Table 2. In some embodiment, the ICL hâve a structure as shown in Table IB. In some embodiments, the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC). In some embodiments, the ionizable cationic lipid to cholestérol molar ratios is from about 65:35 to 40:60. In some embodiments, the ICL to cholestérol molar ratio is from about 60:40 to about 45:55.

In some embodiments, the phosphatidylcholine to cholestérol molar ratio is from about l:5 to about l:2.

In some embodiments, the membrane further comprises a polymer-conjugated lipid.

In some embodiments, the lipidic nanoparticle comprises ICL, DSPC, cholestérol and polymer-conjugated lipid in a about 49.5:10.3:39.6:2.5 molar ratio.

In some embodiments, the polymer-conjugated lipid is PEG(2000)-dimyristoylglycerol (PEGDMG) or PEG(MoL weight 2,000)-dimyristoylphosphatidylethanolamine (PEG-DMPE).

In some embodiments the percentage of oxidative dégradation products for the ionizable lipid is less than 50 % of that for a DLin-KC2-DMA or DLin-MC3-DMA control formulation.

In some embodiments, the composition is a liquid pharmaceutical formulation for parentéral administration.

In some embodiments, the composition is a liquid pharmaceutical formulation for subcutaneous, intramuscular, or intradermal administration.

In some embodiments, the composition is in the form of a lyophilized powder, that is subsequently reconstituted with aqueous medium prier to administration.

Other aspects of the disclosure relate to a method of preventing a bacterial or viral infection, the method comprising administering to a subject in need thereof an effective amount of the composition provided herein to elicit an immune response. Some embodiments provide methods of vaccinating a subject in need thereof, the method comprising administering the composition 59 comprising a nucleic acid encoding an antigenic protein.

In some embodiments, the composition is administered subcutaneously, intramuscularly, or intradermally.

In some embodiments, the bacterial infection is Mycobacterium tuberculosis infection. In some embodiments, the bacterial infection is a form of nontuberculosis mycobacterium.

In some embodiments, the viral infection is a coronavirus. In some embodiments, the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2

In some embodiments, the viral infection is HIV/AIDs.

In some embodiments, the lipidic nanoparticle is administered parenterally.

In some embodiments, the lipidic nanoparticle composition is administered as part of a single injection.

The présent disclosure features a lipid nanoparticle comprising nucleic acids such as DNA, mRNA, siRNA, antisense oligonucleotides, CRISPR components such as a guide RNA (gRNA or sgRNA) and a CRISPR-associated endonuclease (Cas protein) and a lipid. Exemplary lipids include ionizable cationic lipids (ICLs), phospholipids, sterol lipids, alkylene glycol lipids (e.g., polyethylene glycol lipids), sphingolipids, glycerolipids, glycerophospholipids, prenol lipids, saccharolipids, fatty acids, and polyketides. In some embodiments, the LNP comprises a single type of lipid. In some embodiments, the LNP comprises a plurality (e.g. two or more) of lipids. An LNP may comprise one or more of an ionizable cationic lipid, a phospholipid, a sterol, or an alkylene glycol lipid (e.g., a polyethylene glycol lipid).

In an embodiment, the LNP comprises an ionizable cationic lipid. As used herein “ionizable cationic lipid”, “ionizable lipid” and “ICL” are used interchangeably. An ICL is a lipid that comprises an ionizable moiety capable of bearing a charge (e.g., a positive charge e.g., a cationic lipid) under certain conditions (e.g., at a certain pH range, e.g., under physiological conditions). The ionizable moiety may comprise an amine, and preferably a substituted amine. An ionizable lipid may be a cationic lipid or an anionic lipid. In addition to an ionizable moiety, an ionizable lipid may contain an alkyl or alkenyl group, e.g., greater than six carbon atoms in length (e.g., greater than about 8 carbons, 10 carbons, 12 carbons, 14 carbons, 16 carbons, 18 carbons, 20 carbons or more in length). Additional ionizable lipids that may be included in an LNP described herein are disclosed in Jayaraman et al. (Angew. Chem. Int. Ed. 51:8529-8533 (2012)), Semple et al. Nature Biotechnol. 28:172-176 (2010)), and U.S. Patent Nos. 8,710,200 and 8,754,062, each of which is incorporated herein by reference in its entirety.

In some embodiments, an LNP comprises an ionizable lipid having a structure of Formula (IV):

(IV), or a pharmaceutically acceptable sait thereof,

independently alkyl, alkenyl, alkynyl, or heteroalkyl, each of which is optionally substituted with R^B;

each R^b is independently alkyl, halo, hydroxy, amino, cycloalkyl, or heterocyclyl; n is an integer between 1 and 10 (inclusive); and dénotés the attachment point.

In some embodiments, Y is

In some embodiments, an LNP comprises an ionizable lipid having a structure of Formula (IV10 A), or a pharmaceutically acceptable sait thereof,

(IV-B) wherein each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with hydroxyl; v is

' 'c ; a is 1, 2, 3, 4 or 5; and c is 4, 5, 6, 7 or 8.

In some embodiments, v equals 0 for compounds of Formula (IV-B). In some embodiments, v equals 1 for compounds of Formula (IV-B). In some embodiments, v equals 1 and ql equals 1 for compounds of Formula (IV-A). In some embodiments, v equals 1 and ql equals 2 for compounds of Formula (IV-B).

In some embodiments, the sum of a and c is 6, 7, 8 or 9 in R²² for compounds of Formula (IVB). In some embodiments, the sum of a and c is 6 in R²² for compounds of Formula (IV-B). In some embodiments, the sum of a and c is 7 in R²² for compounds of Formula (IV-B). In some embodiments, the sum of a and c is 9 in R²² for compounds of Formula (IV-B).

In some embodiments, v equals 0 and the sum of a and c is 6, 7, 8 or 9 in R²² for compounds of Formula (IV-B). In some embodiments, v equals 0 and the sum of a and c is 6 in R²² for compounds of Formula (IV-B). In some embodiments, v equals 0 and the sum of a and c is 7 in R²² for compounds of Formula (IV-B). In some embodiments, v equals 0 and the sum of a and c is 9 in R²² for compounds of Formula (IV-B).

In some embodiments, Rio and R12 are independently selected from methyl, ethyl, (CH2)(CH2)OH, and -(CH2)2(CH2)OH for compounds of Formula (IV-B). In some embodiments, Rio and R12 are each methyl and the sum of a and c is 6, 7, 8 or 9 in R²² for compounds of Formula (IVB). In some embodiments, Rio and R12 are each methyl, v is 0 and the sum of a and c is 6, 7, 8 or 9 in R²² for compounds of Formula (IV-B).

In some embodiments, v equals 0 and R²² is

B). In some embodiments, v equals 0 and R²² is

for compounds of Formula (IVand the sum of a and c is 8 for

compounds of Formula (IV-B). In some embodiments, v equals 0 and R²² is and a is l and c is 7 for compounds of Formula (IV-B). In some embodiments, v equals 0 and R²² is

and a is 2 and c is 4 for compounds of Formula (IV-B).

In some embodiments, v equals l and R²² is for compounds of Formula (IV-

B). In some embodiments, v equals l and R²² is and the sum of a and c is 8 for

compounds of Formula (IV-B). n some embodiments, v equals l and R²² is and a is l and c is 7 for compounds of Formula (IV-B).

In some embodiments, v equals 0 and R²² is

for compounds of Formula (IV-B). In some embodiments, v equals 0 and R²² is

and the sum of a and c is 7 or for compounds of Formula (IV-B). In some embodiments, v equals 0 and R²² is

and a is 4 and c is 5 for compounds of Formula (IV-B). In some embodiments, v equals 0 and R²² is

R²² , and a is l and c is 8 for compounds of Formula (IV-B). In some embodiments, v equals 0 and R²² is

, and a is 2 and c is 5 for compounds of Formula (IV-B).

O

In some embodiments, v equals 0 and R²² is for compounds of Formula (IV-

B). In some embodiments, v equals 0 and R²2 is and the sum of and c is 7 or 9 for

R²² .0

R²²

compounds of Formula (IV-B). In some embodiments, v equals 0 and R²² is and a is 4 and c is 5 for compounds of Formula (IV-B). In some embodiments, v equals 0 and R²² is ^RY°

R²²

and a is l and c is 8 for compounds of Formula (IV-B).

In some embodiments, v

R²² O equals 0 and R²² is and a is 2 and c is 5 for compounds of Formula (IV-B).

An LNP may comprise an ionizable lipid at a concentration greater than about 0.1 mol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises an ionizable lipid at a concentration of greater than about l mol%, about 2mol%, about 4mol%, about 8mol%, about 20mol%, about 40mol%, about 50mol%, about 60mol%, about 80mol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises an ionizable lipid at a concentration of greater than about 20mol%, about 40mol%, or about 50mol%. In an embodiment, the LNP comprises an ionizable lipid at a concentration between about lmol% to about 95mol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises an ionizable lipid at a concentration between about 2mol% to about 90mol%, about 4mol% to about 80mol%, about I0mol% to about 70mol%, about 20mol% to about 60mol%, about 40mol% to about 55mol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises an ionizable lipid at a concentration between about 20mol% to about 60mol%. In an embodiment, the LNP comprises an ionizable lipid at a concentration between about 40 mol% to about 55 mol%.

In an embodiment, the LNP comprises a phospholipid. A phospholipid is a lipid that comprises a phosphate group and at least one alkyl, alkenyl, or heteroalkyl chain. A phospholipid may be naturally occurring or non-naturally occurring (e.g., a synthetic phospholipid). A phospholipid may comprise an amine, amide, ester, carboxyl, choline, hydroxyl, acetal, ether, carbohydrate, sterol, or a glycerol. In some embodiments, a phospholipid may comprise a phosphocholine, phosphosphingolipid, or a plasmalogen. Exemplary phospholipids include l ,2-dioleoyl-sn-glycero65

3-phosphocholine (DOPC), l,2-dipalmitoyl-ôTî-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE), l,2-distearoyl-s^glycero-3-phosphocholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), I,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-s»-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-s«-glycero-3phosphoethanolamine (DSPE), l-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), 1,2diarachidonoyl-5H-glycero-3-phosphocholine (DAPC), l-palmitoyl-2-linoleoyl-SH-glycero-3phosphatidylcholine (PLPC), l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), l-stearoyl-2myristoyl-sn-glycero-3-phosphocholine (SMPC), l-palmitoyl-2-myristoyl-SH-glycero-3phosphocholine (PMPC), bis(monoacylglycerol)phosphate (BMP), L-a-phosphatidylcholine, 1,2Diheptadecanoyl-5M-glycero-3-phosphorylcholine (DHDPC), and l-stearoyl-2-arachidonoyl-swglycero-3-phosphocholine (SAPC). Additional phospholipids that may be included in an LNP described herein are disclosed in Li, J. et al. (Asian J. Pharm. Soi. 10:81-98 (2015)), which is incorporated herein by référencé in its entirety.

In some embodiments, an LNP comprises a phospholipid having a structure of Formula (V):

ο (V). or a pharmaceutically acceptable sait thereof, wherein each R²³ is independently alkyl, alkenyl, or heteroalkyl; wherein each alkyl, alkenyl, or heteroalkyl is optionally substituted with R^c; each R²⁵ is independently hydrogen or alkyl; R²⁴ is absent, hydrogen, or alkyl; each R^c is independently alkyl, halo, hydroxy, amino, cycloalkyl, or heterocyclyl; m is an integer between 1 and 4 (inclusive); and u is 2 or 3.

In some embodiments, each R²³ is independently alkyl (e.g., C2-C32 alkyl, C4-C28 alkyl, C8-C24 alkyl, C12-C22 alkyl, or C16-C20 alkyl). In some embodiments, each R²³ is independently alkenyl (e.g., C2-C32 alkyl, C4-C28 alkenyl, C₈-C₂4 alkenyl, C12-C22 alkenyl, or C16-C20 alkenyl). In some embodiments, each R²³is independently heteroalkyl (e.g., C4-C28heteroalkyl, C8-C24heteroalkyl, C12C22heteroalkyl, Ci6-C2oheteroalkyl). In some embodiments, each R²³ is independently C16-C20 alkyl. In some embodiments, each R²³ is independently C17 alkyl. In some embodiments, each R²³ is independently heptadecyl. In some embodiments, each R²³ is the same. In some embodiments, each R²³ is different. In some embodiments, each R²³ is optionally substituted with R^c. In some embodiments, R^c is independently alkyl, halo, hydroxy, amino, cycloalkyl, or heterocyclyl.

In some embodiments, one of R²⁵ is hydrogen. In some embodiments, one of R²⁵ is alkyl. In some embodiments, one of R²⁵ is methyl. In some embodiments, each R²⁵ is independently alkyl. In some embodiments, each R²⁵ is independently methyl. In some embodiments, each R²⁵ is independently methyl and u is 2. In some embodiments, each R²⁵ is independently methyl and u is 3.

In some embodiments, R²⁴ is absent, and the oxygen to which it is attached carries a négative charge. In some embodiments, R²⁴ is hydrogen.

In some embodiments, m is an integer between 1 and 10, 1 and 8, 1 and 6, 1 and 4. In some embodiments, m is 1, 2, 3, or 4. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3.

In some embodiments, compositions comprising both a cationic ionizable lipid and an anionic phospholipid targeting moiety are provided. In some embodiments, the anionic phospholipid is a composition of Formula (V-A):

Formula (V-A) wherein a is 14 or 16, and z is an amide, glycol, or amidyl-alkyl-carboxylic acid moiety. In

wherein m is 2 or 3. In some embodiments, the anionic phospholipid targeting moiety is selected from the group consisting of DSPS (L-isomer), DPPS (L67 isomer), DMPS (L-isomer), DOPS (L-isomer), DSPS (D-isomer), DSPG, DPPG, N-Glu-DSPE, and N-Suc-DSPE.

In some embodiments, the phospholipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the phospholipid is l,2-dioleoyl-5n-glycero-3phosphocholine(DOPC). In some embodiments, the phospholipid is l,2-dipalmitoyl-sn-glycero-3phosphocholine(DPPC). In some embodiments, the phospholipid is l,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE).

Incorporation of phosphatidylserine

The LNP (e.g., as described herein) may comprise one or more of the following components: (i) Ionizable cationic lipid (ICL) containing a Cl6 alkyl or Cl6 alkenyl group or C18 alkyl or Cl8 alkenyl group at a concentration between about lmol% to about 95mol% (or any value therebetween, e.g. about 20mol% to about 80mol%); (ii) A phospholipid at a concentration between O.lmol% to about 20 mol% (or any value there between, e.g. between about 2.5 mol% to about 10 mol%) where the phospholipid also contains Cl6 or C18 alkyl or alkenyl groups; (iii) cholestérol at a concentration between about lmol% to about 95mol% (or any value therebetween, e.g. about 20mol% to about 80mol%); (iv) a phosphatidylserine (PS) or phosphatidylglycerol (PG) added to the LNP lipid formulation at a concentration between about 0.5 mol% to about 20 mol%, about 2.5 mol% to about 10 mol%, about 4 mol% to about 8 mol%, or any value therebetween of the total lipid content of the LNP, and (v) a polyethyleneglycol (PEG)-2000-containing lipid (e.g., DPG-PEG2000, DPPEPEG2000, DMPE-PEG2000, DMG-PEG2000) at a concentration between about 0. lmol% to about 5 mol% (or any value therebetween, e.g. between about l mol% to about 2.5 mol%). In an embodiment, the LNP comprises two of (i)-(v). In an embodiment, the LNP comprises three of (i)-(v). In an embodiment, the LNP comprises four of (i)-(v). In an embodiment, the LNP comprises each of (i)(v). In some embodiments, the LNP comprises (i) and (ii). In some embodiments, the LNP comprises (i) and (iii). In some embodiments, the LNP comprises (i) and (v). In some embodiments, the LNP comprises (ii) and (iii). In some embodiments, the LNP comprises (ii) and (v). In some embodiments, the LNP comprises (iii) and (iv). In some embodiments, the LNP comprises (iii) and (v). In some embodiments, the LNP comprises (i), (ii), and (iii). In some embodiments, the LNP comprises (i), (ii), and (v). In some embodiments, the LNP comprises (ii), (iii), and (v). In some embodiments, the LNP comprises (ii), (iii), (iv) and (v). In an embodiment, the LNP consists or consists essentially of 68 four of (i)-(v). In an embodiment, the LNP consists or consists essentially of each of (i)-(v). In some embodiments, the LNP consists or consists essentially of (i) and (ii). In some embodiments, the LNP consists or consists essentially of (i) and (iii). In some embodiments, the LNP consists or consists essentially of (i) and (v). In some embodiments, the LNP consists or consists essentially of (ii) and (iii). In some embodiments, the LNP comprises (ii) and (v). In some embodiments, the LNP consists or consists essentially of (iii) and (iv). In some embodiments, the LNP consists or consists essentially of (iii) and (v). In some embodiments, the LNP consists or consists essentially of (i), (ii), and (iii). In some embodiments, the LNP consists or consists essentially of (i), (ii), and (v). In some embodiments, the LNP comprises (ii), (iii), and (v). In some embodiments, the LNP consists or consists essentially of (ii), (iii), (iv) and (v).

An LNP may comprise a phospholipid at a concentration greater than about 0. lmol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises a phospholipid at a concentration of greater than about 0.5mol%, about lmol%, about l.5mol%, about 2mol%, about 3mol%, about 4mol%, about 5mol%, about 6mol%, about 8mol%, about I0mol%, about I2mol%, about I5mol%, about 20mol%, about 50mol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises a phospholipid at a concentration of greater than about l mol%, about 5mol%, or about I0mol%. In an embodiment, the LNP comprises a phospholipid at a concentration between about 0.lmol% to about 50mol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises a phospholipid at a concentration between about 0.5mol% to about 40mol%, about lmol% to about 30mol%, about 5mol% to about 25mol%, about I0mol% to about 20mol%, about I0mol% to about I5mol%, or about I5mol% to about 20mol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises a phospholipid at a concentration between about 5mol% to about 25mol%. In an embodiment, the LNP comprises a phospholipid at a concentration between about I0mol% to 20mol%.

In an embodiment, the LNP comprises a sterol or ionizable sterol molécule. A sterol is a lipid that comprises a polycyclic structure and an optionally a hydroxyl or ether substituent, and may be naturally occurring or non-naturally occurring (e.g., a synthetic sterol). Sterols may comprise no double bonds, a single double bond, or multiple double bonds. Sterols may further comprise an alkyl, alkenyl, halo, ester, ketone, hydroxyl, amine, polyether, carbohydrate, or cyclic moiety. Sterol may further contain a bioreducible disulfide linkage between the dialkylamino group and the polycyclic portion of the molécule (see Table 2, Compounds 35-38). An exemplary listing of sterols includes cholestérol, dehydroergosterol, ergosterol, campesterol, β-sitosterol, stigmasterol, lanosterol, dihydrolanosterol, desmosterol, brassicasterol, lathosterol, zymosterol, 7-dehydrodesmosterol, avenasterol, campestanol, lupeol, and cycloartenol. In some embodiments, the sterol comprises cholestérol, dehydroergosterol, ergosterol, campesterol, β-sitosterol, or stigmasteroL Additional sterols that may be included in an LNP described herein are disclosed in Fahy, E. et al. (J. Lipid. Res. 46:839-862 (2005).

Ionizable sterols

In some embodiments, an LNP comprises a sterol having a structure of Formula (VI):

(VI) or a pharmaceutically acceptable sait thereof, wherein

R²⁶ is hydrogen, alkyl, heteroalkyl, or-C(O)R^D, R²⁷ is hydrogen, alkyl, or -OR^E; each of R^D and R^E is independently hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroarylis optionally substituted with alkyl, halo, or carbonyl; and each is either a single or double bond, and wherein each carbon atom participating in the single or double bond is bound to 0, l, or 2 hydrogens, valency permitting.

In some embodiments, one of is a single bond. In some embodiments, one of i_{s a} double bond. In some embodiments, two of are single bonds. In some embodiments, two of are double bonds. In some embodiments, each i_s a single bond. In some embodiments, each i_{s a} double bond.

In some embodiments, the sterol is cholestérol. In some embodiments, the sterol is dehydroergosterol. In some embodiments, the sterol is ergosterol. In some embodiments, the sterol is campesterol. In some embodiments, the sterol is β-sitosterol. In some embodiments, the sterol is stigmasteroL In some embodiments, the sterol is a corticosteroid. (e.g., corticosterone, hydrocortisone, cortisone, or aldostérone).

In some embodiments, an LNP comprises a sterol having a structure of Formula (VI-A):

(VI-A) or a pharmaceutically acceptable sait thereof, wherein q is 3 or 4 and R3 is

Another aspect ofthe disclosure provides a composition comprising an anionic phospholipid of Formula (V-A) and a branched ionizable lipid of Formula (VIII) :

Zi

Formula VIII wherein d is 2, 3 or 4; e and f are each independently 5, 6 or 7; and Zi and Z2 are each independently -O-C(O)- or -C(O)-O-; and Ru and R15 are each independently linear or branched (Cio-C2o)alkyl. In some aspects, R14 and R15 in Formula VII are each a C14 or Ci6 branched alkyl. In some aspects, R14 is a Ci 1 linear alkyl and R15 is a C14 or Ci6 branched alkyl. In some aspects, R14 in Formula VII is a Ch linear alkyl and R15 is a C14 or Ci6 linear alkyl. In some aspects, R14 and/or R15 in Formula VII are each independently

, where g and h are each independently 5, 6, or 7. In some aspects, R14 and Ri5 in Formula VII are each independently

, where g and h are both the same and are 5, 6, or 7. In some aspects, R14 in Formula VII is a linear Cn alkyl and R15 in

Formula VII is

, where g and h are both the same and are 5, 6, or 7.

In some embodiments, the ionizable lipid can be a branched ionizable lipid selected from ALC0315 and SM-102:

ALC-0315

SM-102

An LNP may comprise a sterol at a concentration greater than about 0. lmol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises a sterol at a concentration greater 10 than about 0.5mol%, about lmol%, about 5mol%, about 10mol%, about 15mol%, about 20mol%, about 25mol%, about 35mol%, about 40mol%, about 45mol%, about 50mol%, about 55mol%, about 60mol%, about 65mol%, or about 70mol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises a sterol at a concentration greater than about 10mol%, about

I5mol%, about 20mol%, or about 25mol%. In an embodiment, the LNP comprises a sterol at a concentration between about lmol% to about 95mol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises a sterol at a concentration between about 5mol% to about 90mol%, about I0mol% to about 85mol%, about 20mol% to about 80mol%, about 20mol% to about 60mol%, about 20mol% to about 50mol%, or about 20mol% to 40mol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises a sterol at a concentration between about 20mol% to about 50mol%. In an embodiment, the LNP comprises a sterol at a concentration between about 30mol% to about 60mol%.

In some embodiments, the LNP comprises an alkylene glycol-containing lipid. An alkylene glycol-containing lipid is a lipid that comprises at least one alkylene glycol moiety, for example, a methylene glycol or an ethylene glycol moiety. In some embodiments, the alkylene glycol-containing lipid comprises a polyethylene glycol (PEG). An alkylene glycol-containing lipid may be a PEGcontaining lipid. Polymer-conjugated lipids may include poly( ethylene glycol)-conjugated (pegylated)phospholipids (PEG-lipids) such as PEG(MoL weight 2,000) methoxy-poly(ethylene glycol)-l,2-distearoyl-sn-glycerol (PEG-DSG), PEG(Mol. weight 2,000) methoxy-poly(ethylene glycol)-l,2-palmitoyl-sn-glycerol (PEG-DPG), PEG(MoL weight 2,000) l,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-DSPE) or N-palmitoylsphingosine-l-{succinyl[methoxy(polyethylene glycol)2000]} (PEG-ceramide). The molecular weight of the PEG portion in the PEG-lipid component can also vary from 500-10,000 g/mol, from 1,500-6000 g/mol, but is preferably about 2,000 MW. Other polymers used for conjugation to lipid anchors may include poly(2-methyl-2-oxazoline) (PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), polyN-vinylpyrrolidone (PVP), polyglycerol, poly(hydroxyethyl L-asparagine) (PHEA), and poly(hydroxyethyl L-glutamine) (PEIEG).

A PEG-containing lipid may further comprise an amine, amide, ester, carboxyl, phosphate, choline, hydroxyl, acetal, ether, heterocycle, or carbohydrate. PEG-containing lipids may comprise at least one alkyl or alkenyl group, e.g., greater than six carbon atoms in length (e.g., greater than about 8 carbons, 10 carbons, 12 carbons, 14 carbons, 16 carbons, 18 carbons, 20 carbons or more in length), e.g., in addition to a PEG moiety. In an embodiment, a PEG-containing lipid comprises a PEG moiety comprising at least 20 PEG monomers, e.g., at least 30 PEG monomers, 40 PEG monomers, 45 PEG monomers, 50 PEG monomers, 100 PEG monomers, 200 PEG monomers, 300 PEG monomers, 500 PEG monomers, 1000 PEG monomers, or 2000 PEG monomers. Exemplary PEG-containing lipids include PEG-DMG (e.g., DMG-PEG2k), PEG-c-DMG, PEG-DSG, PEG-DPG, 73

PEG-DSPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, and PEG-DLPE. In some embodiments, the PEG-lipids include PEG-DMG (e.g., DMG-PEG2k), PEG-c-DMG, PEG-DSG, and PEG-DPG. Additional PEG-lipids that may be included in an LNP described herein are disclosed in Fahy, E. et al. (J. Lipid. Res. 46:839-862 (2005) which is incorporated herein by reference in its entirety.

In some embodiments, an LNP comprises an alkylene glycol-containing lipid having a structure of Formula (VII):

(VII) or a pharmaceutically acceptable sait thereof, wherein each R²⁸ is independently alkyl, alkenyl, or heteroalkyl, each of which is optionally substituted with R^F; A is absent, O, CH2, C(O), or NH; E is absent, alkyl, or heteroalkyl, wherein alkyl or heteroalkyl is optionally substituted with carbonyl; each R^F is independently alkyl, halo, hydroxy, amino, cycloalkyl, or heterocyclyl; and z is an integer between 10 and 200 (inclusive).

In some embodiments, each R²⁸ is independently alkyl. In some embodiments, each R²⁸ is independently heteroalkyl. In some embodiments, each R²⁸ is independently alkenyl.

In some embodiments, A is O or NH. In some embodiments, A is CH2. In some embodiments,

A is carbonyl. In some embodiments, A is absent.

In some embodiments, E is alkyl. In some embodiments, E is heteroalkyl. In some embodiments, both A and E are not absent. In some embodiments, A is absent. In some embodiments, E is absent. In some embodiments, either one of A or E is absent. In some embodiments, both A and E are independently absent.

In some embodiments, z is an integer between 10 and 200 (e.g., between 20 and 180, between 20 and 160, between 20 and 120, between 20 and 100, between 40 and 80, between 40 and 60, between 40 and 50). In some embodiments, z is 45.

In some embodiments, the PEG-lipid is PEG-DMG (e.g., DMG-PEG2k). In some embodiments, the PEG-lipid is a-(3’-{[l,2-di(myristyloxy)propanoxy] carbonylamino}propyl)-œmethoxy, polyoxyethylene (PEG-c-DMG). In some embodiments, the PEG-lipid is PEG-DSG. In some embodiments, the PEG-lipid is PEG-DPG.

An LNP may comprise an alkylene glycol-containing lipid at a concentration greater than about 0.lmol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration of greaterthan about 0.5mol%, about lmol%, about L5mol%, about 2mol%, about 3mol%, about 4mol%, about 5mol%, about 6mol%, about 8mol%, about 10mol%, about I2mol%, about I5mol%, about 20mol%, about 50mol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration of greater than about lmol%, about 4mol%, or about 6mol%. In an embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration between about O.lmol% to about 50mol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration between about 0.5mol% to about 40mol%, about lmol% to about 35mol%, about l.5mol% to about 30mol%, about 2mol% to about 25mol%, about 2.5mol% to about 20%, about 3mol% to about I5mol%, about 3.5mol% to about I0mol%, or about 4mol% to 9mol%, e.g., of the total lipid content of the LNP. In an embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration between about 3.5mol% to about I0mol%. In an embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration between about 4mol% to 9mol%.

In some embodiments, the LNP comprises at least two types of lipids. In an embodiment, the LNP comprises two of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP comprises at least three types of lipids. In an embodiment, the LNP comprises three of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP comprises at least four types of lipids. In an embodiment, the LNP comprises each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid.

The LNP (e.g., as described herein) may comprise one or more of the following components: (i) an ionizable cationic lipid at a concentration between about lmol% to about 95mol% (e.g. about 20mol% to about 80mol%); (ii) a phospholipid at a concentration between 0. lmol% to about 50mol% (e.g. between about 2.5mol% to about 20mol%); (iii) a sterol at a concentration between about lmol% to about 95mol% (e.g. about 20mol% to about 80mol%); and (iv) a PEG-containing lipid at a concentration between about O.lmol% to about 50mol% (e.g. between about 2.5mol% to about 20mol%). In an embodiment, the LNP comprises one of (i)-(iv). In an embodiment, the LNP comprises two of (i)-(iv). In an embodiment, the LNP comprises three of (i)-(iv). In an embodiment, the LNP comprises each of (i)-(iv). In some embodiments, the LNP comprises (i) and (ii). In some embodiments, the LNP comprises (i) and (iii). In some embodiments, the LNP comprises (i) and (iv). In some embodiments, the LNP comprises (ii) and (iii). In some embodiments, the LNP comprises (ii) and (iv). In some embodiments, the LNP comprises (iii) and (iv). In some embodiments, the LNP comprises (i), (ii), and (iii). In some embodiments, the LNP comprises (i), (ii), and (iv). In some embodiments, the LNP comprises (ii), (iii), and (iv).

The LNP (e.g., as described herein) may comprise one or more of the following components: (i) Ionizable cationic lipid (ICL) at a concentration between about lmol% to about 95mol% (e.g. about 20mol% to about 80mol%); (ii) DSPC at a concentration between 0.1mol% to about 50mol% (e.g. between about 2.5mol% to about 20mol%); (iii) cholestérol at a concentration between about lmol% to about 95mol% (e.g. about 20mol% to about 80mol%); and (iv) DMG-PEG2k at a concentration between about 0. lmol% to about 50mol% (e.g. between about 2.5mol% to about 20mol%). In an embodiment, the LNP comprises two of (i)-(iv). In an embodiment, the LNP comprises three of (i)-(iv). In an embodiment, the LNP comprises each of (i)-(iv). In some embodiments, the LNP comprises (i) and (ii). In some embodiments, the LNP comprises (i) and (iii). In some embodiments, the LNP comprises (i) and (iv). In some embodiments, the LNP comprises (ii) and (iii). In some embodiments, the LNP comprises (ii) and (iv). In some embodiments, the LNP comprises (iii) and (iv). In some embodiments, the LNP comprises (iii) and (iv). In some embodiments, the LNP comprises (i), (ii), and (iii). In some embodiments, the LNP comprises (i), (ii), and (iv). In some embodiments, the LNP comprises (ii), (iii), and (iv).

In an embodiment, the LNP comprises a ratio of ionizable lipid to phospholipid of about 50:1 to about 1:1 (e.g., 40:1, 32:3, 6:1, 7:1, 5:1, 24:5, 26:5, 10:3, 15:2, 16:7, 18:1, 3:1, 3:2, or 1:1). In an embodiment, the LNP comprises a ratio of ionizable lipid to phospholipid of about 15:2. In an embodiment, the LNP comprises a ratio of ionizable lipid to phospholipid of about 5:1. In an embodiment, the LNP comprises a ratio of ionizable lipid to a sterol of about 10:1 to about 1:10 (e.g., 9:1,8:1,8:7,7:1, 7:5,7:3,6:1,6:5,5:1,5:3,4:1,4:3,3:1,2:1, 1:1, 1:2, 1:3,3:4, 1:4,3:5, 1:5,4:5, 1:6, 5:6, 7:6, 7:8, or 8:9). In an embodiment, the LNP comprises a ratio of ionizable lipid to an alkylenecontaining lipid of about 1:10 to about 10:1 (e.g., 1:9, 1:8, 7:8, 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3:1, 2:1, 1:1, 1:2, 1:3, 3:4, 1:4, 3:5, 1:5, 4:5, 1:6, 5:6, 7:6, 7:8, or 8:9). In an embodiment, the LNP comprises a ratio of phospholipid to an alkylene-containing lipid of about 10:1 to about 1:10 (e.g., 9:1, 8:1, 8:7, 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3:1, 2:1, 1:1, 1:2, 1:3, 3:4, 1:4, 3:5, 1:5, 4:5, 1:6, 5:6, 7:6, 7:8, or 8:9). In an embodiment, the LNP comprises a ratio of a sterol to an alkylenecontaining lipid of about 50:1 to about 1:1 (e.g., 40:1,32:3, 6:1, 7:1, 5:1, 24:1, 22:1, 20:1, 22:5, 24:5, 26:5, 10:3, 15:2, 16:7, 18:1, 3:1, 3:2, or 1:1).

In an embodiment, a LNP (e.g., described herein) comprises two of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid (e.g., PEG-containing lipid). In another embodiment, a LNP (e.g., described herein) comprises three of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid (e.g., PEG-containing lipid). In an embodiment LNP (e.g., described herein) comprises each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid (e.g., PEG-containing lipid).

In some embodiments, an LNP described herein has a diameter between 5 and 500 nm, e.g., between 10 and 400 nm, 20 and 350 nm, 25 and 325 nm, 30 and 300 nm, 50 and 250 nm, 60 and 200 nm, 75 and 190 nm, 80 and 180 nm, 100 and 200 nm, 200 and 300 nm, and 150 and 250 nm. The diameter of an LNP may be determined by any method known in the art, for example, dynamic light scattering, transmission électron microscopy (TEM) or scanning électron microscopy (SEM). In some embodiments, an LNP has a diameter between 50 and 100 nm, between 70 and 100 nm, and between 80 and 100 nm. In an embodiment, an LNP has a diameter of about 90 nm. In some embodiments, an LNP described herein has a diameter greater than about 30 nm. In some embodiments, an LNP has a diameter greater than about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about 225 nm, about 250 nm, about 275 nm or about 300 nm. In an embodiment, an LNP has a diameter greater than about 70 nm. In an embodiment, an LNP has a diameter greater than about 90 nm. In an embodiment, an LNP has a diameter greater than about 180 nm.

In some embodiments, a plurality of LNPs described herein has an average diameter ranging from about 40 nm to about 180 nm. In some embodiments, a plurality of LNPs described herein has an average diameter from about 50 nm to about 150 nm. In some embodiments, a plurality of LNPs described herein has an average diameter from about 50 nm to about 120 nm. In some embodiments, a plurality of LNPs described herein has an average diameter from about 60 nm to about 120 nm. In some embodiments, a plurality of LNPs has an average diameter of about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm

In some embodiments, a nanoparticle or plurality of nanoparticles described herein has an average neutral to négative surface charge of less than -100 mv, for example, less than -90 mv, -80 mv, -70 mv, -60 mv, -50 mv, -40 mv, -30 mv, and -20 mv. In some embodiments, a nanoparticle or plurality of nanoparticles has a neutral to négative surface charge of between -I00 mv and 100 mv, between -75 mv to 0, or between -50 mv and -10 mv.

In some embodiments, at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the nanoparticles of a plurality of nanoparticles hâve an average neutral to négative surface charge of less than -100 mv. In some embodiments, a nanoparticle or plurality of nanoparticles has an average surface charge of between -20 mv to +20, between -10 mv and +10 mv, or between -5 mv and +5 mv at pH 7.4. LNPs that are neutral in charge hâve improved pharmacokinetics and biological performance compared to cationic LNPs.

Making Lipid Nanoparticles (LNPs)

The method of making an LNP can comprise mixing a first solution with a second solution. Mixing can be achieved using standard liquid mixing techniques, such as propellor mixing, vortexing solutions or preferably through microfluidic mixing or high efficiency T-mixing. In some embodiments, the first solution comprises a lipid or a plurality of lipids and a nucleic acid, where ail components are solubilized, in water/solvent System. The solvent may be any water miscible solvent (e.g., éthanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethylsulfoxide, dioxane or tetrahydrofuran). In some embodiments, the first solution comprises a small percentage of water or pH buffered water. The first solution may comprise up to at least 60% by volume of water, e.g., up to at least about 0.05%, 0.1%, 0.5%, l%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55% or 60% by volume of water. In an embodiment, the first solution comprises between about 0.05% and 60% by volume of water, e.g., between about 0.05% and 50%, about 0.05% and 40%, or about 5% and 20% by volume of water.

In some embodiments, the first solution comprises a single type of lipid, for example, an ionizable lipid, a phospholipid, a sterol, or a PEG-containing lipid. In some embodiments, the first solution comprises a plurality of lipids. In some embodiments, the plurality comprises an ionizable lipid, a phospholipid, a sterol, or a PEG-containing lipid. In some embodiments, the plurality of lipids comprise cholestérol, l,2-distearoyl-s/7-glycero-3-phosphocholine (DSPC),l,2-dimyristoyl-racglycero-3-methylpolyoxyethylene2000 (DMG-PEG2k) or a-(3’-{[l,2-di(myristyloxy)propanoxy] carbonylamino}propyl)-œ-methoxy, polyoxyethylene (PEG2000- C-DMG), and an ionizable lipid. The plurality of lipids may exist in any ratio. In an embodiment, the plurality of lipids comprises an ionizable lipid or sterol, a phospholipid, a sterol, a PEG-containing lipid of the above lipids or a combination thereof in a particular ratio (e.g., a ratio described herein).

