Lipid nanoparticles comprising sugar-based stabilizing agents for nucleic acid delivery and method of preparation thereof

Abstract

The disclosure provides sugar-based stabilizing agents, nanoparticle compositions, and uses thereof. In one aspect, a nanoparticle composition includes: (a) an ionizable lipid; (b) one or more lipids; and (c) a sugar-based stabilizing agent. The disclosure provides methods for preparing the nanoparticle compositions and uses of the nanoparticle or the pharmaceutical composition for preventing, treating, or ameliorating conditions or diseases.

Description

Docket no: 2024-24253-P-WOSTABILIZING AGENTS, COMPOSITIONS, AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U. S. Provisional Patent Application No.63 / 748,228, filed on January 22, 2025, the content of which is incorporated by reference in its entirety herein.BACKGROUND

[0002] Lipid nanoparticle (LNP) formulations and nucleic acid containing lipid nanoparticle (NALNP) formulations are used for a variety of applications, particularly medical applications such as oligonucleotide-based therapeutics, e.g., vaccines, immunogenic cell incorporation, gene editing, and gene therapy. LNP and NALNP formulations can also be used for antibiotics and vitamins, among other uses.

[0003] However, there is a need for improved LNP and NALNP formulations. The present invention provides for ameliorating at least some of the disadvantages of the prior art. These and other advantages of the present invention will be apparent from the description as set forth below.BRIEF SUMMARY

[0004] In one aspect, the disclosure provides a stabilizing agent including a structure of formula (I):(I) wherein Y is a substituted or unsubstituted monosaccharide, a substituted or unsubstituted disaccharide, a substituted or unsubstituted trisaccharide, a substituted or unsubstituted oligosaccharide, a substituted or unsubstituted galactosamine, a substituted or unsubstituted aminoglycoside, or a derivative thereof; X is null (when Y is directly attached to Ri), a methyl (CH2), an O (ether), a NH (amine), a S (sulfide), a carbonyl (C=O), ester (O-C=O), carbamate, triazole, or a disulfide (S-S); and Ri and R2 are each individually and independently a H, a sterol,Docket no: 2024-24253-P-WOa cholesterol, a tocopherol, a substituted or unsubstituted Ci- C 50 alkyl, a saturated or unsaturated Ci-50 alkyl, a substituted or unsubstituted Ci- C50 acyl, and / or a linear or branched Ci- C50 alkyl.

[0005] In some embodiments, the monosaccharide includes galactose, glucose, ribose, mannose, xylose, or fructose. In some embodiments, the disaccharide is a sucrose, maltose, trehalose, cellobiose, or lactulose. In some embodiments, X is O, R2 is H, and Ri is saturated or unsaturated C3-C15 alkyl and / or a linear or branched C3-C15 alkyl. In some embodiments, the stabilizing agent is used for stabilizing a lipid nanoparticle or liposome.

[0006] In one aspect, the disclosure provides a lipid nanoparticle composition including: (a) an ionizable lipid; (b) one or more lipids; (c) and the stabilizing agent of any preceding embodiment. In some embodiments, the one or more lipids includes a structural lipid, a sterol, or a combination thereof. In some embodiments, the lipid nanoparticle composition consists essentially of: (a) an ionizable lipid; (b) two lipids; (c) and the stabilizing agent of any preceding embodiment. In some embodiments, the lipid nanoparticle composition consists essentially of: (a) an ionizable lipid; (b) a sterol; and (c) and the stabilizing agent of any preceding embodiment. In some embodiments, the lipid nanoparticle composition consists essentially of: (a) an ionizable lipid; (b) a structural lipid; and (c) and the stabilizing agent of any preceding embodiment.

[0007] In some embodiments, the lipid nanoparticle composition is substantially free of PEG or PEG-R, wherein R is any atom or molecule covalently attached to PEG. In some embodiments, the structural lipid is neutrally charged, positively charged, or negatively charged. In some embodiments, the ionizable lipid is DODMA, DLin-MC3-DMA, DLin-KC2-DMA, BOCHD-C3-DMA, C12-200, PNI 127, PNI 516, PNI 550, PNI 560, PNI 580, PNI 659, PNI 660, PNI 714, PNI 721, PNI 722, PNI 723, PNI 726, PNI 728, PNI 730, PNI 761, PNI 762, PNI 768, PNI 769, PNI 771, PNI 825, or a combination thereof. In some embodiments, the ionizable lipid includes a cyclopentyl or a tetrahydrofuranyl head group or scaffold.