In some embodiments, the second solution is water. In some embodiments, the second solution is an aqueous buffer with a pH between 3-6 (e.g., a pH of about 3, about 4, about 5, or about 6). The second solution may comprise a load component, e.g., a nucleic acid (e.g., mRNA). The second solution may comprise a small percentage of water-miscible organic solvent. The second solution may comprise up to at least 60% by volume of at least one water miscible organic solvent, e.g., up to at least about 0.05%, 0.1 %, 0.5%, l%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,l0%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% , 60% or any percent therebetween by volume of at least one organic solvent (e.g., a water miscible organic solvent). In an embodiment, the second solution comprises between about 0.05% and 60% by volume of organic solvent, e.g., between about 0.05% and 50%, about 0.05% and 40%, or about 5% and 20% by volume of organic solvent (e.g., a water miscible organic solvent). The aqueous buffer solution can be an aqueous solution of citrate buffer. In some embodiments, the aqueous buffer solution is a citrate buffer solution with a pH between 4-6 (e.g., a pH of about 4, about 5, or about 6). In an embodiment, the aqueous buffer solution is a citrate buffer solution with a pH of about 6.

In some embodiments, the solution comprising a mixture of the first and second solutions comprising the LNP suspension can be diluted. In some embodiments, the pH of the solution comprising a mixture of the first and second solutions comprising the LNP suspension can be adjusted. Dilution or adjustment of the pH of the LNP suspension can be achieved with the addition of water, acid, base or aqueous buffer. In some embodiments, no dilution or adjustment of the pH of the LNP suspension is carried out. In some embodiments, both dilution and adjustment of the pH of the LNP suspension is carried out.

In some embodiments, excess reagents, solvents, unencapsulated nucleic acid maybe removed from the LNP suspension by tangential flow filtration (TFF) (e.g., diafiltration). The organic solvent (e.g., éthanol) and buffer may also be removed from the LNP suspension with TFF. In some embodiments, the LNP suspension is subjected to dialysis and not TFF. In some embodiments, the LNP suspension is subjected to TFF and not dialysis. In some embodiments, the LNP suspension is subjected to both dialysis and TFF.

In one aspect, the présent disclosure features a method comprising treating a sample of LNPs comprising nucleic acid, with a fluid comprising a detergent (e.g., Triton X-100, or anionic détergents (such as, but not limited to, sodium dodecyl sulfate (SDS), or non-ionic detergent, such as but not limited to β-octylglucoside, or Zwittergent 3-14) for a period of time suitable to dégradé the lipid layer and thereby release the encapsulated and/or entrapped nucleic acid(s). In an embodiment, the method 79 further comprises analyzing the sample for the presence, absence, and/or amount of the released nucleic acid(s).

LNP comprising ligands

Some aspects of the disclosure relate to LNP comprising a ligand (also referred herein as targeting ligand) having a binding specificity for a cell surface antigen, wherein the binding of the ligand to the antigen induces the intemalization of the ligand. Some embodiments relate to compositions comprising LNP comprising a ligand as described herein.

In some embodiment, the targeting ligand is coupled to a lipid conjugale. For example, the lipid conjugale can be a hydrophilic polymer-lipid conjugale such as, but not limited to, PEG(2000)DSPE or PEG(2000)-DSG. Coupling can be achieved by a variety of chemistries know in the art (for example, see Bioconjugates Techniques (Greg T. Hermanson), 3rd Edition, 2013, Elsevier). In some embodiment, the targeting ligand is coupled to the lipid conjugale through a linker. The linker molécule generally contains a hydrophilic polymer chain, such as PEG-terminally linked to a lipid domain (phospholipid or sterol) and contains a thiol-reactive functional group such as a maleimide at the terminus. The linkers comprising PEG spacers of size, phosphatidylethanolamine (PE) lipid anchors of various hydrocarbon chain length, and terminal maleimide or iodoacetate groups are currently commercially available from Avanti Polar Lipids (Alabama, USA) and NOF Corporation (Japan). One such strategy commonly used is to couple protein to a thiol-reactive lipopolymer linker, such as l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000 (Mal-PEG-DSPE). Preferably, the protein of interest is engineered to contain one cysteine in the Cterminus to ensure a single-point conjugation. Altematively, F(ab)2 or Fab' can be enzymatically generated from IgGs and by réduction of disulfide bonds with a reducing agent such as dithiothreitol (DTT), mercaptoethylamine, (tris(2-carboxyethyl)phosphine) TCEP-HCL generate reactive cysteine thiol groups to couple to Mal-PEG-DSPE. The reaction of Mal-PEG-DSPE with reduced cysteine takes place in aqueous buffer at pH 5.5-7.5, for example pH 5.5,. 6, 6.5, 7, 7.5, and preferably pH 6.0. The reaction is typically complété within 4 hours. A small amount of cysteine or mercaptoethanol is added to react with unreacted maleimide groups and quenches the coupling reaction. Although it is not necessary to remove unconjugated protein prior to the subséquent membrane insertion step it is useful to purify the conjugale for the purposes of storage and allow more précisé characterization. Due to the large size of the lipopolymer micelles (équivalent molecular weight 850 kDa; Nellis et al., 2005a), size exclusion chromatography (SEC) is a convenient way to do so. Characterization of such 80 protein-conjugates is achieved by a variety of techniques. For example, the purity is determined by SEC, molecular weight is quantitated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), protein melting points by differential scanning calorimetry (DSC), isoelectric point détermination by capillary electrophoresis and target binding affinity by surface plasmon résonance (BIAcore) and biolayer interferometry (ForteBio).

Examples of targeting ligands may be antibodies or antibody fragments against cell surface receptors, including the Her2 receptor, epidermal growth factor receptor (EGFR) receptor, Ephrin A2 receptor, CLEC9A receptor, DEC205 receptor, CLEC4A receptor, XCR1 receptor, CD 141 receptor, HLA-DR receptor, transferrin receptor type 1, transferrin receptor type 2, VEGF receptor, PDGF receptor, integrin, NGF receptor, CD 19, CD20, CD22, CD33, CD43, CD38, CD56, CD69, prostate spécifie membrane antigen (PSMA) or a variety of other cell surface receptors, or glycoconjugates, proteoglycans, glycoproteins, and glycolipids such as the glycoconjugate N-acetylgalactosamine (GalNAc) ligand which binds the asialoglycoprotein receptor (ASGPR), or small molécule conjugates such as folate-PEG-DSPE which targets the folate receptor.

In one embodiment, the targeting ligand is an anti-DEC205 antibody. DEC-205 (CD205) is a type I cell surface protein expressed primarily by dendritic cells (DC). It is found on interdigitating DC in T cell areas of lymphoid tissues, bone marrow-derived DC, Langerhan’s cells, and at low levels on macrophages and T cell and is significantly up-regulated during the maturation of DC. Expression of DEC-205 is positively correlated with that of CD8a, both being found at high levels on lymphoid DC and at low levels on myeloid DC. DEC-205 is also expressed at moderate levels by B cells and is up-regulated during the pre-B cell to B cell transition. Recombinant anti-human DEC205 antibody is commercially available from Creative Biolabs.

In one embodiment, antigen spécifie targeting on LNPs is achieved by preparing ligandtargeted LNP by co-incubation of LNP with targeting ligand-lipid conjugale. The targeting ligandlipid conjugale may be prepared prior to LNP préparation (see Nellis et al. Biotechnol Prog. 2005 JanFeb;21(l):205-20).

In one embodiment, LNPs are co-incubated with antibody or fragment -PEG-phospholipid micelles or other ligand-conjugate and heated at 37° C ovemight to promote antibody conjugale insertion into the LNP outer membrane (Nellis et al. Biotechnol Prog. 2005 Jan-Feb;21( 1):221-32). In another aspect, insertion can be achieved by heating at elevated températures for shorter time periods, for example 0.5-8h at 37° C, or preferably 0.5-2h at 37° C. Micellar insertion can be quenched by lowering the température quickly by putting the LNPs on ice after which they can be stored in the refrigerator at 4°C. The total amount of lipid conjugale added can be between 0.02%-2% of total lipid, or preferably O.l%-l%, or preferably 0.l%-0.5% total lipid. The incorporation efficiency of antibody-lipid conjugale can be measured by SDS-PAGE after LNP dissociation by SDS or other détergents by comparison to a standard curve of the same protein (Nellis et al. Biotechnol Prog. 2005 Jan-Feb;21(1):205-20). The insertion efficiency of other targeting ligands can be measured by ultra high performance liquid chromatography equipped with evaporative light scattering detector (UPLCELSD) (Gauthier et al., J Mol Sci. 2019 Nov 12;20(22):5669).

FIG. 2 shows the reaction of the reduced c-terminal cysteine of a Fab’ antibody fragment with a maleimide terminated-poly(ethylene glycol) 2000 derivatized distearoylphosphatidylethanolamine. RI and R2 are stearic acid. The final antibody lipopolymer conjugale is an intermediate that is subsequently inserted into the outer lipid layer of lipidic nanoparticle to make it actively targeted.

LNP targeting can also accomplished by adding lipids to the formulation. For example, phosphatidylserine is known to redistribute to the extemal surface of the plasma membrane during apoptosis and is a molecular eue for phagocytotic cell attraction (Fadok et al. Cuit Biol. 2003 Aug 19;13(16):R655-7). Phosphatidylserine (PS) and phosphatidylglycerol (PG) are recognized by dendritic cells and can induce uptake and activation of dendritic cells LNP targeting can also accomplished by adding certain anionic phospholipids to the formulation (Table 3). For example, phosphatidylserine is known to redistribute to the extemal surface of the plasma membrane during apoptosis and is a molecular eue for phagocytotic cell attraction (Fadok et aL Cuit Biol. 2003 Aug 19;13(16):R655-7). Phosphatidylserine (PS) and phosphatidylglycerol (PG) are recognized by dendritic cells and can induce uptake and activation of dendritic cells (Caronni et aL, Nat Comm. 2021 April 14; 12: 2237-2253; Ischihashi et aL, PLOS One 2013). Although anionic phospholipids hâve been used previously in the context of liposomes, their inclusion in lipidic nanoparticles that include condensed nucleic acids is unexpected since anionic headgroups may compete for binding sites of the ionizable cationic lipids with the phosphate backbone of mRNA, may inhibit intracellular escape by altering the surface charge, or may resuit in aggregation of LNPs during formation or storage.

Table 3. Anionic Phospholipid Targeting Moieties

DSPS (L-isomer)

DPPS (L-isomer)

DMPS (L-isomer)

DOPS (L-isomer)

DSPS (D-isomer)

o

DSPG

DPPG

o

N-Glu-DSPE

O

N-Suc-DSPE

In one embodiment, the anionic targeting ligands are selected from the group, phosphatidylserine (PS), phoshatidylglycerol (PG), N-glutaryl-phosphatidylethanolamine (N-glu5 PE), or N-succinyl-phosphatidylethanolamine (N-Suc-PE). In one embodiment, the anionic phospholipid used is phosphatidylserine. In another embodiment, the phosphatidylserine contains the L-isomer of serine. In another embodiment, the acyl chains for the phosphatidylserine are fully saturated, such as the case for dimyristoylphosphatidyl-L-serine (DMPS), dipalmitoylphosphatidylL-serine (DPPS), or distearoylphosphatidyl-L-serine (DSPS). In a preferred embodiment, the PS used is the L-isomer of either DPPS or DSPS. The phosphatidylserine may also contain an asymmetric acyl chain composition, for example where one acyl chain is stearic acid and another is palmitic acid.

In one embodiment, PS or PG are added to the LNP lipid formulation at a concentration between about O.l mol% to about 20 mol%, about 0.1 mol% to about 10 mol%, about 0.1 mol% to about 5 mol%, about 0.5 mol% to about 20 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 5 mol%, about l mol% to about 20 mol%, about l mol% to about 10 mol%, or about l mol% to about 5 mol%, of the total lipid content of the LNP. In one embodiment, the PS is added to the LNP lipid formulation at a concentration between about l mol% to about 20 mol%, about 2.5 mol% to about 10 mol%, about 3 mol% to about 9 mol%, or about 4 mol% to about 8 mol%, of the total lipid content of the LNP.

In one embodiment the PS lipid is included in the LNP composition comprising ionizable cationic lipids known in the art, including DOD AP, AKG-OA-DM2, O-Il 769, DLin-MC3-DMA, DLin-KC2-DMA, DLin-KC3-DMA, ALC-0315, and SM-102.

In another embodiment the PS lipid is included in the LNP composition comprising ICLs of Formula I, II, III, combinations thereof or pharmaceutically salts thereof. In another embodiment the PS lipid is included in the LNP composition using N/P ratios between 3 and 8, between 4 and 7, or between 5 and 6.

In some aspects, a method of delivering a nucleic acid to a cell is provided, the method comprising: contacting the cell with a composition comprising an LNP comprising a ligand (also referred herein as targeting ligand) having a binding specificity for a cell surface antigen, wherein the binding of the ligand to the antigen induces the intemalization of the ligand. In some embodiments, the targeting ligand can be, but is not limited to, an intemalizing antibody, or a fragment thereof, a small molécule conjugates or gylcoconjugates. In some embodiments, the binding of the targeting ligand to a spécifie cell surface antigen induces the intemalization of the LNP with the targeting ligand attached by a cell expressing at least 100,000 or at least 1,000,000 molécules of the antigen when contacted and incubated with the cell under intemalizing conditions.

Table 4. Exemplary dialkyl and branched ionizable cationic lipids

DODAP

AKG-0A-DM2

AKG-OA-DM3

0-11769

Dlin-MC3-DMA

AKG-KC2-0A

AKG-KC3-0A

Dlin-KC2-DMA

Dlin-KC3-DMA

Table 4. Exemplary dialkyl and branched ionizable cationic lipids (Continued)

ALC-0315

SM-102

Compositions

In some embodiments, a lipidic nanoparticle composition comprises lipids and nucleic acids, the lipidic nanoparticles comprising a compound of Formula I, II, III, combinations thereof or pharmaceutically acceptable salts thereof.

In some embodiments, an LNP comprises an ionizable lipid having a structure of Formula (IV).

In some embodiments, the composition further comprises a pharmaceutical excipient.

In some embodiments, the lipidic nanoparticles are in an aqueous medium.

In some embodiments, the nucleic acid is entrapped in the lipidic nanoparticle with a compound of Formula I, II, III, IV or combinations thereof, wherein the nucleic acid is either RNA or DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid is DNA.

In some embodiments, the lipidic nanoparticle comprises a membrane comprising phosphatidylcholine and a sterol. In some embodiments, the sterol is cholestérol. In some embodiments, the lipidic nanoparticle comprises a membrane comprising phosphatidylcholine, ionizable cationic lipid (ICL). In some embodiments, the ICL hâve a structure of Formula I, II, III or IV, and cholestérol, wherein the membrane séparâtes the inside of the lipidic nanoparticles from the aqueous medium. In some embodiment, the ICL hâve a structure as shown in Table 1A and Table 2. In some embodiment, the ICL hâve a structure as shown in Table IB. In some embodiments, the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC). In some embodiments, the ionizable cationic lipid to cholestérol molar ratios is from about 65:35 to 40:60. In some embodiments, the ICL to cholestérol molar ratio is from about 60:40 to about 45:55.

In some embodiments, the phosphatidylcholine to cholestérol molar ratio is from about l :5 to about l:2.

In some embodiments, the membrane further comprises a polymer-conjugated lipid.

In some embodiments, the lipidic nanoparticle comprises ICL, DSPC, cholestérol and polymer-conjugated lipid in a about 49.5:10.3:39.6:2.5 molar ratio.

In some embodiments, the polymer-conjugated lipid is PEG(2000)-dimyristoylglycerol (PEGDMG) or PEG(MoL weight 2,000)-dimyristoylphosphatidylethanolamine (PEG-DMPE).

The compositions of this disclosure may be administered by various routes, for example, to effect systemic delivery via intravenous, parentéral, intraperitoneal, or topical routes. The compositions may be administered intravenously, subcutaneously, or intraperitoneally to a subject. In some embodiments, the disclosure provides methods for in vivo delivery of nucleic acids to a subject.

In some embodiments, the composition is a liquid pharmaceutical formulation for parentéral administration.

In some embodiments, the composition is a liquid pharmaceutical formulation for subcutaneous, intramuscular, or intradermal administration.

In some embodiments, the composition is in the form of a lyophilized powder, that is subsequently reconstituted with aqueous medium prior to administration.

Methods of use

Targeting of dendritic cells

Dendritic cells (DCs) are specialized antigen-presenting cells that play a central rôle in initiating and regulating adaptive immunity. Owing to their potent antigen (Ag) présentation capacity and ability to generate distinct T-cell responses, efficient and spécifie delivery of Ags to DCs is the comerstone for generating Ag-specific effector and memory cells against tumors or pathogens.

Dendritic cells can be generated from human blood monocytes by adding granulocytemacrophage colony-stimulating factor (GM-CSF), IL-4, and IFN-gamma to differentiate monocytederived DC in vitro. Cells in culture exhibit both dendritic and veiled morphologies, the former being adhèrent, and the latter suspended. Phenotypically, they are CDla-/dim, CDlla+, CDllb++, CDUc+, CD14dim/-, CD16a-/dim, CD18+, CD32dim/-, CD33+, CD40+, CD45R0+, CD50+,

CD54+, CD64-/dim, CD68+, CD71+, CD80dim, CD86+/++, MHC class 1++/, HLA-DR++/, HLADP+, and HLA-DQ (Geiseler et al. Dev Immunol. 1998;6( 1-2):25-39).

Altematively, human primary blood dendritic cell lines hâve been developed and are commercially available from Creative Biolabs.

CD8+ T cells can produce IL2, IFN-γ, and TNF, cytokines that are known to hâve critical functions during mycobacterium tuberculosis infection. Importantly, CD8+ T cells hâve cytolytic functions to kill mycobacterium tuberculosis -infected cells via granule-mediated function (via perforin, granzymes, and granulysin) or Fas-Fas ligand interaction to induce apoptosis. In humans, CD8+ T cell can produce granulysin, which can kill mycobacterium tuberculosis directly. Therefore, it is anticipated that antigen generating mRNA LNPs delivered to DC will stimulate a CD8+ T cell response to fight against mycobacterium tuberculosis infection.

CD8+ T cells are able to recognize M. tuberculosis spécifie antigens (as peptides) presented by classical and non-classical MHC molécules. Classically restricted CD8+ T cells hâve been identified that recognize antigens presented by antigen presenting cells in the context of classical MHC la (HLA-A, -B, -C) molécules. Non-classically restricted CD8+ T cells include those CD8+ T cells that are capable of recognizing Mg antigen in the context of HLA-E molécules (non-MHC 1 a), glycolipids associated with group 1 CD1 molécules and MHC I-related molécules (MRI) such as mucosal associated invariant T cells (MAIT). Finally, γδ T cells represent a separate population of CD8 (and CD4) T cells that hâve both innate and adaptive functions in response to mycobacterium tuberculosis infection. CD8+ T cells hâve been shown to play direct functions in response to mycobacterium tuberculosis infection but they also play important rôles in orchestrating many different functions in the overall host immune response (e.g., interaction to provide optimal CD4 T cell function)

In one embodiment, LNPs can be added to cultured human dendritic cells at an appropriate concentration, (e.g. 1-5 pg/mL mRNA). After some time to allow for cellular uptake and antigen expression, human T cells (HemaCare) can be added, and the cell culture media is sampled at various times for INF-γ by Elisa (R&D Systems, DIF50C). Altematively, the cells can be analyzed by flow cytometry for CD8+ marker or intracellular INFy production (PE anti-human IFN- γ antibody, Biolegend).

In one embodiment, LNPs can be administered into a subject at a dose of 0.01-5 mg/kg mRNA by any route of administration outlined above. According to some embodiments, a proportion of LNPs are taken up DC cells, while most will accumulate in the liver and spleen. The DC cells can express the antigenic peptide, process it for MHC I présentation and travel to the lymph node for présentation to naïve T cells inducing an éducation of memory T-cells towards the antigen.

In one embodiment, LNPs that hâve been modified with a targeting ligand such as antiDEC205-PEG-DSPE can be administered into a subject at a dose of 0.01-5 mg/kg mRNA. According to some embodiments, a higher proportion of LNPs can be taken up DC cells, allowing for increased production of antigenic peptide compared to non-targeted LNP and a more efficient vaccination against the pathogen. Additional targeting ligands for dendritic cells include, but are not limited to, CLEC9A, CLEC4A, XCRl, CD141, and HLD-DR. For example, assessing the CD8+ reactivity to the in vivo produced antigen could be accomplished by measuring INFy plasma levels by species spécifie IFN-gamma Quantikine ELISA Kits from R&D Systems.

In some embodiments, LNP compositions provide désirable pharmacokinetic properties such as extended plasma half-life and stable encapsulation of mRNA. The plasma half-life can be measured as the percentage of the injected dose (ID) remaining in blood after 6 or 24 hours following injection intravenously in immunocompétent mice. The stability of the encapsulation of mRNA over 24 hours in plasma can be deteimined by changes in the mRNA-to-lipid ratio (mRNA/L ratio) following iv administration in mice. In some embodiments, the percentage of encapsulated mRNA remaining in blood is greater than 20 %, preferably greater than 30 %, and most preferably greater than 40 % of the injected dose at 6 hours. The percent retained in blood after 24 h is preferably greater than 10 %, and more preferably greater than 20 % of the injected dose.

Disclosed herein are methods for preventing mycobacteria infection, such as Mycobacterium tuberculosis, or gram positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Additional mycobacteria and gram positive bacteria include, but are not limited to, Mycobacterium avium complex, Mycobacterium leprae, Mycobacterium gordonae, Mycobacterium abscessus, Mycobacterium abscessus, Mycobacterium mucogenicum, streptococci, vancomycin-resistant enterococci (VRE), Staphylococcus pneumoniae, Enterococcus faecium, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, the viridans group streptococci, Listeria monocytogenes, Nocardia, and Corynebacterium.

Administration of a vaccine for inducing a second immune response may provide MHC class II - presented epitopes that are capable of eliciting a CD4 + helper T cell response against cells expressing antigens from which the MHC presented epitopes are derived. Altematively or additionally, administration of a vaccine for inducing a second immune response may provide MHC class I - presented epitopes that are capable of eliciting a CD8 + T cell response against cells expressing antigens from which the MHC presented epitopes are derived. Furthermore, administration of a vaccine for inducing a second immune response may provide one or more neo epitopes (including known neo epitopes) as well as one or more epitopes not containing cancer spécifie somatic mutations but being expressed by cancer cells and preferably inducing an immune response against cancer cells, preferably a cancer spécifie immune response. In one embodiment, administration of a vaccine for inducing a second immune response provides neo epitopes that are MHC class II - presented epitopes and / or are capable of eliciting a CD4 + helper T cell response against cells expressing antigens from which the MHC presented epitopes are derived as well as epitopes not containing cancer - spécifie somatic mutations that are MHC class I - presented epitopes and / or are capable of eliciting a CD8 + T cell response against cells expressing antigens from which the MHC presented epitopes are derived. In one embodiment, the epitopes do not contain cancer - spécifie somatic mutations.

A cellular immune response”, a cellular response”, a “cellular response against an antigen” or a similar term is meant to include a cellular response directed to cells characterized by présentation of an antigen with class I or class II MHC . The cellular response relates to cells called T cells or T lymphocytes which act as either “helper cells” or “killer cells”. The helper T cells (also termed CD4 + T cells ) play a central rôle by regulating the immune response and the killer cells (also termed cytotoxic T cells , cytolytic T cells , CD8 + T cells or CTLS ) kill diseased cells such as cancer cells, preventing the production of more diseased cells. In preferred embodiments, the présent disclosure involves the stimulation of an anti-Mycobacterium tiiberculosis CTL response against the mycobacterium expressing one or more expressed antigens and preferably presenting such expressed antigens with class I MHC.

An “antigen” according to the disclosure covers any substance that will elicit an immune response. In particular, an “antigen” relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T-lymphocytes (T cells). As used herein, the term “antigen” comprises any molécule which comprises at least one epitope. Preferably, an antigen in the context of the présent disclosure is a molécule which, optionally after processing, induces an immune reaction, which is preferably spécifie for the antigen (including cells expressing the antigen). According to the présent disclosure, any suitable antigen may be used, which is a candidate for an immune reaction, wherein the immune reaction is preferably a cellular immune reaction. In the context of the embodiments of the présent disclosure , the antigen is preferably presented by a cell, preferably by an antigen presenting cell which includes a diseased cell, in particular a cancer cell, in the context of

MHC molécules, which results in an immune reaction against the antigen. An antigen is preferably a product which corresponds to or is derived from a naturally occurring antigen. Such naturally occumng antigens may include tumor antigens.

As used herein, an antigen peptide ” relates to a portion or fragment of an antigen which is capable of stimulating an immune response, preferably a cellular response against the antigen or cells characterized by expression of the antigen and preferably by présentation of the antigen such as diseased cells, in particular cancer cells. Preferably, an antigen peptide is capable of stimulating a cellular response against a cell characterized by présentation of an antigen with class I MHC and preferably is capable of stimulating an antigen - responsive cytotoxic T - lymphocyte (CTL). Preferably, the antigen peptides according to the disclosure are MHC class I and / or class II presented peptides or can be processed to produce MHC class I and / or class II presented peptides. Preferably, the antigen peptides comprise an amino acid sequence substantially corresponding to the amino acid sequence of a fragment of an antigen. Preferably, said fragment of an antigen is an MHC class I and / or class II presented peptide. Preferably, an antigen peptide according to the disclosure comprises an amino acid sequence substantially corresponding to the amino acid sequence of such fragment and is processed to produce such fragment, i.e., an MHC class I and / or class II presented peptide derived from an antigen. If a peptide is to be presented directly, i.e., without processing, in particular without cleavage, it has a length which is suitable for binding to an MHC molécule, in particular a class I MHC molécule, and preferably is 7 - 20 amino acids in length, more preferably 7 - 12 amino acids in length, more preferably 8 - 11 amino acids in length, in particular 9 or 10 amino acids in length.

The main types of professional antigen - presenting cells are dendritic cells, which hâve the broadest range of antigen présentation, and are probably the most important antigen - presenting cells, macrophages, B - cells, and certain activated épithélial cells. Dendritic cells (DCs ) are leukocyte populations that présent antigens captured in peripheral tissues to T cells via both MHC class II and I antigen présentation pathways . It is well known that dendritic cells are potent inducers of immune responses and the activation of these cells is a critical step for the induction of antitumoral immunity. Dendritic cells are conveniently categorized as “immature” and “mature” cells, which can be used as a simple way to discriminate between two well characterized phenotypes.

However, this nomenclature should not be construed to exclude ail possible intermediate stages of différentiation. Immature dendritic cells are characterized as anti gen presenting cells with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcy receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression 91 of these markers, but a high expression of cell surface molécules responsible for T cell activation such as class I and class II MHC , adhesion molécules (e.g. CD54 and CD11) and costimulatory molécules (e .g., CD40 , CD80 , CD86 and 4 - 1 BB). Dendritic cell maturation is referred to as the status of dendritic cell activation at which such antigen - presenting dendritic cells lead to T cell priming, while présentation by immature dendritic cells results in tolérance. Dendritic cell maturation is chiefly caused by biomolecules with microbial features detected by innate receptors (bacterial DNA, viral RNA, endotoxin, etc), pro-inflammatory cytokines (TNF, IL - 1, IFNs), ligation of CD40 on the dendritic cell surface by CD4OL, and substances released from cells undergoing stressful cell death . The dendritic cells can be derived by culturing bone marrow cells in vitro with cytokines, such as granulocyte - macrophage colony - stimulating factor (GM CSF) and tumor necrosis factor alpha. Non - professional antigen-presenting cells do not constitutively express the MHC class II proteins required for interaction with naive T cells; these are expressed only upon stimulation of the non - professional antigen-presenting cells by certain cytokines such as IFNy. Antigen presenting cells” can be loaded with MHC class I presented peptides by transducing the cells with nucleic acid, preferably mRNA, encoding a peptide or polypeptide comprising the peptide to be presented, e.g. a nucleic acid encoding the antigen.

In some embodiments, a pharmaceutical composition comprising a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo. As used herein, a “ nucleic acid ” is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, most preferably in vitro transcribed RNA (IVT RNA ) or synthetic RNA. Nucleic acids include according to the disclosure genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molécules. According to the disclosure , a nucleic acid may be présent as a single - stranded or double - stranded and linear or covalently circularly closed molécule. A nucleic acid can, according to the disclosure , be isolated. The term “ isolated nucleic acid ” means, according to the disclosure , that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and séparation by gel electrophoresis, or (iv) was synthesized, for example, by Chemical synthesis. A nucleic can be employed for introduction into, i.e. transfection of cells , in particular, in the form of RNA which can be prepared by in vitro transcription from a DNA template. The RNA can moreover be modified before application by stabilizing sequences, capping, and polyadenylation.

As used herein , the tenu “ RNA” relates to a molécule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues. “Ribonucleotide” relates to a nucléotide with a hydroxyl group at the 2 ' - position of a B-Dribofuranosyl group. The term “ RNA ” comprises double - stranded RNA, single - stranded RNA, isolated RNA such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinantly generated RNA such as modified RNA which differs from naturally occurring RNA by addition, délétion, substitution and / or alteration of one or more nucléotides. Such alterations can include addition of non - nucléotide material, such as to the end (s) of a RNA or intemally, for example at one or more nucléotides of the RNA. Nucléotides in RNA molécules can also comprise non - standard nucléotides, such as non-naturally occurring nucléotides or chemically synthesized nucléotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally - occurring RNA.

As used herein, the term “RNA” includes and preferably relates to “mRNA”. The term mRNA” means messenger - RNA” and relates to a transcript ” which is generated by using a DNA template and encodes a peptide or polypeptide. Typically, an mRNA comprises a 5 ' - UTR, a protein coding région, and a 3 ' - UTR . mRNA only possesses limited half - life in cells and in vitro. In the context of the présent disclosure , mRNA may be generated by in vitro transcription from a DNA template. The term “ modification” in the context of the RNA used in the présent disclosure includes any modification of an RNA which is not naturally présent in said RNA. In one embodiment of the disclosure, the RNA used according to the disclosure does not hâve uncapped 5'-triphosphates. Removal of such uncapped 5'- triphosphates can be achieved by treating RNA with a phosphatase. The RNA according to the disclosure may hâve modified ribonucleotides in order to increase its stability and / or decrease cytotoxicity. For example, in one embodiment, in the RNA used according to the disclosure 5-methylcytidine is substituted partially or completely, preferably completely, for cytidine. Altematively or additionally, in one embodiment, in the RNA used according to the disclosure pseudouridine is substituted partially or completely, preferably completely, for uridine.

In one embodiment, the term “modification” relates to providing an RNA with a 5 - cap or 5' - cap analog. The term “5 - cap” refers to a cap structure found on the 5 ' - end of an mRNA molécule and generally consists of a guanosine nucléotide connected to the mRNA via an unusual 5' to 5 triphosphate linkage. In one embodiment, this guanosine is methylated at the 7 - position. The term conventional 5' - cap” refers to a naturally occurring RNA 5 '-cap, preferably to the 7 methylguanosine cap (m'G). as used herein, the term “5' - cap” includes a 5' - cap analog that resembles the RNA cap structure and is modified to possess the ability to stabilize RNA and / or enhance translation of RNA if attached thereto, preferably in vivo and / or in a cell.

According to the disclosure , the stability and translation efficiency of RNA may be modified as required. For example, RNA may be stabilized and its translation increased by one or more modifications having a stabilizing effects and/or increasing translation efficiency of RNA. Such modifications are described, for example, in PCT /EP2006 /009448 incorporated herein by reference. In order to increase expression of the RNA used according to the présent disclosure , it may be modified within the coding région, i.e. the sequence encoding the expressed peptide or protein , preferably without altering the sequence of the expressed peptide or protein, so as to increase the GC content to increase mRNA stability and to perform a codon optimization and , thus, enhance translation in cells.

Aspects of the disclosure relate to a method of preventing a bacterial or viral infection, the method comprising administering to a subject in need thereof an effective amount of the composition provided herein to elicit an immune response.

Aspects of the disclosure provide methods of vaccinating a subject comprising administering to the subject a single dosage of the compositions described herein comprising a nucleic acid (e.g. mRNA) encoding an polypeptide in an effective amount to vaccinate the subject. In some embodiments, the nucleic acid is formulated within a cationic lipidic nanoparticle. In some embodiments, the lipidic nanoparticle composition is administered as a single injection.

In some embodiments, the bacterial infection is Mycobacterium tuberculosis infection.

In some embodiments, the viral infection is a coronavirus. In some embodiments, the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2

In some embodiments, the viral infection is HIV/AIDS.

In some embodiments, the lipidic nanoparticle is administered parenterally.

In general, administration to a patient by intradermal injection is possible. However, injection may also be carried out intranodally into a lymph node (Maloy et al. (2001), Proc Natl Acad Sci USA 98:3299-3033). The resulting cells présent the complex of interest and are recognized by autologous cytotoxic T lymphocytes which then propagate.

In some embodiments, the compositions is administered by inhalation. In some embodiments, the composition is formulated as nasal spray, and/or aérosol

Actual dosage levels of the active agents in the pharmaceutical compositions disclosed herein may be varied so as to obtain an amount of the active agent which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

“Parentéral” as used herein in the context of administration means modes of administration other than enterai and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, épidural and intrastemal injection and infusion.

The phrases “parentéral administration” and “administered parenterally” as used herein refer to modes of administration other than enterai (i.e., via the digestive tract) and topical administration, usually by injection or infusion, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, inhalation, subcapsular, subarachnoid, respiratory mucosal, intraspinal, épidural and intrastemal injection and infusion. Intravenous injection and infusion are often (but not exclusively) used for liposomal drug administration.

Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, one or more doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

In some embodiments, the dose comprises between 0.01 to 5 mg/kg of nucleic acid. In some embodiments, the dose comprises between 0.01 to 5 mg/kg of mRNA. In some embodiments, the dose comprises between 0.01 to 3 mg/kg of nucleic acid. In some embodiments, the dose comprises between 0.01 to 3 mg/kg of mRNA. In some embodiments, the dose comprises between 0.01 to 1 mg/kg of nucleic acid. In some embodiments, the dose comprises between 0.01 to 1 mg/kg of mRNA. In some embodiments, the dose comprises between 0.01 to 0.5 mg/kg of nucleic acid. In some embodiments, the dose comprises between 0.01 to 0.5 mg/kg of mRNA. In some embodiments, the dose comprises between 0.01 to 1 mg/kg of mRNA. In some embodiments, the dose comprises between 0.01 to 0.1 mg/kg of nucleic acid. In some embodiments, the dose comprises between 0.01 to 0.05 mg/kg of mRNA. In some embodiments, the dose comprises between 0.01 to 0.1 mg/kg of nucleic acid. In some embodiments, the dose comprises between 0.01 to 0.05 mg/kg of mRNA.

The dosage of the compounds and/or of their pharmaceutically acceptable salts or the LNPs comprising the compounds and/or of their pharmaceutically acceptable salts may vary within wide 95 limits and should naturally be adjusted, in each particular case, to the individual conditions and to the pathogenic agent to be controlled.

Additional Embodiments

l. A compound of Formula I or pharmaceutically acceptable salts thereof.

2. A compound of Formula II or pharmaceutically acceptable salts thereof.

3. A compound of Formula III or pharmaceutically acceptable salts thereof.

4. A compound of Formula IV or pharmaceutically acceptable salts thereof.

5. A compound having a structure as in Table IA.

6. A compound having a structure as in Table 2, wherein the compound is bioreducible.

7. The compound of anyone of embodiments l-6 above, wherein the compound has a pKa between 6 and 7.

8. A lipidic nanoparticle composition comprising a ionizable lipid of Formula I or pharmaceutically acceptable salts thereof, and a nucleic acid.

9. A lipidic nanoparticle composition comprising a ionizable lipid of Formula II or pharmaceutically acceptable salts thereof, and a nucleic acid.

10. A lipidic nanoparticle composition comprising a ionizable lipid of Formula III or pharmaceutically acceptable salts thereof, and a nucleic acid.

11. A lipidic nanoparticle composition comprising a ionizable lipid of Formula IV or pharmaceutically acceptable salts thereof, and a nucleic acid.

12. The composition of any one of embodiments 8-ll above, wherein the ionizable lipid encapsulate the nucleic acid.

13. The composition of any one of embodiments 8-l l, wherein the nucleic acid is a siRNA.

14. The composition of any one of embodiments 8-l l, wherein the nucleic acid is a DNA.

15. The composition of any one of embodiments 8-l l, wherein the nucleic acid is a mRNA.

16. The composition of any one of embodiments 8-H, further comprising a sterol, phosphatidylcholine, or combinations thereof.

17. The composition of embodiment 16, wherein the sterol is cholestérol.

18. The composition of embodiment 17, wherein the ionizable lipid to cholestérol molar ratios is from about 65:35 to about 40:60.

19. The composition of embodiment 17, wherein the ionizable lipid to cholestérol molar ratios is from about 60:40 to about 45:55.

20. The composition of embodiment 17, wherein the phosphatidylcholine to cholestérol molar ratio is from about l :5 to about l :2.

21. The composition of embodiment 17, further comprising a polymer-conjugated lipid.

22. The composition of embodiment 21, wherein the polymer-conjugated lipid comprises PEG(2000)-dimyristoylglycerol (PEG-DMG) or PEG(MoL weight 2,000)dimyristoylphosphatidylethanolamine (PEG-DMPE).

23. The composition of any one of embodiments 8-H, further comprising a targeting ligand, wherein the targeting ligand is oriented to the outside of the nanoparticle.

24. The composition of embodiment 23, wherein the targeting ligand is an antibody.

25. The composition of any one of embodiments 8-H, wherein the composition is a liquid pharmaceutical formulation.

26. The composition of any one of embodiments 8-H, wherein the percentage of oxidative dégradation products for the ionizable lipid is less than 50 % of that for a DLin-KC2-DMA or DLinMC3-DMA control formulation.

27. A method of preventing a bacterial or viral infection, the method comprising administering to a subject in need thereof an effective amount of the composition of any one of embodiments 9-26, and a pharmaceutical excipient, wherein administration elicits an immune response.

28. The method of embodiment 27, wherein the composition is administered for subcutaneously, intramuscularly, or intradermally.

29. The method of embodiment 27, wherein the bacterial infection is Mycobacterium tuberculosis infection.

30. The method of embodiment 27, wherein the viral infection is a SARS-CoV, MERS-CoV or SARS-CoV-2 infection.

31. The method of embodiment 27, wherein the viral infection is a HIV infection.

32. The method of embodiment 27, wherein the infection is a form of nontuberculosis mycobacterium.

33. A lipid nanoparticle (LNP) composition comprising an ionizable lipid having a Chemical structure consisting of a pair of 16 or 18 carbon linear polyunsaturated lipid tails covalently bound to a head group comprising a dialkyl amino group having a pKa of between 6 and 7;

the head group comprising a heterocyclyl or alkyl portion covalently bound to the dialkyl amino group and optionally further comprising a phosphate group;

each polyunsaturated lipid tail being unsaturated except for the at least two olefins separated by at least two methylene groups along the length of the lipid tail, and optionally comprising a single acyl group at the end covalently bound to the head group.

34. The composition of embodiment 33, wherein each lipid tail is identical, and each lipid tail has a total of two olefins separated only by an unsubstituted ethylene, n-propyl, or n-butyl.

35. The composition of embodiment 34, wherein each lipid tail further comprises an acyl group joined to an oxygen of the headgroup to form an ester.

36. The composition of embodiment 34, wherein each lipid tail has the Chemical structure of

Formula A or Formula B:

Formula A wherein a in Formula A is l, 2, 3 or 4; b is 2, 3 or 4; and c in Formula A is 3, 4, 5, 6, ,7; or

Formula B.

wherein a in Formula B is 5, 6 or 7; and c in Formula B is 3, 4, or 5.

37. The composition of embodiment 36, wherein b is 4.

38. The composition of claim 36, wherein the ionizable lipid comprises a Chemical structure selected from the group consisting of: R O

R²²

wherein R²² is the first end of the lipid tail and ? indicates attachment of the head group to the dialkyl amino portion of the head group.

39. The composition of embodiment 38, wherein the dialkyl amino portion of the head group has a Chemical structure of Formula (IV-A)

Rio

Formula IV-A, wherein n is 2, 3 or 4 in Formula (IV-A); and Rio and R12 in Formula (IV-A) are each independently selected from an alkyl group selected from the group consisting of: methyl, ethyl, and propyl, wherein the alkyl in Rio and R12 is optionally substituted with one or more hydroxyl.

40. The composition of embodiment 39, wherein Rio and R12 in Formula (IV-A) are each independently methyl, ethyl, -(CH2)(CH2)OH, or -(CH2)2(CH2)OH.

41. The composition of embodiment 33, wherein the ionizable lipid has a Chemical structure of Formula (I-A)

a is 1, 2, 3, 4, 5 or 6; b is 2, 3 or 4; c is 3, 4, 5, 6, or 7; and the sum of a, b and c is 10 or 12;

each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with one or more hydroxyl; and

42. The composition of claim 41, wherein v is 0.

43. The composition of claim 41, wherein v is l.

44. The composition of embodiment 33, wherein the ionizable lipid has a Chemical structure of Formula II-A:

wherein a is 1, 2, 3, 4, 5 or 6; b is 2, 3 or 4; c is 4, 5, 6, 7 or 8;

q and q’ are each independently 1 or 2; and

Rio and R12 are each (Ci-Cflalkyl optionally substituted with hydroxyL

45. The composition of embodiment 33, wherein the ionizable lipid has a Chemical structure of Formula II-A:

Formula (Π-Β)

100 wherein a is 5, 6 or 7; and c is 3, 4, or 5;

q and q’ are each independently 1 or 2; and

Rio and R12 are each (Ci-C4)alkyl optionally substituted with hydroxyL

EXAMPLES

While this disclosure has been described in relation to certain embodiments, and many details hâve been set forth for purposes of illustration, it will be apparent to those skilled in the art that this disclosure includes additional embodiments, and that some of the details described herein may be varied considerably without departing from this disclosure. This disclosure includes such additional embodiments, modifications and équivalents. In particular, this disclosure includes any combination of the features, terms, or éléments of the various illustrative components and examples.

Unless explicitly indicated otherwise, the isomer form of the phosphatidylserine lipids used in the Examples is phosphatidyl-L-serine.

Certain examples are provided below to illustrate various embodiments of the embodiments disclosed herein. One of ordinary skill in the art will recognize that the various embodiments disclosed herein are not limited to these spécifie illustrative examples.

Example IA: Synthesis of Ionizable Lipids

Scheme 1 Synthesis of acid intermediates for AKG-UO-1 to AKG-UO-3

101

PPh Θ _Br^^^_OH _Br PhjP ''------ -----------HO'

MsCI

O

H₅IO₄, PCC

PPh₃ „0 ® ------ Br ph₃P

For AKG-UO-1, AKG-UO-2, and AKG-UO-3

MsCI

A A

Br Ph₃P'^^^'OH --------M:

MgBr₂ ------ Br

HslOi PCC

PPh₃ Θ ® ----Br ph₃p

The acid intermediates (6Z,12Z)-6,12-octadecadienoic acid and (6Z, 12Z)-6,12-hexadecadienoic acid were prepared by a general synthesis, shown in (S)-4-(dimethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(octadeca-6,12-dienoate) (AKG-UO-1, 5 0-11956) (S)-4-(diethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(octadeca-6,12-dienoate) (AKG-UO-1 A,

0-11955) (S)-4-(dimethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(hexadeca-6,12-dienoate, AKG-UO-4,

0-12401) (S)-4-(diethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(hexadeca-6,12-dienoate, AKG-UO-4A, 0-12402) (S)-4-(dimethylamino)butane-l,2-diyl (6Z,6'Z,11Z, 1 TZ)-bis(octadeca-6,1 l-dienoate)(AKG-UO-la)

102

Scheme 7

TsOH, CH₂CI₂

1. NiiOAc^ tetrahedrate, NaBH₄. EtOH, H₂ balloon

R, = H 5

CH₂CH₃ 6 nBuLi,HMPA THF

-78 °C-20 °C

Ethylènediamine, H₂ balloon

P-2 Ni, H₂”

TsOH, MeOH

R, = H 11

CH2CH₃ 12

Jones reagent acetone

Scheme 8

Scheme 9

R₂ = CH₃ 21

CH₂CH₃ 22

Compounds	Ri	r2
AKG-UO-1	ch2ch3	ch3
AKG-UO-1A	ch2ch3	ch2ch3
AKG-UO-4	H	ch3
AKG-UO-4A	H	ch2ch3

Experimental Procedure

Synthesis of 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran 2

To a solution of 5-bromo-l-pentanol 1 (3.6 g, 21.6 mmol) in dichloromethane (lOOmL) and pyridinium p-toluene sulfonate (40 mg, 0.16 mmol) at 0 °C was added 3,4-dihydro-2H-pyran (6.54 mL, 71.8 mmol). The resulting solution was stirred at room température for one hour then quenched 103 with water. The mixture was extracted with ethyl acetate (2Xl00mL). The combined organics were washed with brine then dried over magnésium sulfate, fdtered, and the filtrate concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 5-10% ethyl acetate in n-hexane as eluant to give 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran, 2 (4.5 g, 83%) as a clear oil.

1H NMR (300 MHz, CDC13): ô ppm 4.55-4.54 (d, J= 4.3 Hz, 1H), 3.92-3.72 (m, 2H), 3.42-3.38 (m, 3H), 1.88-1.55 (m, 3H), 1.52-1.50 (m, 10H).

Synthesis of 2-(trideca-6,12-diyn-l-yloxy)tetrahydro-2H-pyran 4

To a solution of 1,7-Octadiyne 3 (6 mL, 45.4 mmol) and hexamethylphosphoramide (16 mL, 90.8 mmol) in tetrahydrofuran (100 mL) at -78 °C was added [2.5 M n-butyllithium in n-hexane] (18 mL, 45.4 mmol) dropwise. Upon completion of addition, the solution was stirred at -78 °C for one hour then warmed to -20 °C for an additional hour. The resulting solution was cooled once again to -78 °C whereupon a solution of 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran, 2 (5.67 g, 22.7mmol) in tetrahydrofuran (10 mL) was added. The resulting solution was allowed to warm to room température and stirred for 12 hours. After 12 hours, the reaction was cooled to 0 °C and quenched with water (100 mL). The reaction mixture was then concentrated under vacuum to remove tetrahydrofuran and then diluted with n-hexane. The organics were washed with water and brine (2X100 mL). The organic layer was dried over magnésium sulfate, fdtered, and the fdtrate concentrated under vacuum to give a crude oil weighing 9 g. The crude oil was purified by

104 chromatography on silica using 5-10% ethyl acetate in n-hexane as eluant to give 2-(trideca-6,12diyn-l-yloxy) tetrahydro-2H-pyran, 4 (4.5 g, 72%) as a clear oil.

1H NMR (300 MHz, dô.DMSO): δ ppm 4.544.53 (m, 1H), 3.72-3.61 (m, 1H), 3.60-3.58 (m, 1H), 3.43-3.33 (m, 1H), 3.32-3.29 (m, 1H), 2.77-2.75 (t, J= 5.8 Hz, 1H), 2.16-2.13 (m, 6H), 1.55-1.41 (m, 16H).

Représentative Procedure for alkylation of alkynes

Synthesis of 2-(hexadeca-6,12-diyn-l-yloxy) tetrahydro-2H-pyran 7

.....

To a solution of 2-(trideca-6,12-diyn-l-yloxy) tetrahydro-2H-pyran, 4 (7.14 g, 25.86 mmol) and hexamethylphosphoramide (18 mL, 103.4 mmol) in tetrahydrofuran (100 mL) at -78 °C was added [2.5 M n-butyllithium in n-hexane] (41.3 mL, 103.4 mmol) dropwise. Upon completion of addition, the solution was stirred at -78 °C for one hour then warmed to -20 °C for an additional hour. The resulting solution was cooled once again to -78 °C whereupon a solution of 1-iodopropane 5 (9.9 mL, 103.4 mmol) in tetrahydrofuran (20 mL) was added. The resulting solution was allowed to warm to room température and stirred for 12 hours. After 12 hours, the reaction was cooled to 0 °C and quenched with water (100 mL). The reaction mixture was then concentrated under vacuum to remove tetrahydrofuran and then diluted with n-hexane. The organics were washed with water and brine (2X100 mL). The organic layer was dried over magnésium sulfate, filtered, and the fdtrate concentrated under vacuum to give a crude oil weighing 9 g. The crude oil was purified by chromatography on silica using 5% ethyl acetate in n-hexane as eluant to give 2-(hexadeca-6,12diyn-l-yloxy)tetrahydro-2H-pyran, 7 (5.9 g, 72%) as a clear oil.

105 ^lHNMR(300 MHz, CDC13): 4.57-4.55 (m, IH), 3.86-3.74 (m, IH), 3.73-3.71 (m, IH), 3.50-3.39 (m, IH), 3.37-3.36 (m, IH), 2.16-2.11 (m, 8H), 1.59-1.56 (m, 2H), l.55-1.47 (m, 16H), 0.98-0.93 (t, J = 1.6 Hz, 3H).

2-(octadeca-6,12-diyn-1 -yloxy)tetrahydro-2H-pyran 8

0°.......-—.....

IH NMR (300 MHz, CDC13): 4.57-4.55 (m, IH), 3.85-3.74 (m, IH), 3.73-3.70 (m, IH), 3.50-3.38 (m, IH), 3.36-3.35 (m, IH), 2.23-2.12 (m, 8H), 1.61-1.54 (m, 2H), 1.53-1.48 (m, 16H), 1.47-1.46 (m, 4H), 0.90-0.85 (t, J = 1.6 Hz, 3H).

Représentative Procedure for réduction of alkynes to alkenes using “P-2 Ni” Synthesis of 2-(((6Z, 12Z)-hexadeca-6,12-dien-1 -yl)oxy)tetrahydro-2H-pyran 9

To a solution of Sodium borohydride (0.56 g, 14.8 mmol) in éthanol (80 mL) under hydrogen blanket at 0 °C was added Nickel (II) acetate tetrahydrate (3.22 g, 12.98 mmol). Upon completion of addition, the reaction was evacuated under vacuum and flushed with hydrogen. After 10 minutes of stirring, ethylenediamine (3.7 mL, 65.6 mmol), and a solution of 2-(hexadeca-6,l2-diyn-1yloxy)tetrahydro-2H-pyran, 7 (5.9 g, 18.55 mmol) in éthanol (10 mL) was added. The reaction was stirred at room température under a hydrogen balloon for 4 hours. After 4 hours, the reaction mixture was evacuated of hydrogen and then flushed with nitrogen. The crude mixture was filtered over celite, and the filtrate concentrated under vacuum to give a crude oil weighing 4 g. The crude oil was purified by chromatography on silica using 5-10 % diethyl ether in n-hexane as eluant to give 2-(((6Z,12Z)-hexadeca-6,l 2-dien-l-yl)oxy)tetrahydro-2H-pyran, 9 (4.67 g, 78% yield) as a clear oil.

106

1H NMR (300 MHz, CDC13): 5.35-5.34 (m, 4H), 4.58-4.55 (m, 1H), 3.86-3.74 (m, 1H), 3.73-3.71 (m, 1H), 3.51-3.39 (m, 1H), 3.36-3.35 (m, 1H), 2.03-1.98 (m, 8H), 1.57-1.39 (m, 2H), 1.38-1.36 (m, 6H), 1.35-1.32 (m, 10H), 0.91-0.86 (t, J= 1.6 Hz, 3H).

^13C NMR (300 MHz, CDC13): 129.98, 129.85, 98.93, 77.53, 77.10, 76.68, 67.72, 62.43, 30.86, 29.71, 29.70, 29.45, 29.46, 29.44, 27.20, 27.19, 26.01, 25.59, 22.98, 19.78, 13.91.

2-(((6Z, 12Z)-octadeca-6,12-dien-1 -yl)oxy)tetrahydro-2H-pyran 10

1H NMR (300 MHz, CDC13): 5.39-5.29 (m, 4H), 4.58-4.55 (m, 1H), 3.86-3.76 (m, 1H), 3.74-3.68 (m, 1H), 3.51-3.41 (m, 1H), 3.39-3.36 (m, 1H), 2.14-1.97 (m, 8H), 1.56-1.38 (m, 2H), 1.37-1.35 (m, 6H), 1.34-1.28 (m, 14H), 0.93-0.85 (t, J = 1.6 Hz, 3H).

13C NMR (300 MHz, CDCI3): 130.13, 129.97, 129.84, 129.71, 98.93, 77.53, 77.10, 76.68, 67.71, 62.42, 31.62, 30.86, 29.72, 29.71, 29.47, 29.46, 27.27, 27.18, 26.01, 25.59, 22.67, 19.78, 14.18.

Représentative Procedure for deprotection of tetrahvdropyranyl ether (THP)

Synthesis of (6Z,12Z)-hexadeca-6,l 2-dien-l-ol 11

To a solution of 2-(((6Z, 12Z)-hexadeca-6,l 2-dien- l-yl)oxy)tetrahydro-2H-pyran, 9 (4.67 g, 14.5 mmol) in methanol (20 mL) was added p-Toluenesitlfonic acid monohydrate (300 mg, 1.58 mmol) at room température. The resulting solution was stirred at room température for 3 hours then quenched with water. The mixture was extracted with ethyl acetate (2X50 mL). The combined organics were

107 washed with water then dried over magnésium sulfate, fdtered and the fdtrate concentrated under vacuum to give a crude oil weighing 4 g. The crude oil was purified by chromatography on silica using 5-10 % diethyl ether in n-hexane as eluant to give (6Z, !2Z)-hexadeca-6,l2-dien-l-ol, 11 (2.5 g, 72%) as a clear oil.

1H NMR (300 MHz, CDC13): 5.34-5.33 (m, 4H), 3.65-3.61 (m, 2H), 2.02-2.00 (m, 8H), 1.36-1.34 (m, 2H), 1.34-1.25 (m, 10H), 0.89-0.86 (t, J= 0.82 Hz, 3H).

(6Z, 12Z)-octadeca-6,12-dien-l-ol 12

IH NMR (300 MHz, CDC13): 5.36-5.33 (m, 4H), 3.65-3.61 (m, 2H), 2.02-2.01 (m, 8H), 1.36-1.35 (m, 2H), 1.34-1.25 (m, 14H), 0.88-0.85 (t, J = 0.76 Hz, 3H).

Représentative Procedure for oxidation of alcohol to carboxylic acid using Jones reagent

Synthesis of (6Z, 12Z)-hexadeca-6,12-dienoic acid 13

A mixture of (6Z, 12Z)-hexadeca-6,12-dien-l-ol, 11 (2.5 g, 10.5 mmol) and Jones Reagent [2M in sulfuric acid], (10.5 mL, 21 mmol) in acetone (20 mL) at 0 °C was stirred for 2 hours. The mixture was quenched with water and extracted with ethyl acetate (2X100 mL). The combined organics were dried over magnésium sulfate, fdtered and the fdtrate concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 20 % ethyl acetate in nhexane as eluant to give (6Z, 12Z)-hexadeca-6,12-dienoic acid, 13 (1.7 g, 68%) as a clear oil.

108

IH NMR (300 MHz, CDC13): 5.35-5.33 (m, 4H), 2.37-2.32 (t, 2H), 2.06-1.98 (m, 8H), 1.64-1.39 (m, 2H), l .37-1.32 (m, 8H), 0.91-0.87 (t, J = 0.91 Hz, 3H).

(6Z, 12Z)-octadeca-6,12-dienoic acid 14

O

1H NMR (300 MHz, CDC13): 5.36-5.32 (m, 4H), 2.35-2.33 (t, 2H), 2.06-2.01 (m, 8H), 1.64-1.42 (m, 2H), 1.34-1.28 (m, 12H), 0.90-0.85 (t, 3H).

Synthesis of (5)-2-(2,2-dimethyl-l,3-dioxolan-4-yl)ethyl 4-methylbenzenesulfonate 16

-I θ

To a mixture of (5)-2-(2,2-dimethyl-l,3-dioxolan-4-yl)ethan-l-ol 15 (25 g, 171.1 mmol) in pyridine (30 mL) at 0 °C was addedp-Toluenesulfonylchloride (35.8 g, 188.2 mmol) and DMAP (140 mg, 1.14 mmol) and the reaction was stirred at room température ovemight. The mixture was diluted with CH2C12 (500 mL), washed with sat. NH4C1, water and Brine. The organic layer was dried over anhydrous Na2SO4. The solvent was evaporated, and the crude residue used for the next step without purification. (43.8 g, 85%).

1H NMR (300 MHz, CDC13): δ ppm 7.77 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 4.15-4.01 (m, 3H), 3.65-3.47 (m, 2H), 2.43 (s, 3H), 1.82-1.62 (m, 2H), 1.32 (s, 3H), 1.27 (s, 3H).

Représentative Procedure for di-alkylamine substitution

Synthesis of (S)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)-N,N-dimethylethan-1 -amine 19

I

A mixture of (S)-2-(2,2-dimethyl-l,3-dioxolan-4-yl)ethyl 4-methylbenzenesulfonate 16 (10 g, 33.3 mmol) and dimethylamine solution 17 (166 mL, 333.3 mmol) (2M in THF) was stirred at room température for 2 days. The mixture was concentrated, and the crude residue was diluted with

109

CH2C12 (500 mL), washed with sat. NaHCO3, water and Brine. The organic layer was dried over anhydrous Na2SO4. The solvent was evaporated, and the crude residue was purified by flash chromatography (SiO2: CH2C12 = 100% to 10% of MeOH in CH2C12 with 1%NH4OH) and colorless oil product 19 was obtained (2.1 g, 37%).

1H NMR (300 MHz, CDC13): b'ppm 4.15-4.01 (m, 2H), 3.52 (dd, J=1A, 7.4 Hz, 1H), 2.41-2.23 (m, 2H), 2.21 (s, 6H), 1.82-1.62 (m, 2H), 1.39 (s, 3H), 1.33 (s, 3H).

MS (APCI+): 174.1 (M+l) (S)-2-(2,2-diethyl-1,3-dioxolan-4-yl)-N,N-dimethylethan-1 -amine 20

1H NMR (300 MHz, CDC13): δ ppm 4.15-4.01 (m, 2H), 3.48 (dd, J = 7.4, 7.4 Hz, 1H), 2.48-2.43 (m, 6H), 1.82-1.62 (m, 2H), 1.36 (s, 3H), 1.27 fs, 3H), 0.97 (t, J = 7.2 Hz, 6H).

MS (APCI+): 202.2 (M+l)

Représentative Procedure for ketal hydrolysis

Synthesis of (S)-4-(dimethylamino)butane-l,2-diol hydrochloride sait 21

To a mixture of (S)-2-(2,2-dimethyl-l,3-dioxolan-4-yl)-N,N-dimethylethan-l-amine 19 (2 g, 11.54 mmol) in MeOH (10 mL) was added IN HCl aqueous solution (17 mL, 17.3 mmol) and the reaction was heated at 80 °C for 45 min. TLC (Rf = 0.1, 10%MeOH in CH2C12 with 1%NH4OH) showed the completion of reaction. After concentration of the reaction mixture, the crude residue was dissolved in water (5 mL) and lyophilized ovemight. Sticky syrup product 21 was obtained (2.1 g, quant.) as HCl sait.

1H NMR (300 MHz, D2O): δ ppm 3.77-3.72 (m, 1H), 3.54-3.46 (m, 2H), 3.29-3.22 (m, 2H), 2.85 (s, 6H), 1.92-1.79 (m, 2H).

MS (APCI+): 134.1 (M+l)

110 (5)-4-(diethylamino)butane-l,2-diol hydrochloride sait 22

1H NMR (300 MHz, D2O): b ppm 3.77-3.72 (m, 1H), 3.54-3.46 (m, 2H), 3.22-3.15 (m, 6H), 1.921.74 (m, 2H), 1.24 (t, J = 7.4 Hz, 6H).

MS (APCI+): 162.1 (M+l)

Représentative Procedure for di-esterification

Synthesis of (S)-4-(dimethylamino)butane-l,2-diyl (6Z,6'Z,12Z,12'Z)-bis(octadeca-6,12-dienoate)

AKG-UO-1 (0-11956)

AKG-UO-1 O

Oxalyl chloride (0.33 mL, 3.9 mmol) was added dropwise to a solution of (6Z, 12Z)-octadeca-6,12dienoic acid, 14 (0.36 g, 1.3 mmol) in dichloromethane / DMF (15mLs, 25 DL) at 0 °C. and allowed reaction to warm to room température and stir for one hour. After one hour, the reaction was concentrated under vacuum to dryness. The residue was re-dissolved in dichloromethane (10 mL) and added to a mixture of N,N-Diisopropylethylamine (2.3 mL, 10 mmol), 4Dimethylaminopyridine (317 mg, 2.6 mmol), and (S)-4-(dimethylamino)butane-l,2-diol hydrochloride, 21 (101 mg, 0.6 mmol). The resulting solution was allowed to stir for 24 hours. After 24 hours, the reaction was cooled to OC and quenched with water (10 mL). The reaction mixture was extracted with dichloromethane (2X100 mL) and the organics were washed with water and brine (2X100 mL). The organic layer was dried over magnésium sulfate, filtered, and the filtrate concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 2% methanol in dichloromethane as eluant to give (5)-4(dimethylamino)butane-l,2-diyl (6Z,6'Z,12Z, 12'Z)-bis(octadeca-6,12-dienoate), AKG-UO-1, (0.12 g, 30%) as a yellow oil.

111

IH NMR (300 MHz, CDC13): 5.40-5.29 (m, 8H), 5.14-5.12 (m, IH), 4.25 (dd, J = 11.8, 3.3 Hz, IH), 4.05 (dd, J= I2.l, 6.3 Hz, IH), 2.32-2.26 (m, 6H), 2.20 (s, 6H), 2.06-1.99 (m, 16H), 1.78-1.70 (m, 2H), 1.65-1.58 (m, 4H), 1.42-1.25 (m, 24H), 0.90-0.85 (m, 6H).

MS (APCI+): 658.5 (M+l) (S)-4-(diethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(octadeca-6,12-dienoate) AKG-UO-1A (0-11955)

AKG-UO-1A

IH NMR (300 MHz, CDC13).· 5.37-5.29 (m, 8H), 5.12-5.10 (m, IH), 4.25 (dd, J = 12.1, 3.6 Hz, IH), 4.05 (dd, J = 11.8, 6.3 Hz, IH), 2.52-2.42 (m, 6H), 2.29 (t, J = 7.4 Hz, 4H)), 2.06-1.99 (m, 16H), 1.78-1.70 (m, 2H), 1.64-1.59 (m, 4H), 1.41-1.19 (m, 24H), 0.99 (t, J = 7.1 Hz, 6H), 0.96-0.87 (m, 6H).

MS (APCI+): 686.6 (M+l) (S)-4-(dimethylamino)butane-l,2-diyl (6Z,6'Z,12Z,12'Z)-bis(hexadeca-6,12-dienoate) AKGUO-4 (0-12401)

AKG-UO-4

1HNMR(3OO MHz, CDC13): 5.39-5.29 (m, 8H), 5.13-5.12 (m, IH), 4.24 (dd, J = 11.8, 3.3 Hz, IH), 4.05 (dd, J = 11.8, 6.3 Hz, IH), 2.32-2.27 (m, 6H), 2.19 (s, 6H), 2.01-1.99 (m, 16H), 1.75-1.72 (m, 2H), 1.65-1.58 (m, 4H), 1.36-1.31 (m, 16H), 0.91-0.86 (m, 6H).

MS (APCI+): 602.5 (M+l)

Synthesis of (S)-4-(diethylamino)butane-l,2-diyl (6Z,6’Z,12Z,12'Z)-bis(hexadeca-6,12-dienoate)

AKG-UO-4A (0-12402)

112

AKG-UO-4A

IH NMR (300 MHz, CDC13Y 5.40-5.29 (m, 8H), 5.12-5.11 (m, IH), 4.25 (dd, J = 11.8, 3.3 Hz, l H), 4.05 (dd, J = 11.8, 6.3 Hz, l H), 2.54-2.43 (m, 6H), 2.29 (t, J = 7.4 Hz, 4H), 2.11 -1.96 (m, 16H), 1.74-1.65 (m, 2H), 1.65-1.59 (m, 4H), 1.39-1.31 (m, 16H), 0.99 (t, J = 7.1 Hz, 6H), 0.91-0.89 (m, 6H).

MS (APC1+): 630.5 (M+l), involving i) an initial Witting reaction of triphenyl phosphonium ylide, prepared from 5-bromo pentanol, and the corresponding aldéhyde, ii) conversion of the terminal alcohol to bromide by mesylation and substitution, iii) repeating the sequence of ylide synthesis and Witting reaction, and finally iv) periodic acid oxidation of the terminal alcohol. The resulting acid intermediates were utilized in the synthesis of AKG-UO-1 to AKG-UO-4, vide infra.

Scheme 2 Synthesis of acid intermediate for AKG-UO-5

113

OSIR3

Lindlar

H₂

For AKG-U0-5

The acid intermediate (9Z,l5Z)-9,15-octadecadienoic acid used in the synthesis of AKG-UO-5 was prepared by a general synthesis shown in Scheme 2, involving i) alkylation of silyl protected IOhydroxy-l-decyne with (5Z)-l-bromo-5-octene, ii) catalytic hydrogénation of the alkyne to a cis5 alkene, iii) removal of silyl protection on the alcohol, and finally iv) oxidation of the terminal alcohol to the desired acid.

Scheme 3 Synthesis of acid intermediates for AKG-BDG-Ol and AKG-BDG-02

For AKG-BDG-01 and AKG-BDG-02

114

Synthesis of two disulfide acid intermediates used in the synthesis of AKG-BDG-1 and AKG-BDG2 is shown in

Synthesis of (5}-4-(dîmethylamino)butane-l,2-diyl (6Z,6'Z,llZ,irZ)-bis(octadeca-6,ll5 dienoate)(AKG-UO-la)

1. Ni(OAc)2 tetrahedrate, NaBH_4! EtOH, H2 balloon

2. Ethylènediamine, H2 balloon

TsOH, MeOH

P-2 Ni, H₂

Jones reagent acetone

Oxalyl chloride, DMF. CH₂CI₂

HO H

10a

8a

AKG-UO-1a

Experimental Procedure

Synthesis of 2-((5-bromopentyl)oxy)tetrahydro-2H-pvran 2

To a solution of 5-bromo-1 -pentanol 1 (3.6 g, 21.6 mmol) in dichloromethane (lOOmL) and pyridinium p-toluene sulfonate (40 mg, 0.16 mmol) at 0 °C was added 3,4-dihydro-2H-pyran (6.54 mL, 71.8 mmol). The resulting solution was stirred at room température for one hour then quenched with water. The mixture was extracted with ethyl acetate (2XlOOmL). The combined organics were 15 washed with brine then dried over magnésium sulfate, fdtered, and the fdtrate concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 5-10%

115 ethyl acetate in n-hexane as eluant to give 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran, 2 (4.5 g, 83%) as a clear oiL ^1H NMR (300 MHz, CDC13): δ ppm 4.55-4.54 (d, J= 4.3 Hz, 1H), 3.92-3.72 (m, 2H), 3.42-3.38 (m, 3H), 1.88-1.55 (m, 3H), 1.52-1.50 (m, 10H).

Synthesis of 2-(dodeca-6,l l-divn-l-yloxy)tetrahydiO-2H-pyran 4a

4a

To a solution of 1,6-heptadiyne 3a (5 g, 54.3 mmol) and hexamethylphosphoramide (19 mL, 108 mmol) in tetrahydrofuran (100 mL) at -78 °C was added [2.5 M n-butyllithium in n-hexane] (21.7 mL, 54.3 mmol) dropwise. Upon completion of addition, the solution was stirred at -78 °C for one hour then warmed to -20 °C for an additional hour. The resulting solution was cooled once again to 78 °C whereupon a solution of 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran, 2 (6.8 g, 27.1 mmol) in tetrahydrofuran (10 mL) was added. The resulting solution was allowed to warm to room température and stirred for 12 hours. After 12 hours, the reaction was cooled to 0 °C and quenched with water (100 mL). The reaction mixture was then concentrated under vacuum to remove tetrahydrofuran and then diluted with n-hexane. The organics were washed with water and brine (2X100 mL). The organic layer was dried over magnésium sulfate, filtered, and the filtrate concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 5-10% ethyl acetate in n-hexane as eluant to give 2-(dodeca-6,l 1-diyn-lyloxy)tetrahydro-2H-pyran, 4a (4.1 g, 58%) as a clear oil.

^1H NMR (300 MHz, CDC13): δ ppm 4.57-4.56 (m, 1H), 3.96-3.82 (m, 1H), 3.77-3.69 (m, 1H), 3.503.41 (m, 1H), 3.39-3.34 (m, 1H), 2.29-2.25 (m, 4H), 2.15-2.12 (m, 2H), 1.95-1.94 (t, 7= 5.8 Hz, 1H), 1.73-1.43 (m, 14H).

116

Synthesis of 2-(octadeca-6, l l-diyn-l-yloxy)tetrahydro-2H-pyran 6a

6a

To a solution of 2-(dodeca-6, l l-diyn-l-yloxy)tetrahydro-2H-pyran, 4a (4.1 g, 15.64 mmol) and hexamethylphosphoramide (l l mL, 62.6 mmol) in tetrahydrofuran (100 mL) at -78 °C was added [2.5 M n-butyllithium in n-hexane] (12.5 mL, 31.3 mmol) dropwise. Upon completion of addition, the solution was stirred at -78 °C for one hour then warmed to -20 °C for an additional hour. The resulting solution was cooled once again to -78 °C whereupon a solution of 1-iodohexane 5a (9.5 mL, 62.6 mmol) in tetrahydrofuran (20 mL) was added. The resulting solution was allowed to warm to room température and stirred for 12 hours. After 12 hours, the reaction was cooled to 0 °C and quenched with water (100 mL). The reaction mixture was then concentrated under vacuum to remove tetrahydrofuran and then diluted with n-hexane. The organics were washed with water and brine (2X100 mL). The organic layer was dried over magnésium sulfate, filtered, and the fdtrate concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 5% ethyl acetate in n-hexane as eluant to give 2-(octadeca-6,l 1 -diyn-1 yloxy)tetrahydro-2H-pyran, 6a (3.1 g, 57%) as a clear oil.

¹¹¹ NMR (300 MHz, CDC13): 4.58-4.55 (m, 1H), 3.86-3.82 (m, 1H), 3.77-3.69 (m, 1H), 3.51-3.47 (m, 1H), 3.41-3.34 (m, 1H), 2.26-2.21 (m, 6H), 2.14-2.12 (m, 6H), 1.66-1.26 (m, 18H), 0.93-0.85 (t, J =6.5 Hz, 3H).

Synthesis of 2-(((6Z,l lZ)-octadeca-6,1 l-dien-l-yl)oxy)tetrahydro-2H-pyran 7a

7a

To a solution of Sodium borohydride (0.27 g, 14.8 mmol) in éthanol (50 mL) under hydrogen blanket at 0 °C was added Nickel (II) acetate tetrahydrate (1.55 g, 6.25 mmol). Upon completion of addition, the reaction was evacuated under vacuum and flushed with hydrogen. After 10 minutes of stirring, ethylenediamine (1.8 mL, 26.8 mmol), and a solution of 2-(octadeca-6,l 1-diyn-1

117 yloxy)tetrahydro-2H-pyran, 6a (3.1 g, 8.93 mmol) in éthanol (10 mL) was added. The reaction was stirred at room température under a hydrogen balloon for 4 hours. After 4 hours, the reaction mixture was evacuated of hydrogen and then flushed with nitrogen. The crude mixture was filtered over celite, and the filtrate concentrated under vacuum to give a crude oil weighing 4 g. The crude oil was purified by chromatography on silica using 5-10 % diethyl ether in n-hexane as eluant to give 2-(((6Z,l lZ)-octadeca-6,l l-dien-l-yl)oxy)tetrahydro-2H-pyran, 7a (2.86 g, 92% yield) as a clear oil.

^IH NMR (300 MHz, CDC13): 5.4-5.34 (m, 4H), 4.58-4.55 (m, 1H), 3.86-3.82 (m, 1H), 3.74-3.68 (m, 1H), 3.51-3.49 (m, 1H), 3.41-3.36 (m, 1H), 2.06-1.99 (m, 6H), 1.83-1.67 (m, 2H), 1.59-1.51 (m, 6H),1.48-1.32 (m, 16H), 0.92-0.85 (t, J= 6.6 Hz, 3H).

Synthesis of (6Z, 1 lZ)-octadeca-6,l 1-dien-l-ol 8a

8a

Procedure previously described.

^1H NMR (300 MHz, CDC13): 5.37-5.33 (m, 4H), 3.65-3.61 (m, 1H), 2.06-1.99 (m, 6H), 1.56-1.41 (m, 4H), 1.38-1.27 (m, 14H), 0.88-0.85 (t, J = 6.6 Hz, 3H).

Synthesis of (6Z, 1 lZ)-octadeca-6,l 1-dienoic acid 9a

O

9a

Procedure previously described.

^IH NMR (300 MHz, CDC13): 5.38-5.33 (m, 4H), 2.37-2.33 (t, J = 5.6 Hz, 2H), 2.06-1.99 (m, 6H), 1.67-1.59 (m, 2H), 1.41-1.25 (m, 14H), 0.89-0.85 (t, J= 6.6 Hz, 3H).

Synthesis of (5)-4-(dimethylamino)butane-l,2-diyl (6Z,6'Z,11Z, 1 TZ)-bis(octadeca-6,l 1dienoate)(AKG-UO-la)

118

AKG-UO-la

Procedure previously described.

^1H NMR (300 MHz, CDC13): 5.39-5.29 (m, 8H), 5.14-5.12 (m, 1H), 4.25 (dd, J= 11.8, 3.3 Hz, 1H), 4.06 (dd, J = 11.8,6.3 Hz, 1 H), 2.32-2.28 (m, 6H), 2.20 (s, 6H), 2.03-2.01 (m, 16H), 1.74-1.64 (m, 2H), 1.62-1.60 (m, 6H), 1.38-1.27 (m, 22H), 0.89-0.85 (m, 6H).