[0008] In some embodiments, the lipid nanoparticle composition of claim 14, wherein the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure including a cyclopentyl scaffold, according to the formula (IA):Docket no: 2024-24253-P-WOwherein: Li is a direct bond or C1-C5 alkylene;E1 is –O–, –OC(O)O–, –OC(O)–δ1, –OC(O)N(Q)–δ1, –OC(O)S–δ1, –N(Q)C(O)–δ1, –N(Q)C(O)O–δ1, –C(O)O–δ1, or –C(O)N(Q)–δ1; Q is H or C1-C5 alkyl; δ1designates the bond linked to R1;R1is selected from:and wherein;R4and R5are each independently Ci-Ce alkyl, C2-C6 alkenyl or C2-C6 alkynyl; alternatively R4and R5may join to form 4-6 membered heterocyclic ring containing oxygen (O) or up to 2 nitrogen (N),optionally substituted with 1 or 2 substituents each independently a Ci-Ce alkyl, cyclopropyl, OH, or a C1-C3 alkoxy;R6is Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl or a 2- hydroxyethyl;R7is H, Ci-Ce alkyl, C2-C6 alkenyl, or a C2-C6 alkynyl;a and c’ are independently 1, 2, 3, 4, or 5;b, c and e are independently 0, 1, or 2;d is 1 or 2;R2is H, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or-h™-\L2 is a direct bond or δ2–(CR8R8')k–δ3wherein R8and R8'are each independently H, C1-C12 alkyl, C2-C12 alkenyl or C2-C12 alkynyl; δ2designates the bond linked to E2, and δ3designates the bond linked to the cyclopentyl scaffold described in formula (IA);k is 1, 2, 3, 4, or 5;E2is –O–, –OC(O)O–, –OC(O)–δ4, –OC(O)N(Q)–δ4, –N(Q)C(O)–δ4, –N(Q)C(O)O–δ4, –C(O)N(Q)–δ4or –C(O)O–δ4; Q is H or C1-C5 alkyl; where δ4designates the bond linked to R3;R3is C8-C20 alkyl, C8-C20alkenyl, C8-C20 alkynyl,Docket no: 2024-24253-P-WOwherein: f is 0 or 1;g is 1 or 2;g’ is 1, 2, 3, 4, or 5;h is 0, 1, 2, 3 or 4;R9is a C6-C20 chain having the formula –(CH2)i–[L4-(CH2)]j–R12, wherein:L4 is selected fromi is an integer in the range 6-20;j is 0, 1, 2, or 3;R12is H or C4-C8 alkyl;R9is H, C4-C10 alkyl, C4-C10 alkenyl, or C4-C10 alkynyl;R10and R10'are each independently C4-C10 alkyl, C4-C10alkenyl or C4-C10 alkynyl;L3 is –OC(O)–δ5, –O–δ5, or a direct bond; δ5designates the bond linked to R10and R10'; and R11= R9, or has the formula:

[0009] In some embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IIA):wherein: Li is a direct bond;E1 is –OC(O)O–, –OC(O)–δ1, –OC(O)N(Q)–δ1, or –OC(O)S–δ1; Q is H or C1-C5 alkyl; δ1designates the bond linked to R1;R1is selected fromDocket no: 2024-24253-P-WOR5N-R4Me'awherein:R4and R5are each independently Ci-Ce alkyl; alternatively R4and R5may join to form 5-6 membered heterocyclic ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents each independently a Ci-Ce alkyl;R6is Ci-Ce alkyl or cyclopropyl;R7is H or Ci-Ce alkyl;a is 1, 2, or 3;b is 0 or 1;c is 0, 1, or 2;c’ is 2, 3, or 4;d is 2;e is 1;MeR2is H, C1-C5 alkyl, C2-C5 alkenyl, or;R3is selected from:wherein: f and h are each 0;g is 1 or 2;R9is a C6-C20 chain having the formula –(CH2)i–[L4-(CH2)]j–R12, wherein:L4is selected from\ Z ■and;i is an integer in the range 6-20;j is 0, 1, or 2;R12is H or C4-C8 alkyl;R9is H or C4-C10 alkyl;R10and R10'are each independently C4-C10 alkyl;L3 is a direct bond;Docket no: 2024-24253-P-WOR13is the same as R11.

[0010] In some embodiments, the lipid nanoparticle composition of claim 14, wherein the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IIIA):(IIIA)wherein: R1is selected from:eNR4and R5are each independently Ci-Ce alkyl; alternatively, R4and R5may join to form 5-6 membered heterocyclic ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents each independently a Ci-Ce alkyl;R6is Ci-Ce alkyl or cyclopropyl;R7is H or Ci-Ce alkyl;a is 1, 2, or 3;b is 0 or 1;c is 0, 1, or 2;c’ is 2, 3, or 4;d is 2;e is 1;R2is H, C1-C5 alkyl, C2-C5 alkenyl orHR3is selected from:1 R111 R9'o T 9 |>0 R!>R9 / YR9H hR ■O 0wherein: f and h are 0;Docket no: 2024-24253-P-WOg is 1 or 2;R9is a C6-C20 chain having the formula –(CH2)i–[L4-(CH2)]j–R12, wherein:L4 is selected from; ’;i is an integer in the range 6-20;j is 0, 1, or 2;R12is H or C4-C8 alkyl;R9is H or C4-C10 alkyl;R10and R10'are each independently C4-C10 alkyl;L3 is a direct bond;R11is the same as R9.