MS (APCI+): 658.5 (M+l)

A general synthesis of acid intermediate for AKG-BDG-1 involves i) synthesis of 4mercapto butyric acid from 4-bromo butyric acid, ii) reaction of 4-mercapto butyric acid with DPS resulting in 4-(2-pyridinyldisulfanyl)butanoic acid iii) catalytic hydrogénation of 3-decyn-l-ol to a cA-alkene, iv) tosylation of the primary alcohol, v) displacement of the tosyl group using thiourea resulting in a terminal thiol, and finally vi) coupling of the terminal thiol with 4-(2pyridinyldisulfanyl)butanoic acid prepared in step ii above, resulting in the disulfide containing acid intermediate. Following a similar synthetic sequence starting from 3-dodecyn-l-ol yielded the second acid intermediate used in the synthesis of AKG-BDG-2.

Scheme 4 Synthesis of AKG-UO-1, AKG-UO-4, AKG-UO-5, AKG-BDG-1 and AKG-BDG-2

119

A general synthesis of lipids AKG-UO-l, AKG-UO-4, AKG-UO-5, AKG-BDG-l and AKG-BDG-2 shown in Scheme 4, involves the following steps: i) tosylation of the primary alcohol of the 5 commercially available chiral dioxolane ii) displacement of the tosyl group using dimethylamine resulting in a tertiary amine, iii) acid catalyzed deprotection of the diol, and frnally iv) estérification of the diol with the corresponding acid intermediates synthesized according to Schemes l-3. AKGUO-2 is prepared following a similar synthetic sequence starting from a different dioxolane and a corresponding acid intermediate, as shown in Scheme 5 below.

Scheme 5 Synthesis of AKG-UO-2

o

AKG-UO-2

A general synthesis of trialkyl phosphate containing lipid AKG-UO-3 shown in Scheme 6, involves the following steps: i) reaction of primary alcohol of a commercially available chiral dioxolane with 15 methyl dichlorophosphite resulting in the corresponding dialkyl chlorophosphite ii) displacement of 120 the chloride in dialkyl chlorophosphite by treating it with 3-bromo propanol, resulting in the corresponding trialkyl phosphite iii) acid catalyzed deprotection of the diol iv) estérification of the diol with the corresponding acid intermediate synthesized according to Scheme 1, and finally v) displacement of the bromide group using dimethylamine resulting in a tertiary amine.

Scheme 6 Synthesis of AKG-UO-3

Altematively, acid intermediates having two methylene groups between double bond positions in the hydrocarbon chain are synthesized as described in Caballeira et aL, Chem. Phys. Lipids, vol. 100, p. 33-40, 1999, or as described by D’yakonov et al. (D’yakonov et aL, Med. Chem. Res., 2016, vol. 25, p. 30-39; D’yakonov et aL, Chem. Commun. 2013, vol. 49, p 8401-8403; D’yakonov et al., 2020, Phytochem. Rev.).

(S)-4-(dimethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(octadeca-6,12-dienoate) (AKG-UO-1, 0-11956) (S)-4-(diethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(octadeca-6,12-dienoate) (AKG-UO-1 A, 0-11955)

121 (S)-4-(dimethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(hexadeca-6,12-dienoate, AKG-UO-4, O-12401) (S)-4-(diethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(hexadeca-6,12-dienoate, AKG-UO-4A,

O-12402) (S)-4-(dimethylamino)butane-1,2-diyl (6Z,6'Z, 11 Z, 11 'Z)-bis(octadeca-6,11 -dienoate)(AKG-UO-1 a)

Scheme 7

TsOH, CH₂Cl2 1

Scheme 8

TsCI. pyridine

R₂ = CH₃ 17

CH₂CH3 18

R₂

-ÎÜS- %₂

MeOH 80 ^eC HO H ^HCI

R₂=CH₃ 21

CH₂CH₃ 22

Scheme 9

1. Oxalyl chloride. DMF, CH₂CI₂ ^R2

HÔ H

Ό,

R₂ = CH₃ 21

CH₂CH₃ 22

Compounds	Ri	r2
AKG-UO-1	ch2ch3	ch3
AKG-UO-1A	ch2ch3	ch2ch3
AKG-UO-4	H	ch3
AKG-UO-4A	H	ch2ch3

Experimental Procedure

Synthesis of 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran 2

122

To a solution of 5-bromo-l-pentanol 1 (3.6 g, 21.6 mmol) in dichloromethane (lOOmL) and pyridinium p-toluene sulfonate (40 mg, 0.16 mmol) at 0 °C was added 3,4-dihydro-2H-pyran (6.54 mL, 71.8 mmol). The resulting solution was stirred at room température for one hour then quenched with water. The mixture was extracted with ethyl acetate (2XlOOmL). The combined organics were washed with brine then dried over magnésium sulfate, fdtered, and the filtrate concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 5-10% ethyl acetate in n-hexane as eluant to give 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran, 2 (4.5 g, 83%) as a clear oil.

1H NMR (300 MHz, CDC13): ô ppm 4.55-4.54 (d, J = 4.3 Hz, 1H), 3.92-3.72 (m, 2H), 3.42-3.38 (m, 3H), 1.88-1.55 (m, 3H), 1.52-1.50 (m, 10H).

Synthesis of 2-(trideca-6,12-diyn-1 -yloxy)tetrahydro-2H-pyran 4

To a solution of 1,7-Octadiyne 3 (6 mL, 45.4 mmol) and hexamethylphosphoramide (16 mL, 90.8 mmol) in tetrahydrofuran (100 mL) at -78 °C was added [2.5 M n-butyllithium in n-hexane] (18 mL, 45.4 mmol) dropwise. Upon completion of addition, the solution was stirred at -78 °C for one hour then warmed to -20 °C for an additional hour. The resulting solution was cooled once again to -78 °C whereupon a solution of 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran, 2 (5.67 g, 22.7mmol) in tetrahydrofuran (10 mL) was added. The resulting solution was allowed to warm to room

123 température and stirred for 12 hours. After 12 hours, the reaction was cooled to 0 °C and quenched with water (100 mL). The reaction mixture was then concentrated under vacuum to remove tetrahydrofuran and then diluted with n-hexane. The organics were washed with water and brine (2X100 mL). The organic layer was dried over magnésium sulfate, filtered, and the filtrate concentrated under vacuum to give a crude oil weighing 9 g. The crude oil was purified by chromatography on silica using 5-10% ethyl acetate in n-hexane as eluant to give 2-(trideca-6,12diyn-l-yloxy) tetrahydro-2H-pyran, 4 (4.5 g, 72%) as a clear oil.

1H NMR (300 MHz, dô.DMSO): 0 ppm 4.544.53 (m, 1H), 3.72-3.61 (m, 1H), 3.60-3.58 (m, 1H), 3.43-3.33 (m, 1H), 3.32-3.29 (m, 1H), 2.77-2.75 (t, J= 5.8 Hz, 1H), 2.16-2.13 (m, 6H), 1.55-1.41 (m, 16H).

Représentative Procedure for alkylation of alkynes

Synthesis of 2-(hexadeca-6,12-diyn-l-yloxy) tetrahydro-2H-pyran 7

To a solution of 2-(trideca-6,12-diyn-l-yloxy) tetrahydro-2H-pyran, 4 (7.14 g, 25.86 mmol) and hexamethylphosphoramide (18 mL, 103.4 mmol) in tetrahydrofuran (100 mL) at -78 °C was added [2.5 M n-butyllithium in n-hexane] (41.3 mL, 103.4 mmol) dropwise. Upon completion of addition, the solution was stirred at -78 °C for one hour then waimed to -20 °C for an additional hour. The resulting solution was cooled once again to -78 °C whereupon a solution of 1-iodopropane 5 (9.9 mL, 103.4 mmol) in tetrahydrofuran (20 mL) was added. The resulting solution was allowed to warm to room température and stirred for 12 hours. After 12 hours, the reaction was cooled to 0 °C and quenched with water (100 mL). The reaction mixture was then concentrated under vacuum to remove tetrahydrofuran and then diluted with n-hexane. The organics were washed with water and brine (2X100 mL). The organic layer was dried over magnésium sulfate, fdtered, and the fdtrate concentrated under vacuum to give a crude oil weighing 9 g. The crude oil was purified by

124 chromatography on silica using 5% ethyl acetate in n-hexane as eluant to give 2-(hexadeca-6,l2diyn-l-yloxy)tetrahydro-2H-pyran, 7 (5.9 g, 72%) as a clear oil.

^lH NMR (300 MHz, CDC13): 4.57-4.55 (m, IH), 3.86-3.74 (m, IH), 3.73-3.71 (m, IH), 3.50-3.39 (m, IH), 3.37-3.36 (m, IH), 2.16-2.11 (m, 8H), I.59-1.56 (m, 2H), L55-L47 (m, 16H), 0.98-0.93 (t, J = 1.6 Hz, 3H).

2-(octadeca-6,l2-diyn-l-yloxy)tetrahydro-2H-pyran 8 q-——

IH NMR (300 MHz, CDC13): 4.57-4.55 (m, IH), 3.85-3.74 (m, IH), 3.73-3.70 (m, IH), 3.50-3.38 (m, IH), 3.36-3.35 (m, IH), 2.23-2.12 (m, 8H), 1.6M.54 (m, 2H), 1.53-1.48 (m, 16H), l.47-1.46 (m, 4H), 0.90-0.85 (t, J = 1.6 Hz, 3H).

Représentative Procedure for réduction of alkynes to alkenes using “P-2 Ni” Synthesis of 2-(((6Z, 12Z)-hexadeca-6,12-dien-1 -yl)oxy)tetrahydro-2H-pyran 9

To a solution of Sodium borohydride (0.56 g, 14.8 mmol) in éthanol (80 mL) under hydrogen blanket at 0 °C was added Nickel (II) acetate tetrahydrate (3.22 g, 12.98 mmol). Upon completion of addition, the reaction was evacuated under vacuum and flushed with hydrogen. After 10 minutes of stirring, ethylenediamine (3.7 mL, 65.6 mmol), and a solution of 2-(hexadeca-6,12-diyn-lyloxy)tetrahydro-2H-pyran, 7 (5.9 g, 18.55 mmol) in éthanol (10 mL) was added. The reaction was stirred at room température under a hydrogen balloon for 4 hours. After 4 hours, the reaction mixture was evacuated of hydrogen and then flushed with nitrogen. The crude mixture was filtered

125 over celite, and the filtrate concentrated under vacuum to give a crude oil weighing 4 g. The crude oil was purified by chromatography on silica using 5-10 % diethyl ether in n-hexane as eluant to give 2-(((6Z,12Z)-hexadeca-6,l 2-dien-l-yl)oxy)tetrahydro-2H-pyran, 9 (4.67 g, 78% yield) as a clear oil.

1H NMR (300 MHz, CDC13): 5.35-5.34 (m, 4H), 4.58-4.55 (m, 1H), 3.86-3.74 (m, 1H), 3.73-3.71 (m, 1H), 3.51-3.39 (m, 1H), 3.36-3.35 (m, 1H), 2.03-1.98 (m, 8H), 1.57-1.39 (m, 2H), 1.38-1.36 (m, 6H), 1.35-1.32 (m, 10H), 0.91-0.86 (t,J= 1.6 Hz, 3H).

^13C NMR (300 MHz, CDC13): 129.98, 129.85, 98.93, 77.53, 77.10, 76.68, 67.72, 62.43, 30.86, 29.71, 29.70, 29.45, 29.46, 29.44, 27.20, 27.19, 26.01, 25.59, 22.98, 19.78, 13.91.

2-(((6Z, 12Z)-octadeca-6,12-dien-1 -yl)oxy)tetrahydro-2H-pyran 10

1H NMR (300 MHz, CDC13): 5.39-5.29 (m, 4H), 4.58-4.55 (m, 1H), 3.86-3.76 (m, 1H), 3.74-3.68 (m, 1H), 3.51-3.41 (m, 1H), 3.39-3.36 (m, 1H), 2.14-1.97 (m, 8H), 1.56-1.38 (m, 2H), 1.37-1.35 (m, 6H), 1.34-1.28 (m, 14H), 0.93-0.85 (t, J= 1.6 Hz, 3H).

13C NMR (300 MHz, CDC13): 130.13, 129.97, 129.84, 129.71, 98.93, 77.53, 77.10, 76.68, 67.71, 62.42, 31.62, 30.86, 29.72, 29.71, 29.47, 29.46, 27.27, 27.18, 26.01, 25.59, 22.67, 19.78, 14.18.

Représentative Procedure for deprotection of tetrahvdropyranyl ether (THP)

Synthesis of (6Z,12Z)-hexadeca-6,l 2-dien-l-ol 11

126

To a solution of2-(((6Z, 12Z)-hexadeca-6,l 2-dien-l-yl)oxy)tetrahydro-2H-pyran, 9 (4.67 g, 14.5 mmol) in methanol (20 mL) was added p-Tohienesulfomc acid monohydrate (300 mg, 1.58 mmol) at room température. The resulting solution was stirred at room température for 3 hours then quenched with water. The mixture was extracted with ethyl acetate (2X50 mL). The combined organics were washed with water then dried over magnésium sulfate, fdtered and the fdtrate concentrated under vacuum to give a crude oil weighing 4 g. The crude oil was purified by chromatography on silica using 5-10 % diethyl ether in n-hexane as eluant to give (6Z,12Z)-hexadeca-6,12-dien-l-ol, 11 (2.5 g, 72%) as a clear oil.

1H NMR (300 MHz, CDC13): 5.34-5.33 (m, 4H), 3.65-3.61 (m, 2H), 2.02-2.00 (m, 8H), 1.36-1.34 (m, 2H), 1.34-1.25 (m, 10H), 0.89-0.86 (t, J = 0.82 Hz, 3H).

(6Z, 12Z)-octadeca-6,12-dien-1 -ol 12

1H NMR (300 MHz, CDC13): 5.36-5.33 (m, 4H), 3.65-3.61 (m, 2H), 2.02-2.01 (m, 8H), 1.36-1.35 (m, 2H), 1.34-1.25 (m, 14H), 0.88-0.85 (t, J = 0.76 Hz, 3H).

Représentative Procedure for oxidation of alcohol to carboxylic acid using Jones reagent

Synthesis of (6Z, 12Z)-hexadeca-6,12-dienoic acid 13

A mixture of (6Z,12Z)-hexadeca-6,l 2-dien-l-ol, 11 (2.5 g, 10.5 mmol) and Jones Reagent [2M in sulfuric acid], (10.5 mL, 21 mmol) in acetone (20 mL) at 0 °C was stirred for 2 hours. The mixture was quenched with water and extracted with ethyl acetate (2X100 mL). The combined organics were dried over magnésium sulfate, fdtered and the fdtrate concentrated under vacuum to give a

127 crude oil. The crude oil was purified by chromatography on silica using 20 % ethyl acetate in nhexane as eluant to give (6Z,12Z)-hexadeca-6,12-dienoic acid, 13 (1.7 g, 68%) as a clear oil.

IH NMR (300 MHz, CDC13): 5.35-5.33 (m, 4H), 2.37-2.32 (t, 2H), 2.06-1.98 (m, 8H), 1.64-1.39 (m, 2H), 1.37-1.32 (m, 8H), 0.91-0.87 (t, J = 0.91 Hz, 3H).

(6Z,12Z)-octadeca-6,12-dienoic acid 14

O

IH NMR (300 MHz, CDC13): 5.36-5.32 (m, 4H), 2.35-2.33 (t, 2H), 2.06-2.01 (m, 8H), 1.64-1.42 (m, 2H), 1.34-1.28 (m, 12H), 0.90-0.85 (t, 3H).

Synthesis of (5)-2-(2,2-dimethyl-l,3-dioxolan-4-yl)ethyl 4-methylbenzenesulfonate 16

-i θ

To a mixture of ( 5)-2-(2,2-dimethyl-l,3-dioxolan-4-yl)ethan-l-ol 15 (25 g, 171.1 mmol) in pyridine (30 mL) at 0 °C was addedp-Toluenesulfonylchloride (35.8 g, 188.2 mmol) and DMAP (140 mg, 1.14 mmol) and the reaction was stirred at room température ovemight. The mixture was diluted with CH2C12 (500 mL), washed with sat. NH4C1, water and Brine. The organic layer was dried over anhydrous Na2SO4. The solvent was evaporated, and the crude residue used for the next step without purification. (43.8 g, 85%).

IH NMR (300 MHz, CDC13): δ ppm 7.77 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 4.15-4.01 (m, 3H), 3.65-3.47 (m, 2H), 2.43 (s, 3H), 1.82-1.62 (m, 2H), 1.32 (s, 3H), 1.27 (s, 3H).

Représentative Procedure for di-alkylamine substitution

Synthesis of (S)-2-(2,2-dimethyl-l,3-dioxolan-4-yl)-N,N-dimethylethan-l-amine 19

I

128

A mixture of (S)-2-(2,2-dimethyl-1,3-dioxoIan-4-yl)ethyl 4-methylbenzenesulfonate 16 (10 g, 33.3 mmol) and dimethylamine solution 17 (166 mL, 333.3 mmol) (2M in THF) was stirred at room température for 2 days. The mixture was concentrated, and the crude residue was diluted with CH2C12 (500 mL), washed with sat. NaHCO3, water and Brine. The organic layer was dried over anhydrous Na2SO4. The solvent was evaporated, and the crude residue was purified by flash chromatography (SiO2: CH2C12 = 100% to 10% of MeOH in CH2C12 with 1%NH4OH) and colorless oil product 19 was obtained (2.1 g, 37%).

1H NMR (300 MHz, CDC13): à ppm 4.15-4.01 (m, 2H), 3.52 (dd, J= 7.4, 7.4 Hz, 1H), 2.41-2.23 (m, 2H), 2.21 (s, 6H), 1.82-1.62 (m, 2H), 1.39 (s, 3H), 1.33 (s, 3H).

MS (APCI+): 174.1 (M+l) (S)-2-(2,2-diethyl-1,3-dioxolan-4-yl)-N,N-dimethylethan-1 -amine 20

1H NMR (300 MHz, CDC13): δ ppm 4.15-4.01 (m, 2H), 3.48 (dd, J = 7.4, 7.4 Hz, 1H), 2.48-2.43 (m, 6H), 1.82-1.62 (m, 2H), 1.36 (s, 3H), 1.27 fs, 3H), 0.97 (t, J = 7.2 Hz, 6H).

MS (APCI+): 202.2 (M+l)

Représentative Procedure for ketal hydrolysis

Synthesis of (S)-4-(dimethylamino)butane-l,2-diol hydrochloride sait 21

To a mixture of (S)-2-(2,2-dimethyl-l,3-dioxolan-4-yl)-N,N-dimethylethan-l-amine 19 (2 g, 11.54 mmol) in MeOH (10 mL) was added IN HCl aqueous solution (17 mL, 17.3 mmol) and the reaction was heated at 80 °C for 45 min. TLC (Rf = 0.1, 10%MeOH in CH2C12 with 1%NH4OH) showed the completion of reaction. After concentration of the reaction mixture, the crude residue was dissolved in water (5 mL) and lyophilized ovemight. Sticky syrup product 21 was obtained (2.1 g, quant.) as HCl sait.

129

IH NMR (300 MHz, D2O): δ ppm 3.77-3.72 (m, IH), 3.54-3.46 (m, 2H), 3.29-3.22 (m, 2H), 2.85 (s, 6H), 1.92-1.79 (m, 2H).

MS <APCI+): 134.1 (M+l) (5j-4-(diethylamino)butane-l,2-diol hydrochloride sait 22

1HNMR (300 MHz, D2O): d'ppm 3.77-3.72 (m, IH), 3.54-3.46 (m, 2H), 3.22-3.15 (m, 6H), 1.921.74 (m, 2H), 1.24 (t, J = 7.4 Hz, 6H).

MS (APCI+): 162.1 (M+l)

Représentative Procedure for di-esterification

Synthesis of (5y4-(dimethylamino)butane-l,2-diyl (6Z,6'Z, 12Z, 12’Z)-bis(octadeca-6,12-dienoate)

AKG-UO-1 (0-11956)

AKG-UO-1 O

Oxalyl chloride (0.33 mL, 3.9 mmol) was added dropwise to a solution of (6Z, 12Z)-octadeca-6,12dienoic acid, 14 (0.36 g, 1.3 mmol) in dichloromethane / DMF (15mLs, 25 QL) at 0 °C. and allowed reaction to warm to room température and stir for one hour. After one hour, the reaction was concentrated under vacuum to dryness. The residue was re-dissolved in dichloromethane (10 mL) and added to a mixture of N,N-Diisopropylethylamine (2.3 mL, 10 mmol), 4Dimethylaminopyridine (317 mg, 2.6 mmol), and fS)-4-(dimethylamino)butane-l,2-diol hydrochloride, 21 (101 mg, 0.6 mmol). The resulting solution was allowed to stir for 24 hours. After 24 hours, the reaction was cooled to 0C and quenched with water (10 mL). The reaction mixture was extracted with dichloromethane (2X100 mL) and the organics were washed with water and brine (2X100 mL). The organic layer was dried over magnésium sulfate, fdtered, and the filtrate concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 2% methanol in dichloromethane as eluant to give (Sj-4(dimethylamino)butane-l,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(octadeca-6,12-dienoate), AKG-UO-1, (0.12 g, 30%) as a yellow oil.

130

1H NMR (300 MHz, CDC13): 5.40-5.29 (m, 8H), 5.14-5.12 (m, 1H), 4.25 (dd, 7= 11.8, 3.3 Hz, 1H), 4.05 (dd, 7= 12.1, 6.3 Hz, 1H), 2.32-2.26 (m, 6H), 2.20 (s, 6H), 2.06-1.99 (m, 16H), 1.78-1.70 (m, 2H), 1.65-1.58 (m, 4H), 1.42-1.25 (m, 24H), 0.90-0.85 (m, 6H).

MS (APCI+): 658.5 (M+l) (S)-4-(diethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(octadeca-6,12-dienoate) AKG-UO-1A (0-11955)

AKG-UO-1A

1H NMR (300 MHz, CDC13): 5.37-5.29 (m, 8H), 5.12-5.10 (m, 1H),4.25 (dd, J = 12.1, 3.6 Hz, 1H), 4.05 (dd, J = 11.8, 6.3 Hz, 1H), 2.52-2.42 (m, 6H), 2.29 (t, J = 7.4 Hz, 4H)), 2.06-1.99 (m, 16H), 1.78-1.70 (m, 2H), 1.64-1.59 (m, 4H), 1.41-1.19 (m, 24H), 0.99 (t, J = 7.1 Hz, 6H), 0.96-0.87 (m, 6H).

MS (APCI+): 686.6 (M+l) (S)-4-(dimethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(hexadeca-6,12-dienoate) AK.G-UO-4 (0-12401)

1H NMR (300 MHz, CDC13): 5.39-5.29 (m, 8H), 5.13-5.12 (m, 1H), 4.24 (dd, J = 11.8, 3.3 Hz, 1H), 4.05 (dd, J = 11.8, 6.3 Hz, 1H), 2.32-2.27 (m, 6H), 2.19 (s, 6H), 2.01-1.99 (m, 16H), 1.75-1.72 (m, 2H), 1.65-1.58 (m, 4H), 1.36-1.31 (m, 16H), 0.91-0.86 (m, 6H).

MS (APCI+): 602.5 (M+l)

Synthesis of (S)-4-(diethylamino)butane-l,2-diyl (6Z,6'Z,12Z,12'Z)-bis(hexadeca-6,12-dienoate)

AKG-UO-4A (0-12402)

131

AKG-UO-4A

IH NMR (300 MHz, CDC13Y 5.40-5.29 (m, 8H), 5.12-5.11 (m, IH), 4.25 (dd,J= H.8,3.3 Hz, IH), 4.05 (dd, J= 11.8, 6.3 Hz, IH), 2.54-2.43 (m, 6H), 2.29 (t, 7.4 Hz, 4H), 2.11-1.96 (m,

16H), 1.74-1.65 (m, 2H), 1.65-1.59 (m, 4H), 1.39-1.31 (m, 16H), 0.99 (t, J= 7.1 Hz, 6H), 0.91-0.89 (m, 6H).

MS (APCI+): 630.5 (M+l), and finally v) displacement of the bromide group using dimethylamine resulting in a tertiary amine.

132

Altematively, acid intermediates having two methylene groups between double bond positions in the hydrocarbon chain are synthesized as described in Caballeira et al., Chem. Phys. Lipids, vol. 100, p. 33-40, 1999, or as described by D’yakonov et al. (D’yakonov et al., Med. Chem. Res., 2016, vol. 25, p. 30-39; D’yakonov et al., Chem. Commun. 2013, vol. 49, p 8401-8403; D’yakonov et al., 2020, Phytochem. Rev.).

Example IB: Synthesis of Ionizable Lipids (5^-4-(dimethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(octadeca-6,12-dienoate) (AK.G-UO-1, 0-11956) (S)-4-(diethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(octadeca-6,12-dienoate) (AKG-UO-1 A, 0-11955) (S)-4-(dimethylamino)butane-l,2-diyl (6Z,6'Z,12Z,12'Z)-bis(hexadeca-6,12-dienoate, AK.G-UO-4, 0-12401) (5)-4-(diethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(hexadeca-6,12-dienoate, AKG-UO-4A, 0-12402)

133 (5)-4-(dimethylamino)butane-1,2-diyl (6Z,6'Z,l IZ, l l'Z)-bis(octadeca-6,l l-dienoate)(AKG-UO-la)

Scheme 8

TsCI, pyridine

R₂. _d,NH r₂

THF

r₂

1NHCI Ns

------* HO R₂

MeOH. 80 °C HO' H ^HCI

R₂ = CH₃ 17

CH₂CH₃ 18

R₂ = CH₃ 19

CH₂CH₃ 20

R₂=CH₃ 21

CH₂CH₃ 22

Scheme 9

1. Oxalyl chloride, DMF. CH₂CI₂

R₂ = CH₃ 21

CH₂CH₃ 22

XL

Compounds	Ri	r2
AKG-UO-1	CH2CH3	ch3
AKG-UO-1A	CH2CH3	ch2ch3
AKG-UO-4	H	ch3
AKG-UO-4A	H	CH2CH3

Experimental Procedure

Synthesis of 2-((5-bromopentyl)oxy)tetrahydro-2//-pyran 2

134

To a solution of 5-bromo-l-pentanol 1 (3.6 g, 21.6 mmol) in dichloromethane (lOOmL) and pyridinium p-toluene sulfonate (40 mg, 0.16 mmol) at 0 °C was added 3,4-dihydro-2H-pyran (6.54 mL, 71.8 mmol). The resulting solution was stirred at room température for one hour then quenched with water. The mixture was extracted with ethyl acetate (2XlOOmL). The combined organics were washed with brine then dried over magnésium sulfate, filtered, and the fdtrate concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 5-10% ethyl acetate in n-hexane as eluant to give 2-((5-bromopentyl)oxy)tetrahydro-2/7-pyran, 2 (4.5 g, 83%) as a clear oil.

Ή NMR (300 MHz, CDC1₃): δ ppm 4.55-4.54 (d, J = 4.3 Hz, 1 H), 3.92-3.72 (m, 2H), 3.42-3.38 (m, 3H), 1.88-1.55 (m, 3H), 1.52-1.50 (m, 10H).

Synthesis of 2-(trideca-6,12-diyn-l-yloxy)tetrahvdro-2//-pyran 4

To a solution of 1,7-Octadiyne 3 (6 mL, 45.4 mmol) and hexamethylphosphoramide (16 mL, 90.8 mmol) in tetrahydrofuran (100 mL) at -78 °C was added [2.5 M n-butyllithium in n-hexane] (18 mL, 45.4 mmol) dropwise. Upon completion of addition, the solution was stirred at -78 °C for one hour then warmed to -20 °C for an additional hour. The resulting solution was cooled once again to -78 °C whereupon a solution of 2-((5-bromopentyl)oxy)tetrahydro-2/7-pyran, 2 (5.67 g, 22.7mmol) in tetrahydrofuran (10 mL) was added. The resulting solution was allowed to warm to room température and stirred for 12 hours. After 12 hours, the reaction was cooled to 0 °C and quenched with water (100 mL). The reaction mixture was then concentrated under vacuum to remove tetrahydrofuran and then diluted with n-hexane. The organics were washed with water and brine (2X100 mL). The organic layer was dried over magnésium sulfate, fdtered, and the fdtrate concentrated under vacuum to give a crude oil weighing 9 g. The crude oil was purified by chromatography on silica using 5-10% ethyl acetate in n-hexane as eluant to give 2-(trideca-6,12diyn-1-yloxy) tetrahydro-2/f-pyran, 4 (4.5 g, 72%) as a clear oil.

135

Ή NMR (300 MHz, d⁶ DMSO): δ ppm 4.544.53 (m, 1H), 3.72-3.61 (m, 1H), 3.60-3.58 (m, 1H), 3.43-3.33 (m, 1H), 3.32-3.29 (m, 1H), 2.77-2.75 (t, J= 5.8 Hz, 1H), 2.16-2.13 (m, 6H), 1.55-1.41 (m, 16H).

Représentative Procedure for alkylation of alkynes

Synthesis of 2-(hexadeca-6,12-diyn-l-yloxy) tetrahydro-2/f-pyran 7

To a solution of 2-(trideca-6,12-diyn-l-yloxy) tetrahydro-2/7-pyran, 4 (7.14 g, 25.86 mmol) and hexamethylphosphoramide (18 mL, 103.4 mmol) in tetrahydrofuran (100 mL) at -78 °C was added [2.5 M n-butyllithium in n-hexane] (41.3 mL, 103.4 mmol) dropwise. Upon completion of addition, the solution was stirred at -78 °C for one hour then warmed to -20 °C for an additional hour. The resulting solution was cooled once again to -78 °C whereupon a solution of 1-iodopropane 5 (9.9 mL, 103.4 mmol) in tetrahydrofuran (20 mL) was added. The resulting solution was allowed to warm to room température and stirred for 12 hours. After 12 hours, the reaction was cooled to 0 °C and quenched with water (100 mL). The reaction mixture was then concentrated under vacuum to remove tetrahydrofuran and then diluted with n-hexane. The organics were washed with water and brine (2X100 mL). The organic layer was dried over magnésium sulfate, filtered, and the fdtrate concentrated under vacuum to give a crude oil weighing 9 g. The crude oil was purified by chromatography on silica using 5% ethyl acetate in n-hexane as eluant to give 2-(hexadeca-6,12diyn-1-yloxy)tetrahydro-2/f-pyran, 7 (5.9 g, 72%) as a clear oil.

Ή NMR (300 MHz, CDCh): 4.57-4.55 (m, 1H), 3.86-3.74 (m, 1H), 3.73-3.71 (m, 1H), 3.50-3.39 (m, 1H), 3.37-3.36 (m, 1H), 2.16-2.11 (m, 8H), 1.59-1.56 (m, 2H), 1.55-1.47 (m, 16H), 0.98-0.93 (t, J = 1.6 Hz, 3H).

2-(octadeca-6,12-diyn-l-yloxy)tetrahydiO-2//-pyran 8

136 ——

Ή NMR (300 MHz, CDCl₃): 4.57-4.55 (m, IH), 3.85-3.74 (m, IH), 3.73-3.70 (m, IH), 3.50-3.38 (m, IH), 3.36-3.35 (m, IH), 2.23-2.12 (m, 8H), 1.61-1.54 (m, 2H), l.53-1.48 (m, 16H), 1.47-1.46 (m, 4H), 0.90-0.85 (t, J = 1.6 Hz, 3H).

Représentative Procedure for réduction of alkynes to alkenes usina “P-2 Ni”

Synthesis of 2-(((6Z, 12Z)-hexadeca-6,12-dien-l-yl)oxy)tetrahydro-2/7-pyran 9

To a solution of Sodium borohydride (0.56 g, 14.8 mmol) in éthanol (80 mL) under hydrogen blanket at 0 °C was added Nickel (II) acetate tetrahydrate (3.22 g, 12.98 mmol). Upon completion of addition, the reaction was evacuated under vacuum and flushed with hydrogen. After 10 minutes of stirring, ethylenediamine (3.7 mL, 65.6 mmol), and a solution of 2-(hexadeca-6,12-diyn-lyloxy)tetrahydro-2//-pyran, 7 (5.9 g, 18.55 mmol) in éthanol (10 mL) was added. The reaction was stirred at room température under a hydrogen balloon for 4 hours. After 4 hours, the reaction mixture was evacuated of hydrogen and then flushed with nitrogen. The crude mixture was filtered over celite, and the filtrate concentrated under vacuum to give a crude oil weighing 4 g. The crude oil was purified by chromatography on silica using 5-10 % diethyl ether in n-hexane as eluant to give 2-(((6Z,12Z)-hexadeca-6,12-dien-l-yl)oxy)tetrahydiO-2H-pyran, 9 (4.67 g, 78% yield) as a clear oil.

‘H NMR (300 MHz, CDC1₃): 5.35-5.34 (m, 4H), 4.58-4.55 (m, IH), 3.86-3.74 (m, IH), 3.73-3.71 (m, IH), 3.51-3.39 (m, IH), 3.36-3.35 (m, IH), 2.03-1.98 (m, 8H), 1.57-1.39 (m, 2H), 1.38-1.36 (m, 6H), 1.35-1.32 (m, 10H), 0.91-0.86 (t,J= 1.6 Hz, 3H).

137 ^l3CNMR(3OO MHz, CDCh): 129.98, 129.85, 98.93, 77.53, 77.10, 76.68, 67.72, 62.43, 30.86, 29.71,29.70, 29.45, 29.46, 29.44, 27.20, 27.19, 26.01, 25.59, 22.98, 19.78, 13.91.

2-(((6Z,12Z)-octadeca-6,12-dien-l-yl)oxy)tetrahydro-2//-pyran 10

Ή NMR (300 MHz, CDCh): 5.39-5.29 (m, 4H), 4.58-4.55 (m, IH), 3.86-3.76 (m, IH), 3.74-3.68 (m, IH), 3.51-3.41 (m, IH), 3.39-3.36 (m, IH), 2.14-1.97 (m, 8H), 1.56-1.38 (m, 2H), 1.37-1.35 (m, 6H), 1.34-1.28 (m, 14H), 0.93-0.85 (t, J = 1.6 Hz, 3H).

¹³C NMR (300 MHz, CDCh): 130.13, 129.97, 129.84, 129.71,98.93,77.53,77.10,76.68,67.71, 62.42, 31.62, 30.86, 29.72, 29.71, 29.47, 29.46, 27.27, 27.18, 26.01, 25.59, 22.67, 19.78, 14.18.

Représentative Procedure for deprotection of tetrahydropyranyl ether (THP)

Synthesis of (6Z, 12Z)-hexadeca-6,12-dien-1 -ol 11

To a solution of 2-(((6Z, 12Z)-hexadeca-6,12-dien- l-yl)oxy)tetrahydro-2/7-pyran, 9 (4.67 g, 14.5 mmol) in methanol (20 mL) was added/>-Toluenesulfonic acid monohydrate (300 mg, 1.58 mmol) at room température. The resulting solution was stirred at room température for 3 hours then quenched with water. The mixture was extracted with ethyl acetate (2X50 mL). The combined organics were washed with water then dried over magnésium sulfate, filtered and the fdtrate concentrated under vacuum to give a crude oil weighing 4 g. The crude oil was purified by chromatography on silica using 5-10 % diethyl ether in n-hexane as eluant to give (6Z,12Z)hexadeca-6,12-dien-l-ol, 11 (2.5 g, 72%) as a clear oil.

138 ‘H NMR (300 MHz, CDCl₃): 5.34-5.33 (m, 4H), 3.65-3.61 (m, 2H), 2.02-2.00 (m, 8H), 1.36-1.34 (m, 2H), 1.34-1.25 (m, 10H), 0.89-0.86 (t, 7= 0.82 Hz, 3H).

(6Z, 12Z)-octadeca-6,12-dien-l-ol 12

Ή NMR (300 MHz, CDCI3): 5.36-5.33 (m, 4H), 3.65-3.61 (m, 2H), 2.02-2.01 (m, 8H), 1.36-1.35 (m, 2H), 1.34-1.25 (m, 14H), 0.88-0.85 (t, 7= 0.76 Hz, 3H).

Représentative Procedure for oxidation of alcohol to carboxylic acid using Jones reagent

Synthesis of (6Z, 12Z)-hexadeca-6,12-dienoic acid 13

A mixture of (6Z, 12Z)-hexadeca-6,12-dien-l-ol, 11 (2.5 g, 10.5 mmol) and Jones Reagent [2M in sulfuric acid], (10.5 mL, 21 mmol) in acetone (20 mL) at 0 °C was stirred for 2 hours. The mixture was quenched with water and extracted with ethyl acetate (2X100 mL). The combined organics were dried over magnésium sulfate, fdtered and the fdtrate concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 20 % ethyl acetate in nhexane as eluant to give (6Z, 12Z)-hexadeca-6,12-dienoic acid, 13 (1.7 g, 68%) as a clear oil.

Ή NMR (300 MHz, CDCI3): 5.35-5.33 (m, 4H), 2.37-2.32 (t, 2H), 2.06-1.98 (m, 8H), 1.64-1.39 (m, 2H), 1.37-1.32 (m, 8H), 0.91-0.87 (t, J= 0.91 Hz, 3H).

(6Z, 12Z)-octadeca-6,12-dienoic acid 14

139

HO

Ή NMR (300 MHz, CDCI3): 5.36-5.32 (m, 4H), 2.35-2.33 (t, 2H), 2.06-2.01 (m, 8H), 1.64-1.42 (m, 2H), 1.34-1.28 (m, 12H), 0.90-0.85 (t, 3H).

Synthesis of (5)-2-(2,2-dimethyl-l,3-dioxolan-4-yl)ethyl 4-methylbenzenesulfonate 16 ⁰

7^0 ^H

To a mixture of (5)-2-(2,2-dimethyl-l,3-dioxolan-4-yl)ethan-l-ol 15 (25 g, 171.1 mmol) inpyridine (30 mL) at 0 °C was added/>-Toluenesulfonylchloride (35.8 g, 188.2 mmol) and DMAP (140 mg, 1.14 mmol) and the reaction was stirred at room température ovemight. The mixture was diluted with CH2CI2 (500 mL), washed with sat. NH4CI, water and Brine. The organic layer was dried over anhydrous Na2SO4. The solvent was evaporated, and the crude residue used for the next step without purification. (43.8 g, 85%).

‘H NMR (300 MHz, CDCI3): δ ppm 7.77 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 4.15-4.01 (m, 3H), 3.65-3.47 (m, 2H), 2.43 (s, 3H), 1.82-1.62 (m, 2H), 1.32 (s, 3H), 1.27 (s, 3H).

Représentative Procedure for di-alkylamine substitution

Synthesis of (5)-2-(2,2-dimethyl-l,3-dioxolan-4-yl)-M77-dimethylethan-l-amine 19

A mixture of (5)-2-(2,2-dimethyl-l,3-dioxolan-4-yl)ethyl 4-methylbenzenesulfonate 16 (10 g, 33.3 mmol) and dimethylamine solution 17 (166 mL, 333.3 mmol) (2M in THF) was stirred at room température for 2 days. The mixture was concentrated, and the crude residue was diluted with CH2CI2 (500 mL), washed with sat. NaHCÛ3, water and Brine. The organic layer was dried over

140 anhydrous Na2SO4. The solvent was evaporated, and the crude residue was purified by flash chromatography (SiCh: CH2CI2 = 100% to 10% of MeOH in CH2CI2 with 1%NH4OH) and colorless oil product 19 was obtained (2.1 g, 37%).

Ή NMR (300 MHz, CDCI3): à ppm 4.15-4.01 (m, 2H), 3.52 (dd, J= 7.4, 7.4 Hz, 1H), 2.41-2.23 (m, 2H), 2.21 (s, 6H), 1.82-1.62 (m, 2H), 1.39 (s, 3H), 1.33 (s, 3H).

MS (APCI⁺): 174.1 (M+l) (5)-2-(2,2-diethyl-1.3-dioxolan-4-yl)-/V,_ÎV-dimethvlethan-1 -amine 20

Ή NMR (300 MHz, CDCI3): d'ppm 4.15-4.01 (m, 2H), 3.48 (dd, J = 7.4, 7.4 Hz, 1H), 2.48-2.43 (m, 6H), 1.82-1.62 (m, 2H), 1.36 (s, 3H), 1.27 (s, 3H), 0.97 (t, 7=7.2 Hz, 6H).

MS (APCI⁺): 202.2 (M+l)

Représentative Procedure for ketal hydrolysis

Synthesis of (5)-4-(dimethylamino)butane-l,2-diol hydrochloride sait 21

To a mixture of (5)-2-(2,2-dimethyl-l,3-dioxolan-4-yl)-V,7V-dimethylethan-l-amine 19 (2 g, 11.54 mmol) in MeOH (10 mL) was added IN HCl aqueous solution (17 mL, 17.3 mmol) and the reaction was heated at 80 °C for 45 min. TLC (Rf = 0.1, 10%MeOH in CH2CI2 with 1%NH₄OH) showed the completion of reaction. After concentration of the reaction mixture, the crude residue was dissolved in water (5 mL) and lyophilized ovemight. Sticky syrup product 21 was obtained (2.1 g, quant.) as

HCl sait.

141

Ή NMR (300 MHz, D₂O): d'ppm 3.77-3.72 (m, IH), 3.54-3.46 (m, 2H), 3.29-3.22 (m, 2H), 2.85 (s, 6H), 1.92-1.79 (m, 2H).

MS (APCI⁺): 134.1 (M+l) (5j-4-(diethylamino)butane-L2-diol hydrochloride sait 22

Ή NMR (300 MHz, D₂O): d ppm 3.77-3.72 (m, 1H), 3.54-3.46 (m, 2H), 3.22-3.15 (m, 6H), 1.921.74 (m, 2H), 1.24 (t, J = 7.4 Hz, 6H).

MS (APCI⁺): 162.1 (M+l)

Représentative Procedure for di-esterification

Synthesis of (5j-4-(dimethylamino)butane-l,2-diyl (6Z,6'Z,12Z, 12'Z)-bis(octadeca-6,12-dienoate)

AKG-UO-1 (0-11956)

AKG-UO-1 O

Oxalyl chloride (0.33 mL, 3.9 mmol) was added dropwise to a solution of (6Z, l2Z)-octadeca-6,12dienoic acid, 14 (0.36 g, 1.3 mmol) in dichloromethane / DMF (15mLs, 25 pL) at 0 °C. and allowed reaction to warm to room température and stir for one hour. After one hour, the reaction was concentrated under vacuum to dryness. The residue was re-dissolved in dichloromethane (10 mL) and added to a mixture of N,N-Diisopropylethylamine (2.3 mL, 10 mmol), 4Dimethylaminopyridine (317 mg, 2.6 mmol), and (,Sj-4-(dimethylamino)butane-l,2-diol hydrochloride, 21 (101 mg, 0.6 mmol). The resulting solution was allowed to stir for 24 hours. After 24 hours, the reaction was cooled to 0C and quenched with water (10 mL). The reaction mixture was extracted with dichloromethane (2X100 mL) and the organics were

142 washed with water and brine (2X100 mL). The organic layer was dried over magnésium sulfate, fdtered, and the fdtrate concentrated under vacuum to give a crude oiL The crude oil was purified by chromatography on silica using 2% methanol in dichloromethane as eluant to give (S)-4(dimethylamino)butane-l,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(octadeca-6,12-dienoate), AKG-UO-1, (0.12 g, 30%) as a yellow oil.

Ή NMR (300 MHz, CDCh): 5.40-5.29 (m, 8H), 5.14-5.12 (m, IH), 4.25 (dd, J = 11.8, 3.3 Hz, IH), 4.05 (dd, J = 12.1, 6.3 Hz, IH), 2.32-2.26 (m, 6H), 2.20 (s, 6H), 2.06-1.99 (m, 16H), 1.78-1.70 (m, 2H), 1.65-1.58 (m, 4H), 1.42-1.25 (m, 24H), 0.90-0.85 (m, 6H).

MS (APCI⁺): 658.5 (M+l) (5)-4-(diethylamino)butane-l,2-diyl (6Z,6'Z, 12Z,12’Z)-bis(octadeca-6,12-dienoate) AKG-UO-1A (0-11955)

Ή NMR (300 MHz, CDCh): 5.37-5.29 (m, 8H), 5.12-5.10 (m, IH), 4.25 (dd, J= 12.1, 3.6 Hz, IH), 4.05 (dd, J = 11.8, 6.3 Hz, IH), 2.52-2.42 (m, 6H), 2.29 (t, J= 7.4 Hz, 4H)), 2.06-1.99 (m, 16H), 1.78-1.70 (m, 2H), 1.64-1.59 (m, 4H), 1.41-1.19 (m, 24H), 0.99 (t, J= 7.1 Hz, 6H), 0.96-0.87 (m, 6H).

MS (APCI⁺): 686.6 (M+l) (5)-4-(dimethylamino)butane-1,2-diyl (6Z,6'Z, 12Z, 12'Z)-bis(hexadeca-6,12-dienoate) AKG-UO-4 (0-12401)

143

Ή NMR (300 MHz, CDCh): 5.39-5.29 (m, 8H), 5.13-5.12 (m, 1H), 4.24 (dd, J = 11.8, 3.3 Hz, 1H), 4.05 (dd, J= 11.8, 6.3 Hz, 1H), 2.32-2.27 (m, 6H), 2.19 (s, 6H), 2.01-1.99 (m, 16H), 1.75-1.72 (m, 2H), 1.65-1.58 (m, 4H), 1.36-1.31 (m, 16H), 0.91-0.86 (m, 6H).

MS (APCI⁺): 602.5 (M+l)

Synthesis of (5>4-(diethylamino)butane-l,2-diyl (6Z,6'Z,12Z,12’Z)-bis(hexadeca-6,12-dienoate)

AKG-UO-4A (0-12402)

AKG-UO-4A

Ή NMR (300 MHz, CDCh): 5.40-5.29 (m, 8H), 5.12-5.11 (m, 1H), 4.25 (dd, J= 11.8, 3.3 Hz, 1H), 10 4.05 (dd, J= 11.8, 6.3 Hz, 1H), 2.54-2.43 (m, 6H), 2.29 (t, J= 7.4 Hz, 4H), 2.11-1.96 (m, 16H),

1.74- 1.65 (m, 2H), 1.65-1.59 (m, 4H), 1.39-1.31 (m, 16H), 0.99 (t, J= 7.1 Hz, 6H), 0.91-0.89 (m, 6H).

MS (APCI⁺): 630.5 (M+l)

Synthesis of (5)-4-(dimethylamino)butane-l,2-diyl (6Z,6'Z,llZ,irZ)-bis(octadeca-6,lldienoate)(AKG-UO-la)

144

1. Ni(0Ac)2 tetrahedrate, NaBH₄, EtOH, H₂ balloon

2. Ethylènediamine, H2 balloon

TsOH, MeOH

HO.

8a

AKG-UO-la

P-2Ni, H₂”

Jones reagent acetone

1. Oxalyl chloride, DMF. CH2CI2

N

HO H

10a

Experimental Procedure

Synthesis of 2-((5-bromopentyl)oxy)tetrahydro-2//-pyran 2

To a solution of 5-bromo-l-pentanol 1 (3.6 g, 21.6 mmol) in dichloromethane (lOOmL) and pyridinium p-toluene sulfonate (40 mg, 0.16 mmol) at 0 °C was added 3,4-dihydro-2H-pyran (6.54 mL, 71.8 mmol). The resulting solution was stirred at room température for one hour then quenched with water. The mixture was extracted with ethyl acetate (2X lOOmL). The combined organics were 10 washed with brine then dried over magnésium sulfate, filtered, and the filtrate concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 5-10% ethyl acetate in n-hexane as eluant to give 2-((5-bromopentyl)oxy)tetrahydro-2/f-pyran, 2 (4.5 g, 83%) as a clear oil.

Ή NMR (300 MHz, CDCh): δ ppm 4.55-4.54 (d, J = 4.3 Hz, IH), 3.92-3.72 (m, 2H), 3.42-3.38 (m,

3H), 1.88-1.55 (m, 3H), 1.52-1.50 (m, 10H).

145

Synthesis of 2-(dodeca-6,11 -diyn-1 -yloxy)tetrahydro-2/f-pyran 4a

To a solution of 1,6-heptadiyne 3a (5 g, 54.3 mmol) and hexamethylphosphoramide (19 mL, 108 mmol) in tetrahydrofuran (100 mL) at -78 °C was added [2.5 M n-butyllithium in n-hexane] (21.7 mL, 54.3 mmol) dropwise. Upon completion of addition, the solution was stirred at -78 °C for one hour then warmed to -20 °C for an additional hour. The resulting solution was cooled once again to 78 °C whereupon a solution of 2-((5-bromopentyl)oxy)tetrahydro-2//-pyran, 2 (6.8 g, 27.1 mmol) in tetrahydrofuran (10 mL) was added. The resulting solution was allowed to warm to room température and stirred for 12 hours. After 12 hours, the reaction was cooled to 0 °C and quenched with water (100 mL). The reaction mixture was then concentrated under vacuum to remove tetrahydrofuran and then diluted with n-hexane. The organics were washed with water and brine (2X100 mL). The organic layer was dried over magnésium sulfate, filtered, and the filtrate concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 5-10% ethyl acetate in n-hexane as eluant to give 2-(dodeca-6,l 1-diyn-lyloxy)tetrahydro-2//-pyran, 4a (4.1 g, 58%) as a clear oil.

‘H NMR (300 MHz, CDC1₃): J ppm 4.57-4.56 (m, 1 H), 3.96-3.82 (m, 1 H), 3.77-3.69 (m, 1H), 3.503.41 (m, 1H), 3.39-3.34 (m, 1H), 2.29-2.25 (m, 4H), 2.15-2.12 (m, 2H), 1.95-1.94 (t, J= 5.8 Hz, 1H), 1.73-1.43 (m, 14H).

Synthesis of 2-(octadeca-6,1 l-diyn-l-yloxy)tetrahydro-2//-pyran 6a

146

To a solution of 2-(dodeca-6, l l-diyn-l-yloxy)tetrahydro-2/7-pyran, 4a (4.1 g, 15.64 mmol) and hexamethylphosphoramide (l l mL, 62.6 mmol) in tetrahydrofuran (100 mL) at -78 °C was added [2.5 M n-butyllithium in n-hexane] (12.5 mL, 31.3 mmol) dropwise. Upon completion of addition, the solution was stirred at -78 °C for one hour then warmed to -20 °C for an additional hour. The resulting solution was cooled once again to -78 °C whereupon a solution of 1-iodohexane 5a (9.5 mL, 62.6 mmol) in tetrahydrofuran (20 mL) was added. The resulting solution was allowed to warm to room température and stirred for 12 hours. After 12 hours, the reaction was cooled to 0 °C and quenched with water (100 mL). The reaction mixture was then concentrated under vacuum to remove tetrahydrofuran and then diluted with n-hexane. The organics were washed with water and brine (2X100 mL). The organic layer was dried over magnésium sulfate, fdtered, and the fdtrate concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 5% ethyl acetate in n-hexane as eluant to give 2-(octadeca-6,11 -diyn-1 yloxy)tetrahydro-2H-pyran, 6a (3.1 g, 57%) as a clear oil.

Ή NMR (300 MHz, CDC1₃): 4.58-4.55 (m, 1H), 3.86-3.82 (m, 1H), 3.77-3.69 (m, 1H), 3.51-3.47 (m, 1H), 3.41-3.34 (m, 1H), 2.26-2.21 (m, 6H), 2.14-2.12 (m, 6H), 1.66-1.26 (m, 18H), 0.93-0.85 (t, J =6.5 Hz, 3H).

Synthesis of 2-(((6Z,l lZ)-octadeca-6,1 l-dien-l-yl)oxy)tetrahydro-2/f-pyran 7a

7a

To a solution of Sodium borohydride (0.27 g, 14.8 mmol) in éthanol (50 mL) under hydrogen blanket at 0 °C was added Nickel (II) acetate tetrahydrate (1.55 g, 6.25 mmol). Upon completion of addition, the reaction was evacuated under vacuum and flushed with hydrogen. After 10 minutes of stirring, ethylenediamine (1.8 mL, 26.8 mmol), and a solution of 2-(octadeca-6,11-diyn-1 yloxy)tetrahydro-2/7-pyran, 6a (3.1 g, 8.93 mmol) in éthanol (10 mL) was added. The reaction was stirred at room température under a hydrogen balloon for 4 hours. After 4 hours, the reaction mixture was evacuated of hydrogen and then flushed with nitrogen. The crude mixture was fdtered over celite, and the fdtrate concentrated under vacuum to give a crude oil weighing 4 g. The crude oil was purified by chromatography on silica using 5-10 % diethyl ether in n-hexane as eluant to

147 give 2-(((6Z,l lZ)-octadeca-6,l l-dien-l-yl)oxy)tetrahydro-2/7-pyran, 7a (2.86 g, 92% yield) as a clear oil.

‘H NMR (300 MHz, CDCl₃): 5.4-5.34 (m, 4H), 4.58-4.55 (m, 1H), 3.86-3.82 (m, 1H), 3.74-3.68 (m, 1H), 3.51-3.49 (m, 1H), 3.41-3.36 (m, 1H), 2.06-1.99 (m, 6H), 1.83-1.67 (m, 2H), 1.59-1.51 (m, 6H),1.48-1.32 (m, 16H), 0.92-0.85 (t,7=6.6 Hz, 3H).

Synthesis of (6Z,1 lZ)-octadeca-6,1 1-dien-l-ol 8a

HO.

8a

Procedure previously described.

‘H NMR (300 MHz, CDCI3): 5.37-5.33 (m, 4H), 3.65-3.61 (m, 1 H), 2.06-1.99 (m, 6H), 1.56-1.41 (m,4H), 1.38-1.27 (m, 14H), 0.88-0.85 (t,7=6.6 Hz, 3H).

Synthesis of (6Z, 1 lZ)-octadeca-6,1 1-dienoic acid 9a

HO.

9a

Procedure previously described.

Ή NMR (300 MHz, CDCI3): 5.38-5.33 (m, 4H), 2.37-2.33 (t, 7= 5.6 Hz, 2H), 2.06-1.99 (m, 6H), 1.67-1.59 (m, 2H), 1.41-1.25 (m, 14H), 0.89-0.85 (t, 7= 6.6 Hz, 3H).

Synthesis of (y)-4-(dimethylamino)butane-l,2-diyl (6Z,6'Z,11Z,1 rZ)-bis(octadeca-6,l 1dienoate)(AKG-UO-la)

15 13 10 8 ¹⁸ 16 14 12 11 9 7 6 4 2 A \ /x ^x--\ ^x\ x'--\ x^\ /X\ Aj n

AKG-UO-1a

148

Procedure previously described.

'H NMR (300 MHz, CDCI3): 5.39-5.29 (m, 8H), 5.14-5.12 (m, IH), 4.25 (dd, J = ll.8,3.3Hz, IH),

4.06 (dd, J = 11.8,6.3 Hz, 1 H), 2.32-2.28 (m, 6H), 2.20 (s, 6H), 2.03-2.01 (m, 16H), 1.74-1.64 (m, 5 2H), 1.62-1.60 (m, 6H), 1.38-1.27 (m, 22H), 0.89-0.85 (m, 6H).

MS (APCI⁺): 658.5 (M+l)

Example IC. Synthesis of KC-01 sériés of ionizable lipids

Synthesis of 2-((5)-2,2-di((6Z,12Z)-octadeca-6,12-dien-l-yl)-l,3-dioxolan-4-yl)-Æ,Adimethylethan-l-amine (AKG-KC2-01, 0-12095)

3-((5)-2,2-di((6Z,12Z)-octadeca-6,12-dien-l-yl)-l,3-dioxolan-4-yl)-A,jV-dimethylpropan-lamine (AKG-KC3-01, 0-12096) •OH

MsCI, Et₃N CH₂CI₂

2. MgBr₂ diethyl etherate, Et₂O

1. Mg l₂, Et₂O

2. Ethyl formate KOH H₂O

n = 1 5

6

Pyridinium tosylate toluene 110°C

Synthesis of (6Z, 12Z)-l-bromooctadeca-6,12-diene, 2

149

To a solution of (6Z,l2Z)-octadeca-6,12-dien-l-ol, 1 (3.6 g, 13.7mmol) in dichloromethane (50 mL) at 0 °C was added methane sulfonyl chloride (1.26 mL, 16.4mmol) and triethylamine (3.6 mL, 20.5 mmol). The resulting solution was warmed to room température and stirred for 2 hours. The mixture was quenched with water and extracted with dichloromethane (2X100 mL). The combined organics were washed with brine then dried over magnésium sulfate then fdtered. The fdtrate was concentrated under vacuum to give a crude oil. The resulting oil was dissolved in diethyl ether (50 mL), added to a stirring slurry of magnésium bromide ethyl etherate (7 g, 27.4 mmol) in diethyl ether (50 mL) at 0C. The mixture was warmed to room température and stirred for 2 hours. The reaction mixture was quenched with water and extracted with ethyl acetate (2X100 mL). The combined organics were washed with brine then dried over magnésium sulfate then fdtered. The fdtrate was concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 5-10% ethyl acetate in n-hexane as eluant to give (6Z, 12Z)-1bromooctadeca-6,12-diene, 3 (2.9 g, 8.89 mmol, 65%) as a yellow oil.

Ή NMR (300 MHz, CDCh): 5.36-5.33 (m, 4H), 3.42-3.37 (t, J= 7.5 Hz, 2H), 2.04-1.97 (m, 8H), 1.83-1.83 (m, 2H), 1.37-1.28 (m, 14H), 0.90-0.86 (t, J = 6.6 Hz, 3H).

Synthesis of (6Z, 12Z,25Z,3 lZ)-heptatriaconta-6,12,25,3 l-tetraen-19-ol, 3

A solution of (6Z, 12Z)-l-bromooctadeca-6,12-diene, 2 (2 g, 6.08 mmol) in ether (10 mL) was added to a mixture of magnésium tumings (162 mg, 6.69 mmol) and iodine in ether (2 mL) under argon at room température. The mixture stirred at room température for 90 minutes (magnésium tumings consumed) whereupon ethyl formate (0.24 mL, 3.04 mmol) was added. After stirring for one hour at room température, the reaction was quenched with IN HCl solution. The mixture was extracted with ethyl acetate (2X100 mL) and the combined organics washed with water then brine. The

150 organics were dried under magnésium sulfate, filtered, and the fdtrate concentrated under vacuum to give a crude oil. The resulting oil was dissolved in éthanol (10 mL) and added to a solution of potassium hydroxide (260 mg) in water (3 mL). After stirring for 12 hours, the mixture pH was adjusted 4 with 2N HCl. The aqueous solution was extracted with dichloromethane (2X) and combined. The organics were washed with brine then dried under magnésium sulfate and filtered. The fdtrate was concentrated under vacuum to give a crude oil. Purification of the crude oil on silica using 10-30% ethyl acetate in n-hexane as eluant to give (6Z,12Z,25Z,3 lZ)-heptatriaconta6,12,25,3 l-tetraen-19-ol, 3 (0.29 g, 0.55 mmol, 18%) as a clear oil.

‘H NMR (300 MHz, CDCfi): 5.36-5.32 (m, 8H), 3.57 (bs, 1H), 3.33-3.32, (m, 2H), 2.13-1.97 (m, 16H), 1.36-1.29 (m, 34H), 0.90-0.86 (t, J =6.6 Hz, 6H).

Synthesis of (6Z, 12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-one, 4

13 12 10

To a mixture of (6Z, 12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-ol, 3 (0.29 g, 0.55 mmol) and sodium carbonate (3 mg, 0.03 mmol) in dichloromethane was added pyridinium chlorochromate (236 mg, 1.1 mmol) at 0 °C. The mixture was warmed to room température and stirred for one hour. After one hour, silica gel (1 g) was added to reaction and the mixture filtered. The fdtrate was concentrated, and the resulted oil purified on silica using 10-20% ethyl acetate in n-hexane as eluant to give (6Z,12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-one, 4 (0.12 g, 0.23 mmol, 42%) as a clear oil.

Ή NMR (300 MHz, CDC1₃): 5.36-5.32 (m, 8H), 3.36-3.32, (m, 1H), 2.40-2.35 (t, J= 6.6 Hz, 3H), 2.14-2.00 (m, 16H), 1.58-1.54 (m, 4H), 1.34-1.29 (m, 28H), 0.90-0.86 (t,J=6.6 Hz, 6H).

Synthesis of 2-((5j-2,2-di((9Z, 12Z)-octadeca-9,12-dien-l-yl)-l,3-dioxolan-4-vl) ethan-l-ol, 7

151

A mixture of (6Z, 12Z,25Z,3 lZ)-heptatriaconta-6,12,25,3 l-tetraen-19-one, 4 (0.12 g, 0.23 mmol), (4S)-(+)-4-(2-hydroxyethyl)-2,2-dimethyl-l,3-dioxolane 5 (0.20 g, 1.38 mmol), and pyridinium ptoluene sulfonate (9 mg) in toluene (10 mL) was heated at reflux under nitrogen positive pressure. After 12 hours, the mixture was concentrated under vacuum to give a crude oil. The resulting crude oil was purified by chromatography on silica using 20-40% ethyl acetate in n-hexane as eluant to give 2-((5)-2,2-di((9Z, 12Z)-octadeca-9,12-dien-l-yl)-l,3-dioxolan-4-yl) ethan-l-ol, 7 (0.11 g, 0.17 mmol, 77%) as a clear oil.

Ή NMR (300 MHz, CDC1₃): 5.36-5.32 (m, 8H), 4.25-4.20 (m, IH), 4.10-4.06 (m, IH), 3.82-3.77 (m, IH), 3.54-3.49 (m, 1 H), 2.23-2.19 (t, J = 6.6 Hz, 3H), 2.14-2.00 (m, 16H), 1.84-1.78 (m, 2H), 1.62-1.51 (m, 6H), 1.34-1.29 (m, 28H), 0.90-0.86 (t, J= 6.6 Hz, 6H).

Synthesis of 3-((5)-2,2-di((9Z, 12Z)-octadeca-9,12-dien-1 -yl)-1,3-dioxolan-4-yl)piOpan-1 -ol, 8

A mixture of (6Z, 12Z,25Z,3 lZ)-heptatriaconta-6,12,25,3 l-tetraen-19-one, 4 (0.50 g, 0.95 mmol), (S)-(3)-(2,2-Dimethyl-l,3-dioxolane-4-yl)propanol 6 (0.76 g, 4.75 mmol), and pyridinium p-toluene sulfonate (36 mg) in toluene (10 mL) was heated at reflux under nitrogen positive pressure. After 12 hours, the mixture was concentrated under vacuum to give a crude oil. The resulting crude oil was purified by chromatography on silica using 20-40% ethyl acetate in n-hexane as eluant to3-((5)2,2-di((9Z,12Z)-octadeca-9,l2-dien-l-yl)-l,3-dioxolan-4-yl)propan-l-ol, 8 (0.48 g, 0.76 mmol, 80%) as a clear oil.

152

Ή NMR (300 MHz, CDCl₃): 5.34-5.29 (m, 8H), 4.06-4.02 (m, 2H), 3.67-3.47 (m, 2H), 3.45-3.43 (m, IH), 2.12-2.01 (m, 16H), 1.65-1.62 (m, 8H), l.34-l.29(m, 32H), 0.89-0.85 (t, J= 6.6 Hz, 6H).

Synthesis of 2-((5)-2,2-di((9Z, 12Z)-octadeca-9,12-dien-1 -yl)-1,3-dioxolan-4-yl)-M(V-dimethylethanl-amine, (AKG-KC2-01, 0-12095)

AKG-KC2-01

To a solution of 2-((5)-2,2-di((9Z,12Z)-octadeca-9,l 2-dien-l-yl)-l,3-dioxolan-4-yI) ethan-l-ol, 7 (0.49 g, 0.79 mmol) in dichloromethane (10 mL) at 0 °C was added methanesulfonyl chloride (73 pL, 0.95 mmol) and triethylamine (0.26 mL, 1.2 mmol). The solution was warmed to room température and stirred for an addition hour. The reaction was quenched with water and extracted with dichloromethane (2X100 mL). The organics were washed with brine then dried over magnésium sulfate and filtered. The filtrate was concentrated under vacuum to give a crude oil. A solution of 2M dimethylamine (10 mL) was added to the resulting crude oil and allowed to stir for 24 hours. The mixture was then quenched with water and extracted with dichloromethane (2X100 mL). The combined organics were washed with brine then dried over magnésium sulfate then fdtered. The filtrate was concentrated under vacuum to give a crude oil. The crude oil was purified by chromatography on silica using 5-100% ethyl acetate in n-hexane as eluant to give 2-((5)-2,2di((9Z, 12Z)-octadeca-9,12-dien-1 -yl)-1,3-dioxolan-4-yl)-./V,./V-dimethylethan-1 -amine, (AKG-KC201, 0-12095), (206 mg, 0.32 mmol, 41%) as a clear oil.

Ή NMR (300 MHz, CDCI3): 5.35-5.32 (m, 8H), 4.08-4.03 (m, 2H), 3.47 (t, J= 6.8 Hz, IH), 2.362.27 (m, 2H), 2.21 (s, 6H), 2.01-1.99 (m, 16H), 1.88-1.77 (m, 2H), 1.68-1.53 (m, 6H), 1.42-1.19 (m, 34H), 0.96-0.86 (t, J= 3.7 Hz, 6H).

MS(APCI) for C43H79NO2: 642.6

153

Synthesis of 3-((S)-2,2-di((6Z, l2Z)-octadeca-6-l2-dien-4-yl)-l,3-dioxolan-4-yl)-N,Ndimethylpropan-1 -amine, AKG-KC3-01, 0-12096)

AKG-KC3-01

Procedure previously described.

3-((S)-2,2-di((6Z, 12Z)-octadeca-6-12-dien-4-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1 -amine, (AKG-KC3-01, 0-12096), (255 mg, 0.39 mmol, 51%) as a clear oil.

Ή NMR (300 MHz, CDCh): 5.39-5.32 (m, 8H), 4.06-4.02 (m, 2H), 3.48-3.44 (m, 1H), 2.35-2.30 (m, 2H), 2.25 (s, 6H), 2.01-1.98 (m, 16H), 1.70-1.51 (m, 12H), 1.35-1.25 (m, 32H), 0.90-0.85 (t, J = 6.6 Hz, 6H).

MS(APCI) for C44H81NO2: 656.6

Synthesis of 2-((5)-2,2-di((Z)-octadec-9-en-l-yl)-l,3-dioxolan-4-yl)-jV,7V-dimethylethan-l-amine (AKG-KC2-OA, 0-11880)

2-((5)-2,2-di((Z)-hexadec-9-en-l-yl)-l,3-dioxolan-4-yl)-7V^V-dimethylethan-l-amine (AKGKC2-PA, 0-11879)

3-((5)-2,2-di((Z)-octadec-9-en-l-yl)-l,3-dioxolan-4-yl)-A^V-dimethylpropan-l-amine (AKGKC3-OA, 0-11957)

154

1. MsCI, Et₃N. CH₂CI₂

Ri = CH₂CH₃ 1 :

H 2 :

MgBr₂diethyl etherate, Et₂0 /----------------Ri = CH₂CH₃ 3

H 4

Mg. I₂, Et₂0

2. Ethyl formate. KOH. H₂0

PCC, Na₂CO₃

CH₂CI₂

Ri	n
ch2ch3	1	9
H	1	10
ch2ch3	2	11

Compounds	Ri	n
AKG-KC2-OA	ch2ch3	1
AKG-KC2-PA	H	1
AKG-KC3-OA	ch2ch3	2

Experimental Procedure (Refer to previously described synthesis of AKG-KC2-01)

Synthesis of (Z)-l-bromooctadec-9-ene 3

Br

Procedure previously described.

(Z)-l-bromooctadec-9-ene, (6.4 g, 19.33 mmol) as a clear oil.

‘H NMR (300 MHz, CDCI3): 5.36-5.32 (m, 2H), 3.41 (t, J= 7.5 Hz, 2H), 2.01-1.99 (m, 4H), 1.871.82 (m, 2H), 1.44-1.26 (m, 22H), 0.87 (t, J = 6.6 Hz, 3H).

(Z)-16-bromohexadec-7-ene 4

155

Br

Ή NMR (300 MHz, CDCh): 5.36-5.32 (m, 2H), 3.42 (t, J= 7.5 Hz, 2H), 2.01-1.99 (m, 4H), 1.871.82 (m, 2H), 1.44-1.26 (m, 18H), 0.89 (t, J = 6.6 Hz, 3H).

Synthesis of (9Z,28Z)-heptatriaconta-9,28-dien-19-ol 5

Procedure previously described.

(9Z,28Z)-heptatriaconta-9,28-dien-19-ol (1.2 g, 2.25 mmol, 47%) as a solid.

Ή NMR (300 MHz, CDCh): 5.36-5.29 (m, 4H), 3.57 (bs, 1H), 2.01-1.97 (m, 8H), 1.42-1.26 (m, 53H), 0.89 (t,J=6.6Hz, 6H).

(7Z,26Z)-tritriaconta-7,26-dien-17-ol 6

Ή NMR (300 MHz, CDCh): 5.36-5.29 (m, 4H), 3.57 (bs, 1H), 2.01-1.97 (m, 8H), 1.42-1.26 (m, 45H), 0.89 (t, .7=6.6 Hz, 6H).

Synthesis of (9Z,28Z)-heptatriaconta-9,28-dien-19-one 7

Procedure previously described.

(9Z,28Z)-heptatriaconta-9,28-dien-19-one (0.89 g, 1.67 mmol, 74%) as a clear oil.

156

Ή NMR (300 MHz, CDCl₃): 5.36-5.29 (m, 4H), 2.03-1.98 (m, 8H), I.42-1.26 (m, 52H), 0.90-0.89 (t,7=6.6 Hz, 6H).

(7Z,26Z)-tritriaconta-7,26-dien-17-one 8

Ή NMR (300 MHz, CDCI3): 5.36-5.29 (m, 4H), 2.03-1.98 (m, 8H), 1.42-1.26 (m, 44H), 0.90-0.89 (t, 7= 6.6 Hz, 6H).

Synthesis of 2-((5)-2,2-di((Z)-octadec-9-en-l-yl)-l,3-dioxolan-4-yl) ethan-l-ol 9

Procedure previously described.

2-((5)-2,2-di((Z)-octadec-9-en-l-yl)-l,3-dioxolan-4-yl) ethan-l-ol (0.39 g, 0.63 mmol, 74%) as a clear oil.

Ή NMR (300 MHz, CDC1₃): 5.36-5.28 (m, 4H), 4.22-4.10 (m, 1H), 4.08-4.05 (m, 1H), 3.82-3.79 (m, 2H), 3.48 (t, 7= 6.8 Hz, 1H), 2.24-2.21 (m, 1H), 2.01-1.99 (m, 8H), 1.81-1.80 (m, 2H), 1.591.54 (m, 6H), 1.34-1.26 (m, 45H), 0.87 (t, 7= 6.3 Hz, 6H).

Synthesis of 2-((5)-2,2-di((Z)-hexadec-9-en-l-yl)-l,3-dioxolan-4-yl)ethan-l-ol, 10

157

Procedure previously described.

2-((5)-2,2-di((Z)-hexadec-9-en-l-yl)-l,3-dioxolan-4-yl)ethan-l-ol (l.02 g, 1.65 mmol, 51%) as a clear oil.

Ή NMR (300 MHz, CDCh): 5.36-5.29 (m, 4H), 4.23-4.10 (m, IH), 4.07-4.05 (m, IH), 3.82-3.79 (m, 2H), 3.48 (t, J =6.6 Hz, l H), 2.24-2.12 (m, IH), 2.01-1.97 (m, 8H), 1.84-1.78 (m, 2H), 1.571.55 (m, 8H), 1.34-1.29 (m, 35H), 0.87 (t, J= 6.3 Hz, 6H).

Synthesis of 3-((5)-2,2-di((Z)-octadec-9-en-1 -yl)-1,3-dioxolan-4-yl)propan-1 -ol, 11

Procedure previously described.

3-((5)-2,2-di((Z)-octadec-9-en-l-yl)-l,3-dioxolan-4-yl) propan-l-ol (0.41 g, 0.65 mmol, 76%) as a clear oil

Ή NMR (300 MHz, CDCh): 5.39-5.32 (m, 4H), 4.06-4.03 (m, 2H), 3.71-3.67 (m, 2H), 3.47-3.46 (m, IH), 2.01-1.99 (m, 10H), 1.66-1.59 (m, 4H), 1.56-1.54 (m, 6H), 1.34-1.26 (m, 44H), 0.87 (t,J = 6.3 Hz, 6H).

Synthesis of 2-((5)-2,2-di((Z)-octadec-9-en-1 -yl)-1,3-dioxolan-4-yl)-AGV-dimethylethan-1 -amine, (AKG-KC2-OA, 0-11880)

AKG-KC2-OA

158

Procedure previously described.

2-((5)-2,2-di((Z)-hexadec-9-en-1 -yl)-1,3-dioxolan-4-yl)-2V,jV-dimethylethan-1 -amine, (AKG-KC2OA, 0-11880), (200 mg, 0.31 mmol, 49%) as a clear oil.

Ή NMR (300 MHz, CDCh): 5.38-5.28 (m, 4H), 4.08-4.01 (m, 2H), 3.48 (t, J= 6.8 Hz, 1H), 2.392.24 (m, 2H), 2.21 (s, 6H), 2.01-1.97 (m, 8H), 1.82-1.77 (m, 2H), 1.68-1.52 (m, 6H), 1.34-1.26 (m, 46H), 0.87 (t, J=6.3 Hz, 6H).

MS(APCI) for C43H83NO2: 646.7

Synthesis of 2-((5)-2,2-di((Z)-hexadec-9-en-1 -yl)-1,3-dioxolan-4-yl)-A,A-dimethylethan-1 -amine, (AKG-KC2-PA, 0-11879)

AKG-KC2-PA

Procedure previously described.

2-((5)-2,2-di((Z)-hexadec-9-en-1 -yl)-1,3-dioxolan-4-yl)-A,A-dimethylethan-1 -amine, ( AKGKC2-PA, 0-11879), (195 mg, 0.33 mmol, 18%) as a clear oil.

Ή NMR (300 MHz, CDCh): 5.35-5.28 (m, 4H), 4.08-4.02 (m, 2H), 3.48 (t, J= 6.6 Hz, 1H), 2.382.27 (m, 2H), 2.20 (s, 6H), 2.01-1.99 (m, 8H), 1.97-1.80 (m, 2H), 1.77-1.52 (m, 6H), 1.34-1.29 (m, 38H), 0.87 (t, J= 6.3 Hz, 6H).

MS(APCI) for C39H75NO2: 590.6

Synthesis of 3-((5)-2,2-di((Z)-octadec-9-en-1 -yl)-1.3-dioxolan-4-yl)-A,A-dimethylpropan-1 -amine, (AKG-KC3-OA, 0-11957)

159

AKG-KC3-OA

Procedure previously described.

3-((5)-2,2-di((Z)-octadec-9-en-1 -yl)-1,3-dioxolan-4-yl)-MA-dimethylpropan-1 -amine, (AKG-KC3OA, 0-11957), (160 mg, 0.24 mmol, 37%) as a clear oil.

Ή NMR (300 MHz, CDCh): 5.39-5.28 (m, 4H), 4.06-4.01 (m, 2H), 3.44 (t, J= 6.8 Hz, IH), 2.26 (t, J= 6.8 Hz, 2H), 2.20 (s, 6H), 2.01-1.97 (m, 8H), 1.82-1.77 (m, 2H), 1.60-1.43 (m, 8H), 1.34-1.26 (m, 46H), 0.87 (t, J= 6.3 Hz, 6H).

MS(APCI) for C44H85NO2: 660.6

Example 2. Assay for in vitro cytotoxicity in human hépatocyte or cancer cells

LNPs can be tested in vitro over a sériés of 10 dilutions to détermine IC50 in human hepatocyte/liver (HepG2; ATCC #HB8065) cells. As these formulations are generally expected to be nontoxic, a positive control of Lipofectamine™ 3000 (ThermoFisher &L3000015) -complexed mRNA (2 μΐ reagent/1 pg mRNA) is included in ail studies. The mRNA used is CleanCap FLuc, EGFP, or MCherry reporter gene mRNA (5moU; Trilink #L-7202, #L-7201, or #L-7203). Data is reported out as the full cell viability curve, as well as a calculation of the actual IC50 value for each compound.

Adhèrent cells are grown to ~80% confluency. The cells are trypsinized by adding 0.25 % trypsin-EDTA (Gibco # 25200-072) and the cells subsequently spun down, and 5 ml of growth medium (MEM media; Corning # 10010 CM) added to disperse the cells. The cell density is determined using a hemocytometer. Growth medium (MEM media containing 10% FBS; Corning # 35015 CV) is added to the cells to adjust to an appropriate concentration of cells. Then, 200 μΐ of the cells (5,000 cells/well) is added to a 96-well clear flat-bottom plate (Costar #9804) and incubated in the plate at 37°C in a humidified incubator with 5% CO2 for 24 h.

160

Serial dilutions of LNP formulations using growth medium as solvent are prepared. These compounds are provided as stérile aqueous with a concentration of 1 mg/ml mRNA. For making dilutions, each LNP stock was warmed to room température. These were further diluted to 4x in the growth media to the highest mRNA concentration tested of 250 ug/ml.

LNPs are added to the wells at a sériés of 1:3 dilutions from the initial 250 pg/ml concentration for each LNP by aspirating out the old media and replacing it with 200 μΐ of the LNP containing media. The plates were incubated at 37°C in a humidified incubator with 5% CO2 for 72 h. At the end of the LNP incubation period, replace the media in each well with 100 μΐ of IX PrestoBlue Cell Viability Reagent (ThermoFisher Cat # Al3261). Incubate the plate at 37°C in a humidified incubator with 5% CO2 30 min to 2 h. Take readings at 30, 60, and 120 min. Read fluorescence with 560 nm excitation and 590 nm émission using SpectraMax M5 plate reader (Molecular Devices). Correct background by subtracting the RFU of the control containing only the culture medium (background control well) from ail sample readings. Calculate the percentage of cytotoxicity using the formula below:

% Cytotoxicity [(RFU.Medium ^— RFU Treatment)/ RFU.Medium] ^x 100% The IC50 was determined using GraphPad Prism using the following formula:

Y=100/(l + 10^A((LogIC50-X)*HillSlope)))

The cytotoxicity of Lipofectamine™ 3000 (ThermoFisher #L3000015) -complexed mRNA (2 μΐ reagent/1 pg mRNA) positive control can in some embodiments be 5-100-fold more toxic than compounds disclosed here. This shows that disclosed compounds are less toxic than commercial transfection reagents in an in vitro hepatocytotoxicity assay. In some embodiments, the compounds described herein form less toxic LNPs in vivo than commercially available transfection reagents.

Example 3. Détermination of pKa of ionizable lipid

The pKa of an ionizable cationic lipid can be calculated several ways. For lipids this is sometimes difficult because membrane structure and neighboring lipids in the membrane can influence the dissociation properties of the amino group, potentially giving inaccurate values. An insitu measurement is idéal, where the apparent pKa of the ionizable lipid is measured while the lipid is within its intended environment, in this case as part of an LNP (Jayaraman 2012, Sabins 2018).

For each LNP formulation, amino lipid pKa values are determined by measuring the fluorescence of 2-(p-toluidino)-6-napthalene sulfonic acid (TNS) during titration from pH 3 to 12. TNS is an anionic molécule that does not fluoresce in solution but increases fluorescence when

161 associated with a positive lipid membrane, and this property has been used in the past to probe membrane surface charge. A master buffer stock is prepared (10 mM sodium phosphate, 10 mM sodium borate, 10 mM sodium citrate, 150 mM sodium chloride) which are used to préparé buffers at various pH values for determining apparent pKa. Using l M sodium hydroxide and l M hydrochloric acid, ~20 unique buffers from the master buffer stock are prepared at different pH values between about 3 and 12. 300 mM 6-(p-Toluidino)-2-naphthalenesulfonic acid sodium sait (TNS reagent) solubilized in dimethyl sulfoxide (DMSO) is used as a stock. LNP are prepared and purified into the desired pH buffer with a final mRNA concentration of 0.04 mg/mL. Using a 96-well plate, preloaded with desired buffers, mRNA containing LNP are added to so that the final concentration of mRNA is 0.7 pg/mL. To each well, TNS is added so that the DMSO concentration is l% (v/v). After mixing, the fluorescence of TNS in each well is measured (Ex/Em 33lnm/445nm) and a sigmoidal best fit analysis is applied to the fluorescence data. The pKa is determined as the pH giving rise to halfmaximal fluorescence intensity. The apparent pKa measured for compounds l-36 is within the pH range 6.0-7.0.

Example 4. Measurement of cell uptake of LNPs.

Measurement of LNP cellular uptake is achieved by fluorescent imaging and/or fluorescent quantification. There are many suitable fluorescent tracers available, such as l,T-Dioctadecyl3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (Dil), 3,3'-Dilinoleyloxacarbocyanine Perchlorate (DiO), l,T-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine Perchlorate (DiD) and l, l 'Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine lodide (DiR) (Thermo). These lipids are weakly fluorescent in water but exhibit high fluorescence when incorporated into lipid membranes such as those présent in LNPs. It is important that the lipids chosen are photostable and hâve high extinction coefficients.

LNPs containing these types of lipids are visualized under a fluorescent microscope. In one method, LNP lipid formulation contains a fluorescent lipid tracer such as l,T-Dioctadecyl-3,3,3',3'Tetramethylindodicarbocyanine-5,5'-Disulfonic Acid (DÎI5-DS) at 0.l-0.5mol% total lipid. Cells of interest are grown in a suitable cell culture dish, such as a 24-well plate (Corning). The cells are seeded the day prior to the uptake study at 50% confluency and grown ovemight under appropriate conditions, for example 37 °C, 5% CO2, 90-100% humidity. LNPs are added to cell culture medium at 0.1-100 ug/mL mRNA, allowed to interact with the cells for some time (4-24h), then the cells are washed with media three times to remove non-intemalized LNPs before viewing. The cells are viewed with a

162 microscope with fluorescent détection capabilities. The relative extent of LNP cellular uptake is determined from the fluorescent intensity signal from the cells, using non-treated cells as a background control. Altematively, quantitative measurement of cellular fluorescent lipid may be achieved by pelleting cells, solubilizing them with a detergent such as Triton-XlOO, and quantifying fluorescence by a spectrofluorometer or quantifying fluorescent lipid tracer by HPLC.

In a similar manner, the quantitation of fluorescently labeled mRNA is achieved. For example, dye-labeled enhanced green fluorescent protein (EGFP) and Firefly luciferase (FLuc) mRNAs, both transcribed with Cyanine 5-UTP:5-Methoxy-UTP at a ratio of l:3 is currently available from Trilink Biotechnologies. Cyanine 5 has an excitation maximum of 650 nm and an émission maximum of 670 nm. Substitution in this ratio results in mRNA that is easily visualized and that can still be translated in cell culture. By entrapping fluorescently labeled mRNA one can visualize the intracellular delivery of mRNA by methods described above.

LNP uptake in cells may be achieved by endogenous methods such as ApoE mediated, or through exogenous methods such as active targeting. It was found that the LNP Systems containing ionizable cationic lipids take advantage of a “naturel” targeting process where they adsorb apolipoprotein E (ApoE) in the blood (Cullis et al 2017) and are then actively taken up in hépatocytes by a number of receptors that contain ApoE binding ligands (Williams et al. 2010). By using nonoverlapping fluorophores it is possible to independently track mRNA and LNP intracellular distribution and organelle accumulation kinetics.

mRNA cellular expression levels may be quantified by using reporter Systems such as EGFP, FLuc or mCherry, available from Trilink Biotechnologies. In one embodiment, EGFP mRNA is encapsulated in LNPs, and added to cells of interest at 0. l-l00ug/mL mRNA. The cells may be washed free of non-intemalized LNP by replacing the media after 4-24h. At 24h, GFP signal is quantified by fluorescence microscopy or flow cytometry. In this way it is possible to differentiate between a panel of LNP formulations based upon reporter protein expression levels.

Example 5. Transfection Selectivity Index

A Transfection selectivity Index (TSI) is calculated to détermine the relative transfection efficiency in mammalian cells, compared to the relative toxicity in those same cells. The selectivity index was calculated using the formula below:

TSI EFmammalian/ IC50,mammalian

163 where EF_mammaiian is the transfection efficiency expressed in terms of ng protein/million cells and ICjo.mammaiian relates to the cell viability of the same formulation in terms of half maximal inhibitory concentration.

The LNPs using compounds described here ( l -36) hâve a 50% higher TSI than LNPs made using otherwise identical LNPs made with control molécule DLin-MC3-DMA as the ICL

Example 6. An assay for lipid peroxidation.

The extent of oxidation can be determined using a forced dégradation assay where LNP samples are treated with 3 % H2O2 at 25 °C, and sampled on days 0, l, 3, and 5 for lipid oxidation products (Blessy et al. (2014) Journal of Pharmaceutical Analysis 4, 159-165). The oxidation reaction can be quenched by addition of 0.1 M butylated hydroxytolulene (BHT) in éthanol and stored frozen at -80 °C until measurement. Lipid oxidation products can be measured using a 2-thiobarbituric acid (TBA) reactivity assay (Gutteridge (1982) FEBS Letters 150, 454-458) to detect malondialdehyde (MDA), an end product of lipid peroxidation or by détection using an HPLC assay with evaporative light scattering détection (ELSD) or charged aérosol détection (CAD). Lipid oxidation and isomerization impurity structures can be assigned based on known literature precedent and are expected to be mixtures of isomers.

Generally, it is known in the art that lipids with multiple unsaturations in the acyl chain are more sensitive to oxidation (see Reis and Spickett (2012) Biochim Biophys Acta I8I8, 2374-2387).

It is anticipated that compounds l -36 described herein will hâve less susceptibility to oxidative damage or dégradation when compared to a control LNPs containing the DLin-KC2-DMA lipid or when compared to a control LNPs containing DLin-MC3-DMA. In some embodiments, the compounds provided herein hâve greater than 30 %, greater 50 %, greater 75 %, greater 90 %, and greater 95 % réduction in oxidation byproducts when compared to the control LNP.

Example 7. Preparing ligand-targeted LNPs

Antibody ligands providing for spécifie uptake of LNPs into the cells of interest, such as, immune cells, in the form of antibody Fab’ fragments or single chain Fv fragments are prepared by any method known in the art (for example, as described in Drummond et al. U.S. Pat. Appl. 20180271998; Zhou et al. U.S. Pat. 10,406,225; Marks et al. U.S.Pat. 8,974,792, which are incorporated herein by reference in their entireties). To provide for conjugation of the ligands to LNPs, the ligands are constructed with a C-terminal sequence having a Cysteine residue, such as CAA, or

164

GGSGGC. The ligands are expressed in bacterial or eukaryotic cells and isolated from the cellular mass or growth medium using standard methods such as protein affinity chromatography or métal chélation chromatography. To activate the thiol group of a terminal Cysteine residue, the ligands are incubated in the presence of 15 mM Cysteine in a 10 mM citrate buffer, pH 6.0-6.2, containing 140 mM NaCl, for l hour, and purified by gel-chromatography on a Sephadex G-25 or similar column, eluent 10 mM citrate buffer, pH 6.0-6.2, containing 140 mM NaCl. The protein concentration in the purified, cysteine-activated ligand solution is determined using UV spectrophotometry at 280 nm. The antibody ligand, at l -10 mg/ml in the above named buffer, is mixed with the aqueous solution of a maleimide-terminated PEG-DSPE dérivative (mal-PEG(2000)-DSPE, cat. No. 880126, Avanti Polar Lipids, AL, USA, or Sunbright ® DSPE-020MA, NOF corporation, Japan) at the protein/lipid molar ratio of 4:1. Mal-PEG-lipids having PEG spacer with molecular weight or 3,400 (Sunbright ® DSPE034MA) or 5,000, available from NOF Corporation (Sunbright ® DSPE-050MA), can be used where longer distance between the LNP surface and the ligand moiety is désirable. The solution is incubated at ambient température for 2 hours, adjusted to 0.5 mM Cysteine to block unreacted maleimide groups, and the micellar ligand-PEG-DSPE conjugale is purified by gel chromatography on Ultrogel AcA 34 (if the ligand is a Fab) or Ultrogel AcA 44 (if the ligand is a scFv), eluent - 144 mM NaCl buffered with 10 mM HEPES, pH 7.0-7.4. The conjugated protein is quantified by UV spectrophotometry, and the purity is confirmed by SDS gel-electrophoresis.

Ligand is appended to the surface of LNPs by one of the following methods.

Method 1. Preformed LNPs (obtained as described in Hope et al. US 10,653,780 incorporated herein by référencé in its entirety) are mixed with the micellar solution of the ligand-PEG-DSPE conjugale in a HEPES-buffered saline (10 mM HEPES, 140 mM NaCl, pH 7.0-7.2) to achieve the required ligand/lipid ratio in the range of 5-100 (typically 15-30) ligands per LNP particle. The mixture is incubated with slow agitation 2 hours at 37-40 °C, or ovemight at 2-8 °C, during which time the conjugale is incorporated into the outer lipid layer of the LNPs. The ligand-conjugated LNPs are purified from unincorporated ligand-PEG-DSPE by gel chromatography on Sepharose CL-2B or CL-4B (hydrophilic size exclusion media with the same molecular weight cutoff can be also used); the LNP fraction appearing near the void volume is collected. The amount of ligand conjugated to the particles is determined by SDS gel-electrophoresis with Coomassie Blue or fluorescent staining and concurrently run ligand standards.

Method 2. A solution of the ligand-PEG-DSPE conjugale in 10 mM Na-citrate buffer pH 4.0 containing also the nucleic acid component of the LNP is mixed with the ethanolic solution of the 165

LNP lipids to the final éthanol concentration of 40 % by volume as described by Semple et al., U.S. Pat. 8,021,686, incorporated herein by reference in its entirety. Altematively, a LNP-préparation protocol of Hope et al. U.S. Pat. 10,653,780 (incorporated herein by reference in its entirety) is employed. The amount of ligand-PEG-DSPE is 0.1-1 mol% of the lipid. The mixture is dialyzed against HEPES-buffered saline (10 mM HEPES, 140 mM NaCl, pH 7.0) to remove éthanol. LigandPEG-DSPE is incorporated in the resulting LNPs. Any residual ligand-PEG-DSPE is removed by gel chromatography using Sepharose CL-4B or CL-2B, eluent HEPES-buffered saline, or by buffer exchange for HEPES-buffered saline by tangential flow filtration on a polysulfone membrane (fiat or hollow fiber cartridge) having 500 KD molecular weight cutoff.

Method 3. Mal-PEG-DSPE is combined with preformed LNPs in a citrate-buffered saline (10 mM Na- citrate buffer pH 6.0-6.2, 140 mM NaCl) in the amount of 0.1-1 mol% relative to the LNP lipid in the same manner as ligand-PEG-DSPE of Method 1. LNPs with incorporated mal-PE-DSPE are purified from unincorporated mal-PEG-DSPE by gel chromatography on Sepharose CL-4B in the same buffer, and incubated with thiol-activated antibody ligand (5-100 ligands pre LNP particle) for 2-24 hours. Ligand-conjugated LNP so obtained are purified from unconjugated ligand by Sepharose CL-4B gel chromatography using HEPES-buffered saline pH 7.0 as eluent.

Method 4. Mal-PEG-DSPE is incoiporated into the LNPs at 0.1-1 mol% of the LNP lipid in the same manner as ligand-PEG-DSPE according to Method 2. The resulting Mal-PEG-conjugated LNPs are incubated with the thiol-activated ligand and purified as described in Method 3.

Method 5. The protocol of Method 4 is perfonned with the différence that instead of mal-PEGDSPE a maleimide-conjugated lipid without a PEG spacer (mal-DSPE, Coatsome® FE-808MA3, NOF corporation, Japan) is added to the lipid solution. The resulting maleimide-LNPs are conjugated to thiol-activated ligand as per Method 3.

Method 6. A low-molecular ligand (e.g., mannose) is conjugated to LNP by the Methods 1 or 2 wherein a mannose-PEG-DSPE (Biochempeg Scientific, MA, USA, cat. No. 12169) is substituted for an antibody ligand-PEG-DSPE.

Example 8. Determining optimal ligand density of the ligand-targeted LNPs

A panel of LNPs are prepared with the increasing ligand density in a given range (2-200 ligands per LNP particle, or 5-100 ligands per LNP particle) using any of the methods of Example 7. The LNPs are fluorescently labeled by incorporation of a fluorescently labeled lipid or fluorescently labeled nucleic acid as described in Example 4. The labeled ligand-conjugated LNPs are tested for the 166 cell uptake according to Example 4, and the ligand content corresponding to the maximum of the ligand-specific cell uptake of the LNPs is determined. The nucleic acid intracellular function (such as mRNA expression) can be used as an assay output (Example 4), in which case the presence of a lipid or nucleic acid détectable label is not necessary.

Example 9. Préparation of lipidic nanoparticles (LNPs).

mRNA modified with 5-methoxyuridine (5moU) and coding for mCherry (Cat#L-7203) was obtained from Trilink Biotechnologies (San Diego, CA). Ail uridine nucleosides were substituted with Nl-methyl-pseudouridine. To produce the mRNA, a synthetic gene encoding the mRNA sequence was cloned into a DNA plasmid. The synthetic gene was comprised of an RNA promoter, a 5’ untranslated région, mCherry protein coding sequence, a 3’ untranslated région, and a poly(A) tail région of approximately 120 As. The open reading frame sequence for the mCherry mRNA from TriLink (Cat#L-7203) corresponds to SEQ ID NO: l:

AUGGUGAGCAAGGGCGAGGAGGACAACAUGGCCAUCAUCAAGGAGUUCAUGCGGUU CAAGGUGCACAUGGAGGGCAGCGUGAACGGCCACGAGUUCGAGAUCGAGGGCGAGG GCGAGGGCCGGCCCUACGAGGGCACCCAGACCGCCAAGCUGAAGGUGACCAAGGGCG GCCCCCUGCCCUUCGCCUGGGACAUCCUGAGCCCCCAGUUCAUGUACGGCAGCAAGG CCUACGUGAAGCACCCCGCCGACAUCCCCGACUACCUGAAGCUGAGCUUCCCCGAGG GCUUCAAGUGGGAGCGGGUGAUGAACUUCGAGGACGGCGGCGUGGUGACCGUGACC CAGGACAGCAGCCUGCAGGACGGCGAGUUCAUCUACAAGGUGAAGCUGCGGGGCACC AACUUCCCCAGCGACGGCCCCGUGAUGCAGAAGAAGACCAUGGGCUGGGAGGCCAGC AGCGAGCGGAUGUACCCCGAGGACGGCGCCCUGAAGGGCGAGAUCAAGCAGCGGCUG AAGCUGAAGGACGGCGGCCACUACGACGCCGAGGUGAAGACCACCUACAAGGCCAAG AAGCCCGUGCAGCUGCCCGGCGCCUACAACGUGAACAUCAAGCUGGACAUCACCAGC CACAACGAGGACUACACCAUCGUGGAGCAGUACGAGCGGGCCGAGGGCCGGCACAGC ACCGGCGGCAUGGACGAGCUGUACAAGAGCGGCAACUGA

Stock solutions of each lipid were prepared. Ionizable lipids were weighed out in 4 mL glass vials (Thermo B7999-2) and dissolved in éthanol (Sigma-Aldrich 200 proof, RNase free) to a final concentration of 10 mM. Other lipids such as DSPC, Cholestérol and PEG-DMG were weighed out and dissolved in éthanol to a concentration of l mM. DSPS was dissolved in methanol (Sulpelco, Omnisolve) at a concentration of l mM and briefly heated to 70 °C to complété its dissolution.

Lipid mixtures for each individual LNP were prepared by adding the desired volume of each lipid stock solution to a new vial, adding éthanol if needed to achieve a final volume of 1.2 mL. For example, an LNP formulation of AKG-UO-l/DSPC/DSPS/Chol/PEG-DMG (50/2.5/7.5/38.5/1.5

167 mol%), with an N/P of 5 contained 1500 nmol AKG-UO-l, 75 nmol DSPC, 225 nmol DSPS, 1155 nmol Chol and 45 nmol PEG-DMG for every 100 pg of mRNA used.

mRNA solutions were prepared by thawing frozen mRNA (mCherry mRNA, Trilink) vials and diluting mRNA in 6.25 mM sodium acetate (pH 5.0) to a final concentration of 0.033 mg/mL. To préparé LNPs, a NanoAssemblr Benchtop microfluidic device (from Précision Nanosystems) was used. If LNPs contained DSPS, the heating block accessory set to 70 °C was used, otherwise LNPs were mixed at room température. 3 mL of mRNA solution was loaded into a 3 mL disposable syringe (BD 309656) and l ml of lipid mixture in a l ml syringe (BD309659) and placed in the NanoAssemblr heating block for 4 min prior to mixing. LNP formation was achieved by pumping the liquid streams through a disposable microfluidics cassette at 3: l aqueous: alcohol volume ratio at 6 mL/min mixing speed. After mixing, 3.6 mL of LNP mixture was collected, while the initial mixed volume of 0.35 mL and last 0.05 mL of mix was discarded. Ethanol was removed by buffer exchange using SpectraPor dialysis tubing ( 12-I4k MWCO) in PBS (Cytivia, SH30256.01) or by sequential concentration and dilution using Amicon Ultra-4 centrifugal concentrators ( 10k MWCO, at 500 g).

LNPs were typically exchanged into PBS, pH 7.4 and then 15 mM Tris, pH 7.4, 20% sucrose, concentrated to 20-50 ug/mL mRNA, stérile fdtered (Thermo Nalgene 0.2 um #720-1320) prior to freezing by immersion in liquid nitrogen for 5 min and long-term storage at - 20 °C.

Example 10. LNP Characterization

A. mRNA concentration and relative encapsulation efficiency détermination by fluorescent binding dye

Materials: Ribogreen reagent (Thermo #11491), 3 x 96-well plates with lids, PBS, dissociation buffer (PBS with 10% DMSO and 1% (wt/wt) Zwittergent 3-14 (Sigma-Aldrich #693017), mRNA, general pipette tips & repeater pipette tips.

1. 5 mL of 2 pg/mL mRNA stock were prepared in DPBS or PBS

2. Diluted standards were prepared as follows in single wells in a 96-well plate (Plate A);

Final [mRNA] ng/mL	Vol. stock 2 pg/mL (pL)	Vol. PBS (pL)
2000	400	0
1500	300	100
1000	200	200

168

Final |mRNA] ng/mL	Vol. stock 2 pg/mL (pL)	Vol. PBS (pL)
500	100	300
0	0	400

3. Using different wells in Plate A, samples were diluted to be within the standard curve, you’ll need one well per sample. For example, if the approximate mRNA concentration should be ~ 30 ug/mL in the sample, a 20X dilution was performed (Dilution Factor). (20 uL sample added to 380 pL PBS in a well). No lid was used on plate A. Samples were mixed by gentle pipetting up & down.

Example of Plate A

A

0

500

1000

1500

2000

B

C

S l

S 2

S 3

S4

S 5

S6

S 7

S 8

Etc.

D

E

F

G

H

4. Two more plates, plates B & C were used. Using a multichannel pipettor, 60 pL of each standard 2 were pipetted into wells each (duplicate), and sample into 3 wells each (triplicate)

Example of Plate B and C

A

0

500

1000

1500

2000

B

0

500

1000

1500

2000

C

S l

S 2

S 3

S4

S 5

S 6

S 7

S 8

Etc.

D

S l

S 2

S 3

S4

S 5

S6

S 7

S 8

Etc.

E

S l

S 2

S 3

S4

S 5

S 6

S 7

S 8

Etc.

F

G

H

169

5. The number of wells used on each plate was counted and 4 was added to this number. For plate B, PBS was prepared with Ribogreen diluted 1:100. For example, for 40 wells, 44 was used as the number. 44 X 60 pL = 2.64 mL Ribogreen solution needed, so that would be 2.61 mL PBS with 26.4 pL Ribogreen.

6. For plate C, 2.61 mL Dissociation buffer and 26.4 uL Ribogreen was pipetted.

7. Using a repeater pipette set for 60 pL, PBS+RiboGreen was added to each well on plate B and 60 pL Dissociation Buffer+Ribogreen to plate C. Both plates B and C were mixed on an orbital mixer (120 rpm) for 1 min. Plate B was placed in the dark for 15 min. Plate C was incubated at 37 °C in the dark for 10 min, followed by 5 min at RT.

8. Both plates were read one after the other, using Ex. 465, Em. 530nm

9. Using the standard curve, the slope and intercept were calculated and by extrapolation the mRNA concentrations of the samples on plate B & C were calculated (average and std.dev)

10. Percent encapsulation efficiency (% EE) by [mRNA] plate B/ [mRNA] plate C X 100 was calculated

11. Total [mRNA] by taking [mRNA] plate C X dilution factor was calculated.

B. LNP Particle size

1. 30 pL of LNP was mixed with 1.5 mL PBS in a polystyrène cuvette (Sarstedt, #67.754) and analyzed for size using a ZetaSizer Pro (Malvem) using ZS Xplorer software, version number 1.4.0.105. The Z-average size and polydispersity index value were recorded. Typically, size measurements of LNPs were taken post LNP mixing, post buffer exchange and post stérile fdtering.

C. LNP Zêta Potential

1. 30 pL of LNP was mixed with 1.5 mL PBS and injected into a disposable folded capillary cell (Malvem Nanoseries DTS 1070) and zêta potential measured on a ZetaSizer Pro at 25 °C.

Example 11. Détermination of transfection efficiency in murine dendritic cells of LNPs using mCherry mRNA.

A. Cell Propagation, Transfection, Harvesting and Staining Protocol

1. MutuDC1940 cells (ABM) were grown according to supplier’s instructions in T75 flasks. When required, they were plated at 180,000 cells/well into 24-well plate one day prior to transfection.

170

2. LNPs were added in triplicate to each well at 1 pg/mL in 1 mL media and after 24h the cells were washed once with DPBS (VWR 02-0119-1000).

3. 0.2 mL of DPBS (plus 5 mM EDTA, pH 7.4) was then added to facilitate detachment.

4. The cells were placed at 37 °C for 3 min, until detached.

5. 0.5 ml DPBS added to each well and the liquid transferred to a flow cytometry tube (Falcon mL #352054).

6. The tube was centrifuged at 1100 rpm for 3-5 min and the liquid poured off.

7. 100 pL of Zombie Violet (Biolegend) (diluted 1:500 in PBS) was added to each tube.

8. The tubes were gently tapped to resuspend cells and placed in the dark for 15 min at RT.

9. To the cells 0.5 mL of (paraformaldéhyde 4% in PBS:DPBS 1:1) was added and the cells flicked gently to resuspend and put on ice for 30 min. Another 2 ml PBS was added.

10. The cells were pelleted as above and resuspended in 0.5 mL DPBS with 5% BSA and placed in the fridge until needed.

B. Cell Analysis

1. Cells suspensions were analyzed by an Attune NxT flow cytometer using the VL 1 and YL2 for live/dead and mCherry fluorescence signais respectively. Gating analysis was performed on FloJo software.

Example 12. Impact of DSPS on transfection effïciency of dendritic cells using LNPs with KC2 as ionizable cationic lipid.

171

The aim of this study was to explore the effect of phosphatidylserine targeting using DSPS on transfection efficiency in murine dendritic cells. LNPs were prepared as described in Example 9, characterized for particle size and zêta potential as described in Example 10, and evaluated for transfection efficiency in murine dendritic cells as described in Example 11. The LNPs ail had DLinKC2-DMA constant at an N/P ratio of 5 and 50 mol % of total lipid, the PS lipid was varied initially from 0 - 2.5 mol % and the DSPC phospholipid varied from 0 - 7.5 mol % (Total mol % of DSPC and DSPS was constant at 10 mol %), and the cholestérol constant at 38.5 mol % (ail mol % of total lipid). The particle size, Polydispersity Index (PDI), and entrapment efficiency for ail formulations is shown below in Tables 5 and 6.

Table 5. Physicochemical properties of KC2-containing LNPs used in Example 12 varying from 02.5 mol % used in Example 12 and FIG. 3A.

Mol % DSPS	Particle Size (nm)	PDI	% Encapsulation ± SD
0	77.6	0.075	86.4 ±2.8
0.1	82.0	0,171	87.0 ±4.7
0.5	89.2	0.285	86.3 ±3.2
2.5	72.2	0.263	87.1 ±6.8

Table 6. Physicochemical properties of KC2-containing LNPs varying from 0-7.5 mol % used in

Example 12 and FIG. 3B, FIG. 3C, and FIG. 3D.

Mol % DSPS	Particle Size (nm)	PDI	% Encapsulation ± SD
0	73.9	0.14	92.6 ±6.2
1.25	80.3	0.13	90.9 ±5.6
2.5	70.7	0.25	89.0 ±7.4
5	78.7	0.12	85.1 ±6.8
7.5	76.4	0.14	85.4 ±3.6

An initial set of LNPs containing DLin-KC2-DMA and varying phosphatidylserine in the form of DSPS from 0-2.5 mol %, showed little transfection at 0 or 0.5 mol % DSPS, but increase by 18fold when DSPS was incorporated at 2.5 mol % (FIG. 3A). A second sériés of LNPs prepared with DSPS from 0-7.5 mol % was evaluated at 0.1, 0.3, and l pg/mL mRNA concentrations (FIG. 3B, FIG. 3C and FIG. 3D). The transfection efficiency increased as the mol % of DSPS was increased above 2.5 mol %, with a maximum at 7.5 mol % at l pg/mL mRNA, and 5 mol % at both 0.1 and 0.3 pg/mL mRNA. These data demonstrate that the inclusion of phosphatidyl-L-serine can dramatically

172 increase the transfection efficiency of mRNA-containing LNPs, and that maximal uptake occurs between 5-7.5 mol % of DSPS (as % of total lipid).

Example 13. Impact of ICL and anionic phospholipid targeting ligand on mRNA transfection of dendritic cells.

The aim of this study was to see if other anionic phospholipids could also enhance the transfection efficiency of LNPs and how LNPs prepared with varying ICLs and PS targeting would transfect dendritic cells. LNPs were prepared as described in Example 9, characterized for particle size and zêta potential as described in Example 10, and evaluated for transfection efficiency in murine dendritic cells as described in Example 11. The LNPs had various ICLs (DLin-KC2-DMA, K.C2-OA, KC3-OA, or SM-102) constant at an N/P ratio of 5 and 50 mol % of total lipid, the PS lipid was kept constant at 5 mol % and the DSPC at 5 mol %, and the cholestérol constant at 38.5 mol % (ail mol % of total lipid). The particle size, PDI, and entrapment efficiency for ail formulations is shown below in Table 7.

Table 7. Physicochemical properties of LNPs varying in ICL used and with anionic phospholipid at 5 mol %.

Ionizable Cationic Lipid (ICL)	Anionic lipid	Particle Size (nm)	PDI	% Encapsulation ± SD
KC2	5 % DSPS	90.9	0.13	91.0 ± 5.8
KC2-OA	5 % DSPS	74.8	0.13	92.8 ±8.0
KC3-0A	5 % DSPS	95.5	0.09	96.6 ± 18.3
SM-102	None	77.5	0.05	92.6 ±5.0
SM-102	5 % DSPS	65.9	0.13	84.5 ±7.7
KC2	5 % Glu-DSPE	52.1	0.23	87.9 ±6.8
KC2	5 % Suc-DSPE	64.6	0.19	86.6 ±3.6

The transfection results are shown in FIG. 4 show high transfection rates with three different K.C-series ICLs (KC2, KC2-OA, and KC3-OA), and also with LNPs prepared with the branched ICL, SM-102. The encapsulation efficiency was high and the particle size below 100 nm for ail formulations, including those prepared with altemate anionic phospholipids (Suc-DSPE or GluDSPE). The data demonstrate that DSPS (L-serine) can not be substituted with either N-glutaryldistearoylphosphatidylethanolamine (Glu-DSPE) or N-succinyl-distearoylphosphatidylethanolamine (Suc-DSPE) and provide the same high level of mRNA transfection despite both phospholipids also 173 containing two négative charges and both containing the same distearoyl (Cl8:0) fully saturated acyl chains. These studies also clearly show that the addition of DSPS can give rise to high transfection efficiencies for other ionizable cationic lipids, including those with a single unsaturated acyl gain (KC2-OA or KC3-0A) and those including a branched ICL, like SM-102. The addition of DSPS to

SM-102 containing LNPs gave rise to a 22-fold increase in mCherry expression, for example.

Example 14. Dependence of PS targeting on ICL and PS structure.

The aim of this study was to compare PS-targeted LNPs with KC2 and K.C3 sériés ionizable cationic lipids of varying acyl chain composition. K.C2 sériés lipids having a structure of dimethylaminoethyl headgroup structure were compared to the KC3 sériés containing a dimethylaminopropyl-derivatized head group. The LNPs contained various ICLs (KC2, K.C2-01, KC2-OA, KC2-PA, KC3-OA, and KC3-01) as the ICL at an N/P ratio of 5 and 50 mol % ICL, and a constant 1.5 mol % PEG-DMG. The cholestérol content was held constant at 38.5 mol % and the

DSPC content varied inversely with the mol % of DSPS at either 0 or 5 mol % (ail lipid concentrations 15 were used as mol % of total lipid). Transfection efficiency was evaluated in murine dendritic cells as described in Example 11.

Table 8. Physicochemical properties of LNPs varying in ICL used and with anionic phospholipid at mol %.

Ionizable Cationic Lipid (ICL)	PS content	Particle Size (nm)	PDI	% Encapsulation ± SD
KC2	None	95.6	0.08	91.7±2.0
KC2	5 % DSPS	82.5	0.09	87.1 ±3.8
KC2-01	None	82.4	0.08	91.6 ± 3.9
KC2-01	5 % DSPS	76.0	0.10	90.5 ±3.5
KC2-OA	None	89.5	0.08	88.6 ±4.2
KC2-OA	5 % DSPS	85.9	0.10	92.4 ±4.5
KC2-PA	None/ 10 % DPPC	92.3	0.11	92.4 ±4.4
KC2-PA	5 % DSPS/5 %DPPC	85.2	0.10	95.5 ±3.4
K.C3-01	None	88.2	0.10	94.3 ±7.1
KC3-01	5 % DSPS	101.2	0.16	95.2 ±4.4
KC3-OA	None	105.9	0.08	86.6 ± 1.6
KC3-OA	5 % DSPS	103.0	0.13	93.8 ±3.2
KC2-PA	5 % DPPS/5 % DPPC	77.1	0.06	93.8 ±3.7

174

The transfection results are shown in FIG. 5 and clearly show a positive impact of PS targeting on multiple KC-series ICLs. Here we show that ICLs containing both unsaturated C16 and C18 ICLs could be targeted with phosphatidyl-L-serine and give rise to high transfection rates for dendritic cells. The highest rate of transfection came when the PS and PC contained a mismatched acylchain composition, with 5 mol % DPPC and 5 mol % DSPS, and combined with a C16 ICL (KC2-PA).

Example 15. Impact of phosphatidylserine structure on transfection efficiency of LNPs

The aim of this study was to explore the effect of different anionic phospholipid structures on transfection efficiency in dendritic cells. LNPs were prepared as described in Example 9, characterized for particle size and zêta potential as described in Example 10, and evaluated for transfection efficiency in murine dendritic cells as described in Example 11. The LNPs ail had AKG-UO-1 constant at an N/P ratio of 5 and 50 mol % of total lipid, the anionic lipid was varied depending on the formulation and inversely to the DSPC phospholipid, and the cholestérol constant at 38.5 mol % except for the 20 mol % DSPS LNP (ail mol % of total lipid). For samples that contained up to 10% phosphatidylserine component, the phosphatidylcholine composition was reduced accordingly from 10mol%. For example, an LNP with 5 mol% DSPS, contained 5 mol% DSPS and 5 mol% DSPC, and those that contained 10 mol% DSPS had no DSPC. However, for the sample that contained 20mol% DSPS, there was no DSPC in the formulation and the mol% cholestérol was reduced by 10 mol% to 28.5 mol%.

The entrapment efficiency for ail formulations was between 84 and 90 %, indicating highly efficiency mRNA entrapment in the LNP.

Table 9. Physicochemical properties and encapsulation for LNPs prepared with AKG-UO-1 as the ICL and varying anionic phospholipids.

LNP Formulation (anionic lipid)	Particle Size (nm)	PDI	Zêta Potential at pH 5 (mV)	Zêta Potential at pH 7 (mV)	% Encapsulation
No Anionic lipid	98.1	0.03	13.01	-0.2	84.2 ±4.2
2.5% L-DSPS	96.8	0.03	3.4	-1.7	86.1 ±5.0
5% L-DSPS	95.7	0.05	14.9	-2.9	88.3 ±3.2
7.5% L-DSPS	95.1	0.10	17.0	-4.2	89.1 ±3.6
10% L-DSPS	100.5	0.08	15.2	-5.1	89.6 ±3.1
7.5% L-DPPS	96.3	0.07	17.8	-7.6	88.1 ±2.7
7.5% D-DSPS	100.1	0.14	19.5	-9.6	87.4 ±4.4
7.5% L-DOPS	110.4	0.07	15.1	-3.5	89.5 ±3.5
7.5% L-DMPS	97.6	0.08	12.7	-3.8	88.4 ± 1.3

175

20% L-DSPS	97.7	0.07	7.0	-7.0	88.5 ± 1.9
5% DSPG	86.6	0.04	17.5	-4.7	87.1 ±3.1
7.5% DSPG	85.0	0.08	16.6	-4.1	90.1 ±0.9
7.5% Glu-DSPE	94.0	0.07	16.9	-6.4	89.6 ±9.1
7.5% Suc-DSPE	96.9	0.05	14.3	-5.6	88.4 ±9.0

The effect of different phosphatidylserine Chemical forms was evaluated in FIG. 6A showing the importance of saturation, acyl chain length, and serine stereochemistry on LNP transfection activity in murine dendritic cells. PS analogs with oleic acid acyl chains (DOPS) or where the stereochemistry as (D-serine) rather than L-serine gave rise to LNPs with transfection activity similar to that of LNPs prepared without any phosphatidylserine. However, LNPs prepared with PS containing the L-isomer of serine and saturated acyl chains are significantly better at transfecting dendritic cells compared to LNPs without any PS. Of the saturated sériés, those with C16 (DPPS) or C18 (DSPS), showed the highest activity, while those with C14 (DMPS) was lower, although still improved compared to dendritic cells treated with LNPs without any PS. In FIG. 6B the impact of other anionic phospholipids were evaluated with DSPG-containing formulations (5 or 7.5 mol %) showing activity similar to background, and N-glutaryl- or N-succinyldistearoylphosphatidylethanolamine (Glu-DSPE or Suc-DSPE) showing a more modest 3-5-fold enhancement of activity when included in these AKG-UO1 containing LNPs. This example shows that LNPs prepared with saturated phosphatidyl-L-serines transfect dendritic cells significantly better than those containing the unsaturated dioleoylphosphatidyl-L-serine (DOPS) or the D-isomer of DSPS. LNPs containing the L-isomers of DSPS and DPPS showed the highest level of transfection compared to other forms of PS, anionic N-glutaryl or N-succinyl DSPE analogs, or distearoylphosphatidyglycerol (DSPG) at either 5 or 7.5 mol %.

Example 16. Optimizing the DSPS density on AKG-UO-1 containing LNPs

The aim of this study was to explore the impact of DSPS density on transfection efficiency of LNPs. LNPs were prepared as described in Example 9. The LNPs contained AKG-UO-1 as the ICL at an N/P ratio of 5 with between 0 and 20 mol % DSPS and a constant 1.5 mol % PEG-DMG. The cholestérol content was held constant at 38.5 mol % and the DSPC content varied inversely with the mol % of DSPS between 0-10 mol % (ail lipid concentrations were used as mol % of total lipid). In the 20 mol % DSPS formulation, there was no DSPC and the cholestérol content decreased in the total

176 by ΙΟ mol % (from 38.5 to 28.5 mol %). Transfection efficiency was evaluated in murine dendritic cells as described in Example 11.

Table 10. Physicochemical properties and encapsulation for LNPs prepared with AKG-UO-1 as the ICL and varying anionic phospholipids.

LNP Formulation	Particle Size (nm)	PDI	Zêta Potential at pH 5 (mV)	Zêta Potential at pH 7 (mV)	% Encapsulation
UO1 + 0% DSPS	98.1	0.03	13.0	-0.2	84.2 + 4.2
UOl+2.5% DSPS	96.8	0.03	3.4	-1.7	86.1 ±5.0
UO1 +5% DSPS	95.7	0.05	14.9	-2.9	88.3 + 3.2
UO 1+7.5% DSPS	95.1	0.10	17.0	-4.2	89.1+3.6
UO1 +10% DSPS	100.5	0.08	15.2	-5.1	89.6 + 3.1
UO1 +20% DSPS	97.7	0.07	7.0	-7.0	88.5+ 1.9

The dependence of the LNP composition on DSPS concentration is shown in FIG. 7 for the AKG-UO1 containing LNPs. This study suggests that the presence of DSPS in the formulation allows for a high level of transfection of dendritic cells between 2.5 and 10 mol % DSPS, and an apparent peak around 5-7.5 mol % DSPS for AKG-UO-I containing LNPs.

Example 17. Impact of PEG on transfection efficiency of AKG-UO-1 containing LNPs.

The aim of this study was to explore the impact of PEG-lipid density on transfection efficiency of nontargeted and phosphatidyl-L-serine targeted LNPs. LNPs were prepared as described in Example 9. The LNPs contained AKG-UO-1 as the ICL at an N/P ratio of 5 with either 0 or 5 mol % DSPS and between 0.5-4.5 mol % PEG-DMG. The cholestérol content was held constant at 38.5 mol % and the DSPC content was 10 mol % for the formulations with no DSPS and 5 mol % for those with 5 mol % DSPS. At PEG-DMG content above 1.5 mol%, the total cholestérol content was reduced by the amount of PEG-DMG added, for example with a PEG-DMG content of 3.5 mol%, the cholestérol content was reduced to 36.5 mol% from 38.5 mol%. The particles with 0.5 % PEG-DMG showed a négative zêta potential at pH 7.4, and a significant shift to a positive zêta potential at pH 5. The LNPs with 1.5-3.5 mol % PEG-DMG were essentially neutral at pH 7.

Table 11. Physicochemical properties of LNPs used in Example 17 and containing AKG-UO-1, 0 or 5 mol% DSPS

177

% DSPS	% PEG-DMG	Particle Size (nm)	Zêta Potential at pH 7 (mV)	Zêta Potential at pH 5 (mV)
0	0.5	98.9	0.27	25.92
0	1.5	75.0	-0.87	12.07
0	2.5	73.8	-0.51	11.86
0	3.5	81.0	-0.30	6.34
0	4.5	143	-1.47	14.51
5	0.5	106.1	-1.35	9.30
5	1.5	73.7	-0.21	3.23
5	2.5	70.7	-0.20	4.75
5	3.5	65.6	-0.10	7.54
5	4.5	63.1	-1.12	3.73

The impact of PEG-DMG on transfection of dendritic cells by DSPS-targeted LNPs containing the AKG-UO-1 ICL is shown in FIG. 8. The formulation with 0.5 % PEG-DMG showed a transfection efficiency in the presence of 5 % DSPS that was between 6-7 fold higher than observed at 1.5-2.5 % PEG-DMG. Transfection at 1.5 and 2.5 % PEG-DMG was similar, but decreased dramatically at 3.5 and 4.5 mol % PEG-DMG. The ratio of targeted to nontargeted transfection at each PEG-density varied, and was 12-fold at 0. 5 % PEG, 7-fold at l .5 % PEG, 37-fold at 2.5 % PEG, and below 5-fold at 3.5 and 4.5 % PEG, likely because of high PEG-shielding of the PS targeting moiety. The combination of these data show that the optimum range of PEG-densities is between 0.5-2.5 % PEGDMG, with the lower end of the range being optimum for overall transfection efficiency, while the 2.5 % being optimal for target specificity.

Example 18. Oxidative stability of ICLs

The aim of this studies was to compare stability of ionizable cationic lipids with conjugated olefins (such as KC2, KC3 and O-l 1769) and those with conjugated olefins (such as KC2-01, KC301 and UO-1) under accelerated oxidation.

Individual lipid stocks (10 mM) were prepared in éthanol and stored at -20°C. Prior to the experiment, 5 mM suspensions (KC2, KC2-01, KC3, KC3-01, O-l 1769 and UO-1) were prepared by mixing 45 μΐ of 10 mM lipid stock in éthanol (Sigma-Aldrich, cat# 459836) with 45 μΐ of ultra-pure water (Rx Biosciences, cat# P01-UPW02-1000). Liposomal formulations based on AKG-UO-1 and O-l 1769 ionizable cationic lipids (Table 12) were prepared by combining lipid mixtures of desired composition in 1 mL éthanol with 3 mL 6.25 mM sodium acetate, pH 5.0 at 6 mL/min on a NanoAssemblr (Précision Nanosystems). 3.6 mL of the mixture was retained, while the initial 0.35

178 mL of the mixture and final 0.05 mL were discarded. Ethanol was removed by buffer exchange into PBS, pH 7.4 by concentrating each liposome préparation using an Aminon-Ultra 4 centrifugal concentrator at 500g for 10 min at 4 °C and diluting back to the original volume with PBS. This cycle was repeated multiple times until the éthanol concentration was < l%. Finally, liposomes were stérile fdtered through 0.2 pm PES (Nalgene) syringe filters and size measured by a ZetaSizer (Malvem). The AKG-UO-l containing formulation (Lot#l0202l-6) had an average size of 81.8 nm and a PDI of 0.09, whereas the O-l 1769 containing formulation had an average size of 86.6 nm and a PDI of 0.10.

Table 12. LNP formulations used in the accelerated oxidation study.

Lot#	Composition	MW	mol%	Estimated total lipid [mM]
102021-6	AKG-UO-l	658.1	50	1.5
	DSPC	790	10
	Cholestérol	387	38.5
	PEG2000-DMG	2500	1.5
102021-7	0-11769	658.1	50	1.5
	DSPC	790	10
	Cholestérol	387	38.5
	PEG2000-DMG	2500	1.5

Aliquots of the liposomal formulations were stored at -80°C and thawed before the experiment. A combined stock in water of 10% H2O2 (Sigma-Aldrich, cat# H1009) and 1 mM ofFe(III)Cl (SigmaAldrich, cat# 372870) was freshly prepared prior to the treatment. To make 1% final concentration of H2O2 and 100 μΜ Fe(III)Cl, 10 μΐ of 10% H2O2/I mM Fe(III)Cl stock was added to 90 μΐ of both liposomal formulations and individual lipids The liposomes and individual lipids were incubated with H202/Fe(III)Cl at 37°C and then 5 μΐ from each sample was taken at different time points (0, 3, 24, 48 and 72 hours) and dissolved in 90 μΐ of MeOH for HPLC analysis. Dégradation of the main lipid peak was analyzed using Thermo Scientific Vanquish Flex UHPLC occupied with Charged Aérosol Detector (CAD) and Thermo Scientific Accucore™ Cl8+ UHPLC column (L= 50 mm, D= 2.1 mm, Particle Size = 1.5 pm). The UHPLC operating conditions are listed in Table 13.

Table 13. Chromatographie Conditions

Thermo Scientific Vanquish Flex UHPLC Accucore™ Vanquish™ Cl8+ UHPLC column 55 °C

HPLC Instrument

HPLC Column Column Température

179

Flow Rate Injection Volume Absorbance détection CAD Run Time Sample Température Sample Solvent Mobile Phase	0.5 mL/min 5 pL 210 nm 10Hz 15 min 21°C MeOH Mobile Phase A: 5 mM ammonium acetate in water (pH 4) Mobile Phase B: Methanol
Mobile phase program:	Time, min Mobile Phase A,% Mobile Phase B,% -0.5 15 85 0 15 85 2 10 90 4 2 98 8 2 98 12 0 100 14 15 85 15 15 85

The data are presented as a percentage of the main lipid peak measured at different time points relative to the lipid peak measured at time zéro.

Table 14. Dégradation of ICLs with two olefïns separated by one (KC2, KC3, or 0-11769) or more (KC2-01, K.C3-01, UO-1, UO-6, and UO-7) methylenes.

Lipid/Formulation	% of parent lipid peak at time 0
24 h	48 h	72 h
ICL Lipid Suspensions
KC2	35 ± 0.7	6± 1.9	0±0.0
KC2-01	91 ±0.3	87 ±0.9	83 ±0.3
KC3	46 ± 1.8	1 ±0.2	0±0.0
KC3-01	83 ±4.3	80 ±0.4	74 ±2.2
0-11769	31 ±0.9	1 ±0.2	0.3 ±0.1
UO-1	78 ± 0.6	74 ±0.1	65 ±2.5
UO-6	58 ±0.8	32 ± 1.3	22 ± 1.8
UO-7	80 ± 1.7	71 ±0.8	62 ± 1.0
Liposome préparations
UO-1 (102021-6)	86 ±6.2	85 ±4.2	73 ±0.1
0-11769 (102021-7)	27 ±2.2	8± 1.4	4 ± 0.1

180

As shown in FIG. 9A and FIG. 9B, ail ICLs with olefins that had four methylenes between the two olefins (KC2-01, KC3-01 and UO-1) demonstrate dramatically superior stability under accelerated oxidation with hydrogen peroxide comparing to their counterparts with a single methylene separating the two olefins (KC2, KC3 and 0-11769 respectively). Even after 72 h treatment with hydrogen peroxide, the main peaks of KC2-01, KC3-01 and UO-1 stay above 70% relative to the start of the treatment, while KC2, KC3 and O-l 1769 are fully degraded after 48 hours of incubation with hydrogen peroxide. Two other polyunsaturated ICLs (UO-6 and UO-7) similarly showed good stability to oxidation, although the UO-7 with a hydroxyethyl substituent in the head group degraded more rapidly than then dimethylamino ICLs (Table 14).

The stability of ICLs with olefins separated by more than one methylene were studied as part of mRNA-free liposomal formulations using the structurally related UO-1 and 0-11769 ICLs. Other ionizable cationic lipids (KC2, KC2-01, KC3 and KC3-01) were excluded from this study since their chromatographie peaks overleap with the peak of DSPC which compromises the data interprétation. The stability data of UO-1 and 0-11769 based liposomal formulations are shown in FIG. 9A. UO-1 formulated in liposomes has 73 ± 0.05% of main UO-1 peak after 72 hours of incubation in the presence of 1% hydrogen peroxide. In contrast, 0-11769 based liposomes show only 3.9 ± 0.06 % of the 0-11769 peak after 72 hours of the treatment.

The totality of this data suggests that in addition to the improved transfection efficiency demonstrated in Examples 14 and 20 or the ICLs with more than one methylene between their olefins, these lipids also display significantly improved stability to oxidative dégradation.

Example 19. Impact of N/P ratio on transfection efficiency of mCherry mRNA containing LNPs.

The aim of this study was to explore the effect of different N/P ratios on transfection efficiency in dendritic cells. LNPs were prepared as described in Example 9, characterized for particle size and zêta potential as described in Example 10, and evaluated for transfection efficiency in murine dendritic cells as described in Example 11. The LNPs ail used K.C2-01 as the ICL but varied the cationic lipidto-mRNA phosphate (N/P) ratio from 4-7, the PS lipid was constant at 5 mol % and the DSPC phospholipid constant at 5 mol %, and the cholestérol constant at 38.5 mol %. The entrapment efficiency for ail formulations was between 84 and 90 %, indicating highly efficiency mRNA entrapment in the LNP. Transfection efficiency was evaluated in murine dendritic cells as described in Example 11.

Table 15. Physicochemical properties of LNPs used in Example 19

181

% DSPS	N/P	Particle Size (nm)	Zêta Potential at pH 7 (mV)	Zêta Potential at pH 5 (mV)
0	4	85.4	-10.8	16.3
0	5	84.9	-6.9	20.9
0	6	81.1	-7.0	21.6
0	7	75.7	-6.1	21.5
5	4	84.1	-17.5	13.8
5	5	80.9	-17.2	20.4
5	6	82.2	-15.5	15.8
5	7	79.5	-13.9	16.6

Transfection activity was evaluated for both DSPS-targeted and nontargeted KC-Ol LNP formulations at both l ug/ml (FIG. 10A) and 0.33 ug/ml (FIG. 10B). These data show high DSPSmediated transfection efficiency for KC2-01 containing LNPs over a broad range of N/P ratios with 5 the greatest transfection efficiency being observed at an N/P of 7.

Example 20. Impact of ionizable lipid structure on transfection efficiency containing LNPs.

The aim of this study was to explore the effect of different ICLs on transfection efficiency in dendritic cells. LNPs were prepared as described in Example 9, characterized for particle size and zêta potential as described in Example 10, and evaluated for transfection efficiency in murine dendritic 10 cells as described in Example l L The LNPs used the ionizable lipids in Table 16 in the ICL with a constant N/P ratio of 5, the PS lipid was either 0 or 7.5 mol % and the DSPC phospholipid constant at 10 or 2.5 mol % (total of DSPC and DSPC was 10 mol %), and the cholestérol constant at 38.5 mol

Table 16. Physicochemical properties of LNPs used in Example 20.

ICL	% DSPS	Particle Size (nm)	Particle Size (nm) PostFreeze/Thaw	Encapsulation Efficiency (%)
AKG-UO1	0	98.0	122.6	75.4 ±2.2
AKG-UO1	7.5	93.9	94.2	91.6 ± 2.9
AKG-UO1A	0	84.4	103.2	77.5 ±0.9
AKG-UO1A	7.5	89.2	91.1	88.7 ±4.1
0-11769	0	83.0	89.6	87.5 ±2.7

182

0-11769	7.5	130.8	126.4	91.5 ±6.2
AKG-UO4	0	102.3	169.4	46.5 ± 1.8
AKG-UO4	7.5	89.9	91.3	89.0 ±7.6
AKG-UO4A	0	112.1	123.4	73.1 ±4.1
AKG-UO4A	7.5	92.4	91.81	85.8 ±4.3
AKG-DM2-OA	0	77.6	111.5	64.9 ±3.4
AKG-DM2-OA	7.5	106.5	123.0	86.2 ±5.5
DODAP	0	69.8	68.2	83.4 ± 7.7
DODAP	7.5	90.1	88.0	84.1 ±2.3

The effect of targeting and ICL choice on transfection activity was evaluated using both nontargeted and 7.5 mol % DSPS-targeted LNPs (FIG. 11). Ail ICLs used in this study had a related diacyl structure that varied either in the degree and position of unsaturations in the acyl chains or the spécifie ionizable amine used, and thus it’s apparent pKa. This data shows that the AKG-UO1 lipid with the two olefins separated by four methylenes (C 18 total length) showed the highest activity when incorporated into LNPs. The 0-11769 lipid (dilinoleic acid) showed activity that was approximately half of the AKG-UO-1 containing LNP, the latter having similar activity to the AKG-UO4 lipid with C16 acyl chains and the ICL with the same head group but oleic acid (single olefin) at both acyl chains (AKG-0A-DM2). However, the largest réductions in activity came from changing the dimethyl amine substituent in AKG-UO-1 to diethylamino in U0-1A, or in decreasing the number of methylenes between the two esters and dimethylamino group (DODAP). These two changes gave rise to large réductions in transfection activity, the latter even in the presence of DSPS targeting.

It is also worth noting that among this sériés, those LNPs that contained 7.5 mol% DSPS were less susceptible to adverse changes in particle size after they underwent a freeze-thaw process than those that did not contain DSPS. On average, those LNPs that did not contain DSPS changed 28.4 nm in diameter, whereas those that contained DSPS changed 3.9 nm. Since, storage stability is of major concem for mRNA vaccines, the addition of DSPS provides an important stabilizing effect to these LNPs.

Example 21. Measuring the effect of adding 10 and 25 mol% DOPS on LNP particle formation and activity in MutuDC1940 dendritic cell line.

183

The aim of this study was to explore the impact of including DOPS into mRNA LNP formulations at compositions at or below the mol % previously shown in the literature (Gaitonde et al. (20ll) Clin Immunol 138, 135-145; Rodriquez-Femandez (2018) Front Immunol 9, 253) to enhance liposome (with) uptake into dendritic cells. LNPs were prepared as described in Example 9 at 25 °C and analyzed as in Example 10. The LNPs contained KC2 as the ICL at an N/P ratio of 5 with between 0, 10 and 25 mol % DOPS and a constant l.5mol % PEG-DMG. The cholestérol content was held constant at 38.5 mol % for the 0% and 10% DOPS formulations and the DSPC content varied inversely with the mol % of DOPS between 0-10 mol % (ail lipid concentrations were used as mol % of total lipid). In the 25 mol % DOPS formulation, there was no DSPC and the cholestérol content decreased in the total by 15 mol % (from 38.5 to 23.5 mol %). Transfection efficiency was evaluated in murine dendritic cells as described in Example 11.

Table 17. Physicochemical properties of DOPS containing LNP formulations

LNP Formulation	Particle Size (nm)	PDI	Encapsulation Efficiency (%)
KC2/DSPC/DOPS/Chol/PEG- DMG (50/10/0/38.5/1.5)	68.8	0.136	90.0 ±3.5
KC2/DSPC/DOPS/Chol/PEG- DMG (50/0/10/38.5/1.5)	80.8	0.163	90.9 ±3.5
KC2/DSPC/DOPS/Chol/PEG- DMG (50/0/25/23.5/1.5)	Peak 1 = 62.6, Peak 2 = 416.3	0.398	77.3 ±3.5

The impact of 10-25 mol % DOPS on targeting of LNPs to dendritic cells was evaluated in FIG. 12. This study shows that including 10 mol% DOPS in a KC2-based LNP formulation had a positive effect on mCherry expression levels in MutuDC1940 cells, while not adversely affecting either particle size or encapsulation of mRNA. However, when the DOPS content was increased to 25 mol% the expression of mCherry was lower than the formulation that had no DOPS and the size distribution widened as demonstrated by an increase in the PDI and gave rise to a distribution that contained particles > 400 nm. Taken together, 25 mol% may hâve been shown in the literature to enhance liposome uptake into dendritic cells, while in an LNP formulation with mRNA it had a deleterious effect on both particle size and transfection activity. Importantly, the DOPS used here and in the literature contained unsaturated acyl chains, in this case oleic acid. This is similar to what is 184 typical in many cells, where the phosphatidylserine acyl chains are often unsaturated in the sn-2 position, in many instances with multiple olefins (2-4). Although there is a small enhancement with a lower concentration of DOPS, this enhancement was shown in Example 15 to be significantly higher when the PS was comprised of saturated acyl chains, most preferably dipalmitoyl (Cl6) or distearoyl (C18).

Example 22. Impact of pegylation and phosphatidylserine targeting on immunogenicity of SARS-CoV-2 spike protein mRNA vaccine constructs.

Mice and study design. The in vivo study was carried out. Female BALB/c mice were purchased from Jackson Labs, allowed to acclimate in the vivarium for at least 7 days, and were 6-8 weeks at the start of the study. On study day 0 mice were injected intramuscularly in the right quadricep with l ug of vaccine candidate (quantity refers to mRNA) in a volume of 50 pL. Study groups consisted of 5 mice and included vehicle control, comparator vaccines, and experimental vaccine candidates. Mice were given a second injection of the same vaccine candidate 21 days later. Blood was collected and sérum was isolated from 5 randomly selected control mice at the start of the study and from ail mice on study day 21 and 34. Sérum was stored at -80°C until analysis for antibody titers. On study day 34, mice were euthanized and spleens were harvested.

Design and préparation of mRNA. mRNA encoding the SARS-CoV-2 full length spike protein and flanked with the same UTRs used in the BNTl62b2 (Comimaty) vaccine was purchased from Vemal Biosciences. Ail uridine nucleosides were substituted with Nl-methyl-pseudouridine. To produce the mRNA, a synthetic gene encoding the mRNA sequence (VRN029; SEQ ID NO: 2, FIG. 13A) was cloned into a DNA plasmid. The synthetic gene was comprised of an RNA promoter, a 5’ untranslated région, the SARS-COV2 Spike protein receptor binding domain, a 3’ untranslated région, and a poly(A) tail région of approximately 120 As. The plasmid was propagated and expanded in a culture of E. coli and then isolated from the clarified E. coli lysate via anion exchange chromatography. The purified plasmid was linearized using a type Ils restriction enzyme that eut at a site at the end of the poly(A) tail encoding région. That plasmid was then incubated in a buffer with nucléotide triphosphates, RNA polymerase, and RNase inhibitor. To stop the reaction, DNase I was added to digest the linear plasmid template. The uncapped RNA was then purified using chromatography and then incubated in another buffer with GTP, S-adenosylmethionine, a guanalyltransferase, 2’-O-methyltransferase, and RNase inhibitor. The capped mRNA was then purified using chromatography, buffer exchanged into water, and filled into vials.

185

Génération of lipid nanoparticles (LNP) containing mRNA. Stock solutions of each lipid were prepared. Ionizable lipids were weighed out in 4 mL glass vials (Thermo B7999-2) and dissolved in éthanol (Sigma-Aldrich 200 proof, RNase free) to a final concentration of 10 mM. Other lipids such as DSPC (Avanti Polar Lipids), Cholestérol (Dishman) and PEG-DMG (NOF) were weighed out and dissolved in éthanol to a concentration of 1 mM. DSPS-Na (NOF) was dissolved in methanol (Sulpelco, Omnisolve) at a concentration of 1 mM and briefly heated to 70 °C to complété its dissolution.

Lipid mixtures for each individual LNP were prepared by adding the desired volume of each lipid stock solution to a new vial, adding éthanol if needed to achieve a final volume of 1.2 mL. For example, a LNP formulation of AKG-UO-l/DSPC/DSPS/Chol/PEG-DMG (50/2.5/7.5/38.5/1.5 mol%), with an N/P of 5 contained 1500 nmol AKG-UO-l, 75 nmol DSPC, 225 nmol DSPS, 1155 nmol Chol and 45 nmol PEG-DMG for every 100 pg of mRNA used. mRNA solutions were prepared by thawing frozen mRNA (SARS-CoV-2 spike mRNA, Vemal) vials and diluting mRNA in 6.25 mM sodium acetate (pH 5.0) to a final concentration of 0.033 mg/mL, where the concentration is confirmed by absorbance on a Nanodrop.

To préparé LNPs, a NanoAssemblr Benchtop microfluidic device (from Précision Nanosystems) was used. If LNPs contained DSPS, the heating block accessory set to 70 °C was used, otherwise LNPs were mixed at room température. 3 mL of mRNA solution was loaded into a 3 mL disposable syringe (BD 309656) and 1 ml of lipid mixture in a 1 ml syringe (BD309659) and placed in the NanoAssemblr heating block for 4 min prior to mixing. LNP formation was achieved by pumping the liquid streams through a disposable microfluidics cassette at 3:1 aqueous: alcohol volume ratio at 6 mL/min mixing speed. After mixing, 3.6 mL of LNP mixture was collected, while the initial mixed volume of 0.35 mL and last 0.05 mL of mix was discarded. Ethanol was removed by buffer exchange using SpectraPor dialysis tubing (12-14k MWCO) in PBS (Cytivia, SH30256.01). LNPs were typically exchanged into PBS, pH 7.4 and then 15 mM Tris, pH 7.4, 20% sucrose, concentrated to 20-50 ug/mL mRNA, stérile fdtered (Thermo Nalgene 0.2 um #720-1320) prior to freezing by immersion in liquid nitrogen for 5 min and long-term storage at -20°C. For this study, samples were concentrated to >40 pg/mL mRNA, and diluted with varying volumes of 15 mM Tris, 20% Sucrose, pH 7.4 to a target concentration of 40 pg mRNA and then frozen on LN2. Characterization of LNPs was undertaken after an aliquot of the LNPs were thawed and diluted 1:1 (vokvol) with 15 mM Tris, pH 7.4 such that the final concentration was 20 pg/mL mRNA in 15 mM Tris, 10% sucrose, pH 7.4.

186

This simulated the conditions of sample préparation that were performed prior to dosing the animais with an injection of l pg mRNA in 50 pL volume via IM injection into a hind limb.

LNP Characterization. mRNA encapsulation and mRNA concentration within the LNPs was measured using a Ribogreen assay. Nanoparticle size and zêta potential were measured by a zetasizer (Malvem).

SARS-CoV-2 anti-spike antibody titers. A standard indirect ELISA was performed to analyze sérum samples for total IgG binding antibodies to the SARS-CoV-2 spike protein. For this assay, Nunc MaxiSorp 96-well plates were coated with 100 pL of SARS-CoV-2 spike protein (Sino Biological, cat. no. 40589-V08B1) diluted to 2 pg/mL in Ix PBS, pH 7.4. Plates were incubated statically for 12 hrs at 37°C. Unbound coating antigen was removed by washing plates 3x with 100 pL PBS + 0.05% Tween-20. Plates were then blocked in PBS + 5% skim milk for l hr at 37°C. Test and positive control samples were diluted in assay diluent (PBS, Tween-20, l% skim milk) to starting point dilution 1:20 followed by four-fold serial dilutions using U-bottom dilution plates. Once blocking was completed, blocking buffer was removed by inversion and each sample was plated in duplicates. Plates were statically incubated for 2 hr at 37°C, followed by washing 3x with 100 pL of PBS + 0.05% Tween-20 to remove unbound sera. 100 pL of secondary détection antibody (goat antimouse-HRP IgG, Abcam) was added to each well at a dilution of 1:10,000. Plates were incubated statically for 30 min at RT, and unbound antibodies were subsequently removed and plates were washed as described above. To develop, 100 pL of 1-Step Ultra TMB substrate was added to each well and the reaction was stopped after - 10 min with 50 pL of TMB stop solution (SeraCare, cat. no. 5150-0019). The plates were read within 30 min at 450 nm with a Thermo LabSystems Multiskan spectrophotometer. Titers were defined as the reciprocal of the dilutions that generated a spécifie cutoff value for OD 450 on the linear part of the titration curve.

Ex vivo T cell responses. On study day 34, spleens were mechanically dissociated to single-cell suspensions. Cells were resuspended in cell-stimulation media (RPMI with L-Glutamine and HEPES buffer, heat-inactivated fêtai bovine sérum, and Pen/Strep) and 2x10⁶ cells were aliquoted in a volume of 100 pL into 96-well plates. Splénocytes from each mouse were stimulated for approximately 18 hrs at 37°C with 100 pL of media alone, treated with positive control Cell Stimulation Cocktail (ThermoFisher, cat. # 00-4970-93) containing PMA and ionomycin, or 1 pg/mL of a peptide pool covering the SARS-CoV-2 spike protein (JPT, cat. # PM-WCPV-S-2). After Ihr of stimulation, Golgi Stop (BD Biosciences, cat. # 554724) was added to each well.

187

Flow cytometry. After the stimulation, cells were washed with PBS and transferred to 96-well deep-well plates. Cells were stained with LIVE/DEAD near IR viability dye (ThermoFisher, cat. # L10H9) diluted in PBS for 20 min at 4°C. Cells were washed with FBS staining buffer (BD Biosciences, cat. # 554656) and incubated with Fc Block (BD Biosciences, cat. 553142) for 10 min 5 at 4°C. Cells were then stained for 40 min at 4°C with a cell surface antibody cocktail consisting of CD3 BV605 (BD, cat. # 564009), CD4 BV421 (BD, cat. # 562891), and CD8 APC (BD, cat. # 553035). BD Brilliant Stain Buffer (cat. # 563794) was included in the staining buffer. Cells were washed with FBS staining buffer and then fixed for 20 min at room température with Fix/Perm buffer (ThermoFisher, cat. # 00-5523-00). Cells were washed with Ix Perm buffer, incubated with Fc Block, 10 and then stained for 40 min at 4°C with an intracellular cytokine antibody cocktail consisting of IFNg PE/Cy7 (BD, cat. # 557649), IL-2 PE (BioLegend, cat. # 503808) and TNF-a FITC (BD, cat. # 554418). Cells were then washed, resuspended in FBS stain buffer and acquired on a MACSQuant 16 flow cytometer (Miltenyi Biotec). Flow data was analyzed using FlowJo vl0.8.1 (BD Biosciences).

Table 18. Physicochemical properties of LNPs used in evaluating the immunogenicity of SARSCoV-2 spike protein mRNA vaccine construct

LNP Formulation	Particle Size (nm)	Zêta Potential (mV) pH 5	Zêta Potential (mV) pH 7	Encapsulation Efficiency (%)
7.5% DSPS_UO1_PEG-DMG 1.5%	80.3	5.3	-2.6	90.7 + 7.1
7.5% DSPSUO1PEG-DMG 2.5%	71.0	5.5	-1.0	89.1+9.3
7.5% DSPS_UO1_PEG-DMG 3.5%	61.9	6.6	-3.6	89.3 + 10.0
7.5% DSPS_UO1_PEG-DPPE 1.5%	80.7	7.9	-5.1	90.7 + 14.2
7.5% DSPS_UO1_PEG-DPPE 2.5%	68.4	2.7	-2.4	91.0 + 6.9
7.5% DSPS_UO1_PEG-DSG 1.5%	100.7	9.6	-6.7	88.8 + 5.1
7.5% DPPSUO1 PEG-DMG 1.5%	89.8	14.4	-1.4	93.8 + 9.2
UO1_PEG-DMG 1.5%	116.0	20.2	2.5	86.5 + 9.3
7.5% DSPS_KC2OA_PEG-DMG 1.5%	80.0	11.7	-3.5	90.9 + 5.5
7.5% DSPS_KC2OA_PEG-DSG 1.5%	88.5	9.0	-3.9	92.2 + 9.7
ALC0315_1.5% PEG-DMG	79.5	2.0	-2.4	86.7 + 9.3
SM 102_L5% PEG-DMG	92.6	11.8	-0.1	92.0 + 7.1

To evaluate the impact of PEG-lipid concentration on vaccine immunogenicity, BALB/c mice were immunized with mRNA-LNPs containing increasing amounts of PEG-lipid (PEG-DMG or PEG188

DPPE). Blood sérum was collected on day 21 post prime and on day 13 post boost (day 34 of study) for antibody analysis. Splénocytes were stimulated with Spike peptide pools and the percent of CD4 T cells producing IL-2 was quantified using flow cytometry (FIG. 13B and FIG. 13C). Both forms of PEG, PEG-DMG (FIG. 13B) or PEG-DPPE (FIG. 13C), inversely impacted mRNA-LNPA vaccine immunogenicity, with lower concentrations of PEG-lipid showing higher levels of both Bcell and T-cell responses.

To evaluate the impact of PEG-lipid acyl chain composition on mRNA-LNP immunogenicity, BALB/c mice were immunized with mRNA-LNPs containing different PEG formats (PEG-DMG or PEG-DSG). Blood sérum was collected on day 21 post prime and on day 13 post boost (day 34 of study). Splénocytes were stimulated with Spike peptide pools and the percent of CD4 T cells producing IL-2 was quantified using flow cytometry. Antibody titer data were log-transformed prior to statistical analysis. Groups were compared using an unpaired t test. For LNPs made with the ionizable lipid KC2OA, either PEG format performed similarly (FIG. 13D). In contrast, LNPs using the ionizable lipid UOl and PEG-DMG induced a superior antibody response than LNPs containing PEG-DSG (FIG. 13E).

To assess how the incorporation of phosphatidylserine influences mRNA-LNP immunogenicity, BALB/c mice were immunized with mRNA-LNPs containing various ionizable lipids with or without DSPS. On Day 34 ( 13 days post-boost), sérum was collected for quantification of total IgG anti-spike antibodies by ELISA. Splénocytes were stimulated with peptide and the percent of CD4 T cells producing IL-2 was quantified using flow cytometry.

The inclusion of DSPS with comparator ionizable lipid ALC-0315 negatively impacted total IgG anti-spike antibody levels (FIG. 13F). DSPS had the opposite effect on LNPs containing the ionizable lipids UOl and K.C2OA and significantly increased géométrie mean antibody levels 36- and 46-fold, respectively. DSPS also had an effect on CD4 T cell responses, with responses trending higher for LNPs containing UOl and K.C2OA, and significantly higher for SM-102. Taken together, inclusion of DSPS in lipid nanoparticles can substantially influence the immunogenicity of mRNA vaccines, and that the impact of DSPS is influenced by the type of ionizable cationic lipid.

The effects of different phosphatidylserine acyl chain composition on sérum antibody titers (FIG. 13G, panel A) and the magnitude of the spike-specific CD4 T cell response in the spleen (FIG. 13G, panel B) was evaluated. Mice were immunized with LNPs using the ionizable lipid UOl and either the DSPS or DPPS forms of phosphatidyl serine. On Day 34 (13 days post-boost), sérum was

189 collected for quantification of total IgG anti-spike antibodies by ELISA. Splénocytes were stimulated with peptide and the percent of CD4 T cells producing IL-2 was quantified using flow cytometry.

With regards to the impact of the PS acyl chain composition, both forms of PS comparably increased antibody levels over the base formulation lacking PS (FIG. 13G, Panel A). Both forms of 5 PS also had a positive effect on the CD4 T cell response (FIG. 13G, Panel B), although only the formulation containing DPPS was significantly higher than the based formulation without PS. Taken together, both forms of PS included in our LNPs, DPPS or DSPS, had a similar positive effect on increasing the immunogenicity of mRNA-LNP vaccines.

Example 23. Measuring the effect of adding either DSPS D-isomer or L-isomer at 7.5 mol% in KC2-01 based LNPs by particle characteristics and activity in MutuDC1940 dendritic cell line.

The aim of this study was to compare the impact of including the D and the L-isomers of DSPS into mRNA LNP formulations at 7.5 mol%. LNPs were prepared as described in Example 9 at 25 °C and analyzed as in Example 10. The LNPs contained K.C2-01 as the ICL at an N/P ratio of 5 with 2.5 mol% DSPC, 7.5 mol % DSPS (D or L) and a constant l .5 mol % PEG-DMG. Transfection efficiency was evaluated in murine dendritic cells as described in Example l L

Table 19. Physicochemical properties of KC2-01 LNPs with mCherry mRNA and distearoylphosphatidyl-L-serine or distearoylphosphatidyl-D-serine.

LNP Formulation	DSPS (serine isomer)	Particle Size (nm)	PDI	Zêta Potential mV, pH 5	Zêta Potential mV, pH 7	Encapsulation Efficiency (%)
KC2-01/DSPC/Chol/PEG- DMG (50/10/38.5/1.5)	N/A	83.92	0.03	7.7	-0.47	89.2 ±5.7
KC2-01/DSPC/DSPS/ Chol/PEG-DMG (50/2.5/7.5/38.5/1.5)	D	85.4	0.09	7.8	-4.8	88.7 ±3.5

190

KC2-01/DSPC/DSPS/ Chol/PEG-DMG (50/2.5/7.5/38.5/1.5)

L

87.4

0.04

7.7

-2.7

90.9 ±6.0

This study shows that including 7.5 mol% DSPS (either isomer) in a KC2-01 based LNP formulation had no effect on the particle size or mRNA encapsulation. The zêta potential values are similar at pH 5, but the DSPS containing formulation hâve more négative values at pH 7 than the nonDSPS containing formulation, likely a resuit of the additional négative charge added by the DSPS. The impact of the stereochemistry on transfection was evaluated in FIG. 14A and FIG. 14B. An 8fold increase in mCherry expression was observed when the D-isomer of DSPS was used compared to the L isomer at 1 pg/mL mRNA (and 4.7-fold at 0.33 pg/mL mRNA), indicating that the uptake or expression mechanism(s) of DSPS containing LNPs is likely stereospecific.

Example 24. Measuring the effect of inclusion of DSPS in KC2-based LNPs at 25 °C and 65 °C on particle characteristics and activity in MutuDC1940 dendritic cell line.

The aim of this study was to compare the impact of including the D-isomer of DSPS in K.C2 based LNPs produced at two different températures. LNPs were prepared as described in Example 9 where those that contained DSPS were made at 65 °C at those that did not include DSPS were made at 25 °C and ail LNPs were analyzed as in Example 10.

Ail LNPs contained ICL at an N/P ratio of 5 with a constant 1.5 mol % PEG-DMG. The cholestérol content was held constant at 38.5 mol % and the DSPC content varied inversely with the mol % of DSPS at 5 and 7.5 mol % (ail lipid concentrations were used as mol % of total lipid). These mRNA LNPs ail contained mCherry mRNA and the composition of comparator formulations using SM-102 based lipid formulation, the same lipid composition as that used in mRNA-1273 and that using ALC-0315, similar to that used in BNT162b2 were taken from their respective prescribing information. Note, the BNT162b2 comparator using ALC-0315 was made with an N/P of 5.0 in keeping with the other formulations in this study, which is different than the approved Covid vaccine Comimaty.

Table 20. Physicochemical properties of KC2 containing LNPs with 5 or 7.5 % DSPS to similar LNPs prepared with SM 102 or ALC-0315 ionizable cationic lipid.

191

LNP Formulation	Mixing Temp °C	Particle Size (nm)	PDI	Zêta Potential mV, pH 5	Zêta Potential mV, pH 7	Encapsulation Efficiency (%)
KC2	25	70.4	0.21	4.5	-1.1	87.3 + 3.2
KC2 with 5% DSPS	25	78.3	0.12	2.5	-2.2	87.0 + 2.2
KC2 with 5% DSPS	65	88.2	0.11	7.4	-4.2	87.0 + 3.8
KC2 with 7.5% DSPS	25	73.5	0.11	1.0	-2.3	86.9 + 3.8
KC2 with 7.5% DSPS	65	74.3	0.07	0.5	-8.2	87.8 + 2.2
SM102	25	84.0	0.17	6.3	-0.7	81.9 + 3.9
ALC-0315	25	72.1	0.08	3.3	-3.0	80.7 + 2.2

This study shows that heating the lipid solution and mRNA solution in a heating block set to 65 °C had no deleterious effect on particle diameter or, zêta potential or mRNA encapsulation readings at either 5 or 7.5 mol% DSPS content. The impact of température on transfection of DSPS-targeted KC2 LNPs, and the comparison to SMI02 and ALC-0315 containing LNPs is shown in FIG. 15. The 5 mol% DSPS formulation had a 2-fold higher mCherry expression than the exact same formulation made at 25 °C (p < 0.05, T-Test). A comparison of the formulations with 7.5 mol% DSPS made at either température showed no significant différence. The 5 mol% DSPS containing LNP, made at 65 °C gave a 6.5-fold increase in mCherry expression over the SM 102 formulation. It is likely that higher températures increase the solubility of DSPS in alcohol and allow better incorporation of the lipid in the particle, which allows for enhanced uptake in this in vitro study, while nor adversely affecting many of the LNP crucial particle characteristics such as size, encapsulation or mRNA expression.

Example 25. Comparing UO1, UO6 & 7 in LNPs with and without DSPS

The aim of this study was to evaluate AK.G-UO-6 (“UO-6”) and AKG-UO-7 (“UO-7”) in LNPs with and without 7.5 mol% DSPS. Previously, it was found that 7.5 mol% DSPS produced the most optimal LNPs from a UO-1 sériés. Here 7.5 mol% DSPS was chosen as an initial amount of DSPS to include to compare the structurally similar UO-6 and 7 LNPs were prepared as described in Example 9 and analyzed as in Example 10.

The LNPs contained either UO-1, UO-6 or UO-7 as the ICL at 50 mol%. The formulations also contained 38.5 mol% cholestérol, and 1.5 mol% PEG-DMG. The non DSPS containing samples hâve 10 mol% DSPC, and to the 7.5 mol% DSPS containing LNPs, DSPS was added at 7.5 mol%

192 aand DSPC was included at 2.5 mol%. The N/P in ail formulations was 5. Transfection efficiency was evaluated in murine dendritic cells as described in Example 11.

Table 21. Physicochemical properties and encapsulation efficiency of UO-1, UO-6, and UO-7 LNPs with and without DSPS-targeting.

LNP Formulation	Particle Size (nm)	PDI	Zêta Potential mV, pH 5	Zêta Potential mV, pH 7	Encapsulation Efficiency (%)
UO1	72.8	0.11	14.5	5.6	89.6 ±4.8
UO1_7.5%_DSPS	80.5	0.03	13.9	-2.4	91.4±4.2
UO6	72.0	0.22	14.9	4.2	90.5 ± 11.1
UOUO6_7.5%_DSPS	86.1	0.13	14.3	1.7	92.8 ± 11.1
UO7	79.3	0.16	18.2	-2.6	91.4 ± 2.5
UO7_7.5%_DSPS	94.3	0.13	16.9	-5.3	92.3 ±5.0

This study shows that including DSPS in LNPs based on UO1, UO6 and UO7 caused an increase in expression of mCherry, without adverse changes in particle size or mRNA entrapment. The impact of 7.5 % DSPS incorporation into LNPs on dendritic cell transfection activity was shown for formulations prepared with three different ICLs (UO-1, UO-6, and UO-7) (FIG. 16). For UO-1 with 7.5mol% DSPS, the mCherry expression was 11-fold higher than UO6 with 7.5mol% DSPS and 7-fold higher than UO7 with 7.5mol% DSPS. UO-7 with DSPS had 1.5-fold more mCherry expression than UO6 with DSPS. Of the non DSPS containing formulations, UO7 was 2.2-fold more active than UO1 and 1.9-fold more active than UO6. However, LNPs prepared with ail three ICLs maintained good encapsulation efficiencies, particle size, and zêta potential, while benefiting from targeting with DSPS inclusion in the formulation.

Example 26. Evaluating the effect of adding DSPS to UO1, SM102 and ALC-0315 based LNPs by comparing particle characteristics and activity in MutuDC1940 dendritic cell line.

The aim of this study was to evaluate the effect of including the L-isomers of DSPC into mRNA LNP formulations at 0, 5% and 7.5 mol%. LNPs were prepared as described in Example 9 at 25 °C and analyzed as in Example 10.

The LNPs contained either AKG-UO-1 (“UO-1”), SM 102 as the ICL at 50 mol%. For UO1 and SM 102 the formulations contained 38.5 mol% cholestérol, and 1.5 mol% PEG-DMG. The non DSPS containing samples hâve 10 mol% DSPC, and to the DSPS containing LNPs, DSPS was added

193 at 5 or 7.5 mol% with a concomitant réduction in DSPC by the same mol%. For the ALC-0315 formulation, the ICL was 46.3 mol%, DSPC 9.4 mol%, cholestérol 42.7 mol% and PEG-DMG 1.5 mol%. DSPS was added with concomitant réduction in the DSPC mol% as above. The N/P in ail formulations was 5. Transfection efficiency was evaluated in murine dendritic cells as described in

Example 11.

Table 22. Physicochemical properties and encapsulation efficiency of PS-targeted formulations of UOl, SMI02, and ALC-0315 containing LNPs

LNP Formulation	Particle Size (nm)	PDI	Zêta Potential mV, pH 5	Zêta Potential mV, pH 7	Encapsulation Efficiency (%)
UOl	72.8	0.11	10.2	3.6	89.6 ±4.8
UO1_5%_DSPS	83.0	0.11	9.9	-2.7	91.4±4.2
UO1_7.5%_DSPS	80.5	0.03	11.0	1.4	91.1 ±6.3
SM 102	80.7	0.04	13.0	1.6	90.1 ±5.6
SM102_5%_DSPS	81.0	0.02	13.1	-6.1	90.5 ± 1.4
SM102_7.5%_DSPS	80.1	0.05	13.0	-4.0	90.1 ±2.7
ALC0315	74.6	0.06	6.2	-0.9	72.3 ±0.9
ALCO315_5%_DSPS	68.1	0.10	8.0	-8.8	84.2 ± 1.3
ALC0315_7.5%_DSPS	75.0	0.02	7.9	-11.8	84.9 ±3.8

This study shows that including DSPS in LNPs based on UO l, SM 102 and ALC-0315 caused 10 an increase in expression of mCherry (FIG. 17), without adverse changes in particle size or mRNA entrapment. For UO-1 and SM 102, 5 mol% DSPS looked maximal, while 7.5 mol% DSPS was maximal for the ALC-0315 formulation. For UO-1 and SM 102 the increase in expression was 18.1 and 58.7-fold respectively for the 5mol% DSPS samples. The increase was 80.9-fold for the ALC0315 based sample for 7.5 mol% DSPS.

Example 27. Additional exemplary phosphatidyl-L-serine targeted LNP formulations.

Additional formulations shown below could also be prepared using the methods described in the Examples above. Ail lipid concentrations are shown as mol % as a percentage of the total lipid in the LNP. The below compositions vary in conjugated lipid content from 0.5 to 2.5 mol %, in sterol 20 content from 25-45 mol %, in ICL content from 40-65 mol %, in saturated phosphatidyl-L-serine content from 2-10 mol %, and in total noncationic phospholipid content from 5-20 mol %. Most compositions would contain two phospholipids, typically phosphatidyl-L-serine (DSPS or DPPS 194 being preferred) and phosphatidylcholine, although some exemplary formulations may contain more than two phospholipids, including phosphatidylethanolamines, like dioleoylphosphatidylethanolamine (DOPE).

Table 23. Exemplary phosphatidyl-L-serine containing LNP formulations

Formulation	Ionizable Cationic	PS (mol %)	Additional	Sterol	Conjugated
(#)	Lipid (mol %)		Phospholipid(s)	(mol %)	lipid
			(mol %)		(mol %)
1	AKG-UO-1 (47.5)	DSPS (7.5)	DSPC (3.5)	Chol(40)	PEG-DMG (1.5)
2	AKG-UO-1 (45)	DSPS (8)	DSPC (4)	Chol (42)	PEG-DMG (1)
3	AKG-KC2-01 (40)	DSPS (5)	DSPC (5) DOPE (5)	Chol (44)	PEG-DMG (1)
4	AKG-KC2-01 (42.5)	DSPS (7.5)	DSPC (6.5)	Chol (42)	PEG-DMG (1.5)
5	AKG-KC2-01 (65)	DSPS (7.5)	DSPC (2.5)	Chol (24.5)	PEG-DMG (0.5)
6	AKG-KC2-01 (60)	DSPS (6)	DSPC (4)	Chol(29)	PEG-DMG (1)
7	AKG-KC2-01 (55)	DSPS (7)	DSPC (3)	Chol (34)	PEG-DMG (1)
8	AKG-KC2-01 (57)	DSPS (6.5)	DSPC (3.5)	Chol (28)	PEG-DMG (1)
9	AKG-KC2-01 (60)	DSPS (7.5)	DSPC (2.5) DOPE (4)	Chol (28.5)	PEG-DMG (1.5)
10	AKG-KC2-01 (48)	DSPS (6)	DSPC (4)	Chol (41.5)	PEG-DMG (0.5)
11	AKG-KC2-01 (48)	DPPS (6)	DSPC (4)	Chol (41.5)	PEG-DMG (0.5)
12	AKG-KC2-01 (48)	DSPS (6)	DPPC (4)	Chol (41.5)	PEG-DMG (0.5)
13	AKG-KC2-PA (47.5)	DSPS (6)	DSPC (4)	Chol (41.5)	PEG-DMG (1)
14	AKG-KC2-PA(47.5)	DPPS (6)	DSPC (4)	Chol (41.5)	PEG-DMG (1)
15	AKG-KC2-PA(47.5)	DSPS (6)	DPPC (4)	Chol (41.5)	PEG-DMG (1)
16	AKG-UO-04 (48)	DSPS (7.5)	DSPC (2.5)	Chol (41.5)	PEG-DMG (0.5)
17	AKG-UO-04 (48)	DPPS (7.5)	DSPC (2.5)	Chol (41.5)	PEG-DMG (0.5)
18	AKG-UO-04 (48)	DSPS (7.5)	DPPC (2.5)	Chol (41.5)	PEG-DMG (0.5)
19	AKG-UO-1 (47.5)	DSPS (7.5)	DSPC (4.5)	Chol (39)	PEG-DMG (1.5)
20	AKG-UO-1 (47.5)	DPPS (7.5)	DSPC (4.5)	Chol (39)	PEG-DMG (1.5)
21	AKG-UO-1 (47.5)	DSPS (7.5)	DSPC (2.5)	β-sitosterol (42)	PEG-DMG (0.5)
22	AKG-UO-1 (50)	DSPS (7.5)	DSPC (2.5)	β-sitosterol (39)	PEG-DMG (1)
23	AKG-UO-1 (52.5)	DSPS (7.5)	DSPC (2.5)	β-sitosterol (39)	PEG-DMG (1)
24	AKG-UO-1 (55)	DSPS (7.5)	DSPC (2.5)	β-sitosterol (39)	PEG-DMG (1)
25	AKG-UO-1 (55)	DSPS (7.5)	DSPC (2.5)	Chol (34)	PEG-DMG (1)
26	AKG-UO-1 (57)	DSPS (6.5)	DSPC (3.5) DOPE (4)	Chol (28)	PEG-DMG (1)
27	AKG-UO-1 (52.5)	DSPS (7.5)	DSPC (6.5) DOPE (4)	Chol (28)	PEG-DMG (1.5)
28	AKG-UO-la (48)	DSPS (8)	DSPC (2)	Chol (41.5)	PEG-DMG (0.5)
29	AKG-UO-7 (48)	DSPS (7)	DSPC (3)	Chol (41)	PEG-DMG (1)
30	AKG-KC3-01 (45)	DSPS (6)	DSPC (4)	Chol (44)	PEG-DMG (1)
31	AKG-KC2-01 (42.5)	DSPS (7.5)	DSPC (6.5)	Chol (42)	PEG-DMG (1.5)
32	AKG-UO-2 (48)	DPPS (8)	DSPC (2)	Chol (41.5)	PEG-DMG (0.5)
33	Compound 29 (48)	DSPS (5)	DSPC (5)	Chol (41.5)	PEG-DMG (0.5)
34	Compound 33 (48)	DSPS (7.5)	DSPC (2.5)	Chol (41)	PEG-DMG (1)

195

Table 23. Exemplary phosphatidyl-L-serine containing LNP formulations (continued)

Formulation (#)	Ionizable Cationic Lipid (mol %)	PS (mol %)	Additional Phospholipid(s) (mol %)	Sterol (mol %)	Conjugated lipid (mol %)
35	Compound 29 (50)	DSPS (5)	DSPC (5)	Chol (39)	PEG-DMG (1)
36	Compound 33 (50)	DSPS (7.5)	DSPC (2.5)	Chol (39)	PEG-DMG (1)
37	KC3-OA (48)	DSPS (5)	DSPC (5)	Chol (41.5)	PEG-DMG (0.5)
38	ALC-0315 (46)	DSPS (5)	DSPC (5)	Chol (43)	PEG-DMG (1)
39	ALC-0315 (48)	DSPS (5)	DSPC (5)	Chol (41)	PEG-DMG (1)
40	SM-102 (50)	DSPS (5)	DSPC (5)	Chol (39.5)	PEG-DMG (0.5)
41	SM-102 (50)	DSPS (7.5)	DSPC (2.5)	Chol (39)	PEG-DMG (1)
42	SM-102 (48)	DSPS (5)	DSPC (5)	Chol (41)	PEG-DMG (1)
43	AKG-UO-1 (48)	DSPS (7.5)	-	Chol (43.5)	PEG-DMG (1)
44	AKG-UO-1 (48)	DSPS (7.5)	-	Chol (43.5)	PEG-DMG (1)
45	AKG-KC3-01 (48)	DSPS (5)	DSPC (5) DOPE (15)	Chol (26)	PEG-DMG (1)
46	AKG-KC3-01 (48)	DSPS (5)	DSPC (5) DOPE (10)	Chol (31)	PEG-DMG (1)
47	AKG-KC3-01 (48)	DSPS(6)	DSPC (4)	Chol (40)	PEG-DMG (2)
48	AKG-KC3-01 (48)	DSPS (6)	DSPC (4)	Chol (39.5)	PEG-DMG (2.5)
49	AKG-UO-1 (46)	DSPS (7)	DSPE (5)	Chol (41.5)	PEG-DMG (0.5)
50	AKG-UO-1 (48)	DSPS (7.5)	eggSM (2.5)	Chol (41.5)	PEG-DMG (0.5)
51	AKG-KC3-01 (48)	DSPS (5)	HSPC (5)	Chol (41.5)	PEG-DMG (0.5)
52	AKG-UO-1 (47.5)	DSPS (7.5)	DSPC (2.5)	Chol (42)	PEG-DPG (0.5)
53	AKG-UO-1 (47)	DSPS (7.5)	DSPC (2.5)	Chol (42)	PEG-DPG (1)

Various aspects of the présent disclosure may be used alone, in combination, or in a variety of 5 arrangements not specifically discussed in the embodiments described in the foregoing and is therefore not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

While spécifie embodiments of the subject disclosure hâve been discussed, the above 10 spécification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this spécification. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of équivalents, and the spécification, along with such variations.

Ail publications, patents and patent applications referenced in this spécification are 15 incorporated herein by reference in their entirety for ail purposes to the same extent as if each individual publication, patent or patent application were specifically indicated to be so incorporated by reference.

Claims (32)

1. l. A lipid nanoparticle (LNP) composition comprising an ionizable lipid having a Chemical structure consisting of a pair of linear polyunsaturated lipid tails covalently bound to a head group, the head group comprising a dialkyl amino group ;

   the head group comprising a heterocyclyl or alkyl portion covalently bound to the dialkyl amino group and optionally further comprising a phosphate group;

   each polyunsaturated lipid tail being unsaturated except for at least two olefins separated by at least two methylene groups along the length of the lipid tail, and optionally comprising a single acyl group at the end of the lipid tail covalently bound to the head group.
2. 2. The composition of claim l, wherein each lipid tail is identical, and each lipid tail has a total of two olefins separated only by an unsubstituted ethylene, n-propyl, or n-butyl, optionally wherein each lipid tail further comprises an acyl group joined to an oxygen of the headgroup to form an ester, and has a total of 16 or 18 carbon atoms including the acyl group.
3. 3. The composition of claim 2, wherein

   a. the dialkyl amino portion of the head group has a Chemical structure of Formula (IV-A)

   Rio

   AT''Formula IV-A, wherein n is 2, 3 or 4 in Formula (IV-A); and

   Rio and R12 in Formula (IV-A) are each independently selected from an alkyl group selected from the group consisting of: methyl, ethyl, and propyl, wherein the alkyl in Rio and R12 is optionally substituted with one or more hydroxyl; and

   197

   b. the ionizable lipid further comprises the Chemical structure O comprising the acyl group of each lipid tail covalently bound to the portion of the head group distal to the dialkyl amino portion of Formula (IV-A), wherein indicates attachment to Formula IV-A within the head group, and R²² indicates a portion of each lipid tail covalently bound to the acyl group and having the Chemical structure of Formula A:

   wherein S in Formula A indicates attachment of Formula A to R²² within each lipid tail; and a is 4, l, 2, or 3;

   b is 4, 2, or 3; and c is 4, 3, 5, 6, or 7, provided that the sum of a, b and c is in Formula A is 12, 10, 11, or 13, optionally wherein Rio and R12 in Formula (IV-A) are each independently methyl, ethyl, -(CH₂)(CH₂)OH, or -(CH2)₂(CH₂)OH, and optionally wherein b is 4 and wherein Rio and R12 in Formula (IV-A) are each methyl.
4. 4. The composition of claim l, wherein the ionizable lipid has a Chemical structure of Formula (ΙΑ)

   198

   Formula I-A wherein a is 4, l, 2, 3, 5 or 6;

   5 b is 4, 3 or 2;

   c is 4, 3, 5, 6, or 7; and the sum of a, b and c is 12 or 10;

   q is l, 2, 3 or 4; and each of Rio and R12 is independently (Ci-C4)alkyl optionally substituted with one or more

   IO hydroxyl; and

   wherein v is 0 or l ; and q2 is 2 or 1, optionally wherein v is 0 and q is 1, 2 or 3.
5. 5. The composition of claim 4, wherein the ionizable lipid is selected from the group consisting of AKG-UO-l, AKG-UO-l A, AKG-UO-lB, AKG-UO-2, AKG-UO-4, AKG-U0-4A, AKG-UO-5, AKG-UO-6, AKG-UO-7, AKG-UO-8, AKG-UO-9, and AKG-UO-10:

   199

   AKG-UO-4;

   200

   AKG-UO-4A;
6. 6. A composition comprising:

   201

   a. a nucleic acid, optionally wherein the nucleic acid is mRNA;

   b. the ionizable lipid of any one of claims 1-5;

   c. a sterol, optionally wherein the sterol is cholestérol;

   d. one or more phospholipids comprising a phosphatidylserine (PS) lipid, optionally wherein the one or more phospholipids consist of (i) one or more phospholipids selected from the group consisting of: DSPC, HSPC, DPPC, egg SM, and DOPC, and (ii) a PS lipid selected from the group consisting of: DPPS, DSPS and DOPS; and

   e. optionally further comprising a conjugated lipid.
7. 7. The composition of claim 6, wherein the one or more phospholipids consist of:

   a. DSPC; and

   b. one or more PS lipids selected from the group consisting of (L-Serine) DPPS and (LSerine) DSPS, wherein optionally the composition comprises the PS lipid in a total amount of 2.5-10 mol% of the total lipid in the composition, optionally wherein the conjugated lipid comprises PEG.
8. 8. A nucleic acid lipid nanoparticle (LNP) composition comprising:

   a. a nucleic acid;

   b. an ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition;

   c. a sterol in a total amount of 25-45 mol% of the total lipid content of the LNP composition;

   d. one or more phospholipids in a total amount of phospholipids of 5-25 mol% of the total lipid content of the LNP composition, and comprising a phosphatidylserine (PS) in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition; and

   e. optionally further comprising a conjugated lipid in a total amount of 0.5 - 2.5 mol% of the total lipid content of the LNP composition.
9. 9. The composition of claim 8, wherein the nucleic acid is mRNA, optionally wherein the sterol is cholestérol, optionally herein the one or more phospholipid consists of: DSPC and a L-serine PS.

   202

   ΙΟ. The composition of claim 9, wherein the LNP composition comprises the PS in a total amount of 5.0-7.5 mol% of the total lipid in the composition, optionally wherein the conjugated lipid comprises PEG, optionally wherein the conjugated lipid is PEG-DMG, optionally wherein the LNP composition comprises the conjugated lipid in a total amount of 0.5-L5 mol% of the total lipid content of the LNP composition or wherein the LNP composition comprises the conjugated lipid in a total amount of less than l mol% of the total lipid content of the LNP composition.

   I L The composition of claim 8, wherein:

   a. the nucleic acid is a mRNA,

   b. the ionizable cationic lipid in a total amount of 45-55 mol% of the total lipid content of the LNP composition;

   c. a sterol is cholestérol in a total amount of 35-45 mol% of the total lipid content of the LNP composition;

   d. the total amount of phospholipid of 7-15 mol% of the total lipid content of the LNP composition;

   e. the one or more phospholipids consist of DSPC and the PS lipid is one or more lipids selected from the group consisting of the L-serine configuration of DPPS and DSPS; and

   f. the total amount of the PS lipid is 3-9 mol% of the total lipid content of the LNP composition.
10. 12. The composition of claim 11, wherein the composition comprises the PS lipid in a total amount selected from 1.25 mol%, 2.5 mol%, 5 mol%, 7.5 mol%, and 10 mol% of the total lipid content of the LNP composition.
11. 13. A nucleic acid lipid nanoparticle (LNP) composition comprising:

    a. a nucleic acid, wherein the nucleic acid is mRNA;

    b. an ionizable cationic lipid, the ionizable cationic lipid in a total amount of 45-55 mol% of the total lipid content of the LNP composition;

    c. a sterol, wherein the sterol is cholestérol in a total amount of 35-45 mol% of the total lipid content of the LNP composition;

    203

    d. one or more phospholipids, wherein the one or more phospholipids in a total amount of phospholipids of 10 mol% of the total lipid content of the LNP composition, and comprising an anionic phospholipid targeting moiety in a total amount of 3-9 mol% of the total lipid content of the LNP composition; and

    e. a conjugated lipid, the conjugated lipid in a total amount of 0.5 - l .5 mol% of the total lipid content of the LNP composition, optionally (i) wherein the anionic phospholipid targeting moiety is selected from the group consisting of: DSPS (L-isomer), DPPS (L-isomer), DMPS (L-isomer), DOPS (L-isomer), DSPS (Disomer), DSPG, DPPG, N-Glu-DSPE, and N-Suc-DSPE, optionally wherein the conjugated lipid is PEG-DMG; and the PS lipid is selected from the group consisting of: DSPS (L-isomer) and DPPS, (i i) wherein the ionizable cationic lipid is one or more compounds selected from the group consisting of: Compounds l-28 (Table l), Compounds 29-38 (Table 2), AKG-UO-l, AKG-UO-l A, AKG-UO-l B, AKG-UO-lB, AKG-UO-2, AKG-UO-3, AKG-UO-4, AKG-UO-4A, AKG-UO-5, AKG-BDG-01, AKG-BDG-02, AKG-UO-6, AKG-UO-7, AKG-UO-8, AKG-UO-9, and AKG-UOio, (i ii) wherein the ionizable cationic lipid is one or more compounds selected from the group consisting of Compounds 1-3, 5-8, 9-12, 14-28, or (i v) wherein the ionizable cationic lipid is one or more compounds selected from the group consisting of Compounds 29-38.
12. 14. The composition of any one of daims 8-13, wherein the ionizable cationic lipid is one or more compounds selected from the group consisting of: KC2-OA, KC3-OA, Dlin-KC2-DMA , DlinKC3-DMA, KC2-PA, DODAP, AKG-OA-DM2, AKG-OA-DM3, 0-11769, Dlin-MC3DMA, ALC-0315 and SM-102.
13. 15. The nucleic acid lipid nanoparticle (LNP) composition of claim 8, wherein the LNP composition comprises:

    a. a nucleic acid;

    b. an ionizable cationic lipid in a total amount of 50 mol% of the total lipid content of the LNP composition;

    c. cholestérol in a total amount of 38.5 mol% of the total lipid content of the LNP composition;

    204

    d. one or more phospholipids in a total amount of 7-15 mol% of the total lipid content of the LNP composition, and comprising a phosphatidylserine (PS) lipid in a total amount of 3-9 mol% of the total lipid content of the LNP composition, optionally wherein the phospholipids consist of one or more phospholipids selected from the group consisting of: DSPC, HSPC, egg SM, DPPC and DOPC, optionally wherein the PS lipid is one or more L-serine lipids selected from the group consisting of DPPS and DSPS; and

    e. a PEG-containing lipid in a total amount of 0.5 -l .5 mol% of the total lipid content of the LNP composition; or wherein the LNP composition comprises one or more phospholipids comprising at least two (L-Serine) PS lipids having mismatched acyl chain lengths, optionally wherein the PS lipids are DPPC and DSPS, and optionally wherein the DPPC and DSPS are each présent in the LNP at a total amount of 5 mol% each, based on the total lipid content of the LNP composition.
14. 16. A nucleic acid lipid nanoparticle (LNP) composition comprising:

    a. a nucleic acid, the ionizable cationic lipid AKG-UO-l, and a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition, optionally wherein the nucleic acid is mRNA, the PS lipid is (L-Serine) DSPS, (L-Serine) DPPS, or a mixture thereof, and the LNP composition further comprises cholestérol and a second phospholipid selected from the group consisting of: DSPC, DPPC and DOPC, and optionally wherein the LNP composition further comprises 0.5-L5 mol% PEG-DMG or PEG-DSG, based on the total lipid content in the LNP composition; or

    b. a nucleic acid, an ionizable cationic lipid selected from K.C2OA, KC2, KC2-01, ALC0315, and SMI02; and a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition, optionally wherein the LNP composition has a N/P ratio of 3 to 8, optionally wherein the LNP composition has a N/P ratio of 5 to 7, optionally wherein the LNP composition has a N/P ratio of 6, optionally wherein the ionizable cationic lipid is KC2OA, KC2, KC2-01, ALC0315, or SMI02.
15. 17. A nucleic acid lipid nanoparticle (LNP) composition comprising: a nucleic acid, an ionizable cationic lipid selected from AKG-UO-6 and AKG-UO-7; and a (L-Serine) PS lipid in a total amount of 2.5-I0 mol% of the total lipid content of the LNP composition, optionally wherein the LNP composition has a N/P ratio of 3 to 8, optionally the LNP composition has a N/P ratio of 5

    205 to 7, optionally wherein the LNP composition has a N/P ratio of 5, optionally wherein the LNP composition has a N/P ratio of 7.
16. 18. The composition of any one of daims 8-13 and 15-17, wherein the nucleic acid is a mRNA encoding the SARS-CoV-2 spike protein.
17. 19. A nucleic acid lipid nanoparticle (LNP) vaccine composition, wherein the LNP vaccine composition comprises:

    a. a mRNA nucleic acid with a N/P ratio of 3 to 8;

    b. an ALC-0315 ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition;

    c. cholestérol in a total amount of 25-40 mol% of the total lipid content of the LNP composition;

    d. a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition;

    e. DSPC phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition; and

    f. PEG-DMG in a total amount of 0-2.5 mol% of the total lipid content of the LNP composition; or wherein the LNP vaccine composition comprises:

    a. a mRNA nucleic acid with a N/P ratio of 3 to 8;

    b. a Dlin- KC3-01 ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition;

    c. cholestérol in a total amount of 25-40 mol% of the total lipid content of the LNP composition;

    d. a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition;

    e. DSPC phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition; and

    f. PEG-DMG in a total amount of 0-2.5 mol% of the total lipid content of the LNP composition.

    206
18. 20. A nucleic acid lipid nanoparticle (LNP) vaccine composition comprising:

    a. a mRNA nucleic acid with a N/P ratio of 3 to 8;

    b. a KC3-OA ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition;

    c. cholestérol in a total amount of 25-40 mol% of the total lipid content of the LNP composition;

    d. a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition;

    e. DSPC phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition; and

    f. PEG-DMG in a total amount of 0-2.5 mol% of the total lipid content of the LNP composition.
19. 21. A nucleic acid lipid nanoparticle (LNP) vaccine composition comprising:

    a. a mRNA nucleic acid with a N/P ratio of 3 to 8;

    b. an ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition;

    c. cholestérol in a total amount of 25-40 mol% of the total lipid content of the LNP composition;

    d. a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition;

    e. DSPC phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition; and

    f. PEG-DMG in a total amount of 0-2.5 mol% of the total lipid content of the LNP composition, optionally wherein the nucleic acid is a mRNA of SEQ ID NO:2.
20. 22. Use of a (L-Serine) PS lipid in a LNP in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition for targeting of the LNP to dendritic cells.
21. 23. The use of claim 22, wherein the LNP comprises a mRNA, optionally wherein the LNP further comprises cholestérol, optionally wherein the LNP further comprises an ionizable cationic lipid

    207 (ICL), optionally wherein the LNP further comprises one or more additional phospholipids including DSPC, optionally wherein the LNP further comprises a conjugated lipid.
22. 24. The use of claim 23, wherein the LNP comprises:

    a. a mRNA nucleic acid with a N/P ratio of 3 to 8;

    b. an ionizable cationic lipid (ICL) in a total amount of 40-65 mol% of the total lipid content of the LNP composition;

    c. cholestérol in a total amount of 25-40 mol% of the total lipid content of the LNP composition;

    d. a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition;

    e. DSPC phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition; and

    f. a conjugated lipid in a total amount of 0-2.5 mol% of the total lipid content of the LNP composition, optionally wherein the ICL is selected from the compounds of any one of daims 1-5.
23. 25. A nucleic acid lipid nanoparticle (LNP) vaccine composition, wherein the LNP vaccine composition comprises:

    a. a mRNA nucleic acid;

    b. an ALC-0315 ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition;

    c. cholestérol in a total amount of 25-40 mol% of the total lipid content of the LNP composition;

    d. a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition;

    e. DSPC phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition; and

    f. PEG-DMG in a total amount of 0-2.5 mol% of the total lipid content of the LNP composition.

    208
24. 26. A nucleic acid lipid nanoparticle (LNP) composition comprising a Dlin-KC2-DMA ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition, in combination with a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition.
25. 27. A nucleic acid lipid nanoparticle (LNP) composition comprising a KC3-O1 ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition.
26. 28. A nucleic acid lipid nanoparticle (LNP) composition comprising a KC3-OA ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition.
27. 29. The composition of claim 6, wherein the one or more phospholipids consist of:

    a. HSPC;and

    b. one or more PS lipids selected from the group consisting of (L-Serine) DPPS and (LSerine) DSPS, wherein optionally the composition comprises the PS lipid in a total amount of 2.5-10 mol% of the total lipid in the composition, optionally wherein the conjugated lipid comprises PEG.
28. 30. A nucleic acid lipid nanoparticle (LNP) vaccine composition, wherein the LNP vaccine composition comprises:

    a. a mRNA nucleic acid with a N/P ratio of 3 to 8;

    b. an ALC-0315 ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition;

    c. cholestérol in a total amount of 25-40 mol% of the total lipid content of the LNP composition;

    d. a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition;

    e. DSPC phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition; and

    f. PEG-DMG in a total amount of 0-2.5 mol% of the total lipid content of the LNP composition; or

    209 wherein the LNP vaccine composition comprises:

    g. a mRNA nucleic acid with a N/P ratio of 3 to 8;

    h. a Dlin-KC2-DMA ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP composition;

    i. cholestérol in a total amount of 25-40 mol% of the total lipid content of the LNP composition;

    j. a (L-Serine) PS lipid in a total amount of 2.5-10 mol% of the total lipid content of the LNP composition;

    k. DSPC phospholipid in a total amount of 5-25 mol% of the total lipid content of the LNP composition; and

    1. PEG-DMG in a total amount of 0-2.5 mol% of the total lipid content of the LNP composition.
29. 31. A nucleic acid lipid nanoparticle (LNP) vaccine composition comprising an ionizable cationic lipid selected from the group consisting of: KC3-OA, Dlin-KC3-DMA, and KC3-O1, wherein the vaccine composition comprises the ionizable cationic lipid in a total amount of 40-65 mol% of the total lipid content of the LNP vaccine composition.
30. 32. The composition of claim 31, wherein the ionizable cationic lipid is KC3-OA.
31. 33. The composition of claim 31, wherein the ionizable cationic lipid is Dlin-KC3-DMA.
32. 34. The composition of claim 31, wherein the ionizable cationic lipid is KC3-O1.