[0011] In some embodiments, the lipid nanoparticle composition of claim 14, wherein the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IB):wherein p is 0 or 1;E1 is –O–δ1, –OC(O)O–δ1, –OC(O)–δ1, –OC(O)N(Q)–δ1, –OC(O)S–δ1, –C(O)N(Q)–δ1, –C(O)O–δ1, –N(Q)C(O)–δ1, –N(Q)C(O)O–δ1, –N(Q)C(O)S–δ1, or –N(Q)C(O)N(Q)–δ1; wherein Q is H or C1-C5 alkyl; δ1designates the bond linked to R1; R1is selected from:wherein: R3and R4are each independently Ci-Ce alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; alternatively R3and R4may join to form 4-6 membered ring containing oxygen (O) or up to 2 nitrogen (N),Docket no: 2024-24253-P-WOoptionally substituted with 1-2 substituents, each independently a Ci-Ce alkyl, cyclopropyl, OH, or a C1-C3 alkoxy;R5is Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, or a 2-hydroxyethyl;R6is H or Ci-Ce alkyl;a is 1, 2, 3, 4 or 5;b and c are independently 0, 1, or 2;c’ is 1, 2, 3, 4, or 5;d is 1 or 2;e is 0, 1, or 2;E2is –OC(O)–δ2, –OC(O)O–δ2, –OC(O)N(Q)–δ2, –O–δ2, –OCH2CH2O–δ2, or –OC(O)(CH2)6C(O)O–δ2; Q is H or C1-C5 alkyl; δ2designates the bond linked to R2;R2isf L2 r8or has the formula (CH2)g[L3-(CH2)]h R9, wherein:L1 and L2 are each independently a direct bond, –O–δ3, –CH2OC(O)–δ3, or –CH2O–δ3; δ3designates the bond linked to R7and R8;R7and R8are each independently C4-C10 alkyl, C4-C10 alkenyl or C4-C10 alkynyl;f is 0, 1, 2, 3, 4, or 5;L3 is selected fromR9is H or C4-C8 alkyl;g is an integer in the range of 1-18;h is 0, 1, 2, or 3.

[0012] In some embodiments, the lipid nanoparticle composition of claim 14, wherein the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IIB):E E nR2-’'t2 C2-R2Docket no: 2024-24253-P-WOwherein E1 is –OC(O)O–δ1, –OC(O)–δ1, –OC(O)N(Q)–δ1, or –OC(O)S–δ1; Q is H or C1-C5 alkyl; and δ1designates the bond linked to R1;R1is selected from:CN'wherein:R3and R4are each independently a Ci-Ce alkyl; alternatively R3and R4may join to form 5-6 membered ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents selected from a Ci-Ce alkyl;R5is a Ci-Ce alkyl or C3-C6 cycloalkyl;R6is an H or Ci-Ce alkyl;a is 1, 2, 3, or 4;b and c are independently 0, 1, or 2;c’ is 2, 3, or 4;d is 2;e is 0 or 1;E2is –O–δ2, –OC(O)–δ2, –OCH2CH2O–δ2, or –OC(O)(CH2)6C(O)O–δ2; where δ2designates the bond linked to R2;R2isor has the formula (CH2)g[L3-(CH2)]h R9, wherein:L1 and L2are each independently a direct bond, –O–δ3, –CH2OC(O)–δ3, or –CH2O–δ3; δ3designates the bond linked to R7and R8;R7and R8are each independently C4-C10 alkyl, C4-C10 alkenyl or C4-C10 alkynyl;f is 0, 1, 2, 3, 4, or 5;H H H V H A HL3is selected fromand X / .R9is H or a C4-C8 alkyl;g is an integer in the range of 1-18;h is 0, 1, or 2.Docket no: 2024-24253-P-WO

[0013] In some embodiments, the lipid nanoparticle composition of claim 14, wherein the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IIIB):R2–E2E2–R2(IIIB) R1is selected from:R3and R4are each independently a Ci-Ce alkyl group; alternatively R3and R4may join to form 5-6 membered ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents selected from a Ci-Ce alkyl;R5is Ci-Ce alkyl or cyclopropyl;R6is H or Ci-Ce alkyl;a is 1, 2, 3, or 4;b is 0 or 1;c is 0, 1, or 2;c’ is 2, 3, or 4;d is 2;e is 1;E2is –O–δ2, –OC(O)–δ2, –OCH2CH2O–δ2, or –OC(O)(CH2)6C(O)O–δ2; where δ2designates the bond linked to R2;R2isor has the formula –(CH2)g–[L3-(CH2)]h–R9, wherein:Li and L2are each a direct bond;R7and R8are each independently a C4-C10 alkyl;f is 0 or 1;Docket no: 2024-24253-P-WOH H H A H\ / , and \.L3 is selected fromR9is H or a C4-C8 alkyl;g is an integer in the range of 1-18;h is 0, 1, or 2.

[0014] In some embodiments, the structural lipid includes diacylphosphatidylcholines, diacylphosphatidylethanolamines, diacylphosphatidylglycerols, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, cerebrosides, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylethanolamine, palmitoyloleoylphosphatidylcholine, 1 -stearoyl -2-oleoyl-sn-gly cero-3 -phosphocholine, palmitoyloleoyl-phosphatidylethanolamine, di oleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate, dipalmitoyl phosphatidyl ethanolamine, dimyristoylphosphoethanolamine, distearoylphosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine-N-methyl, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N, N-dimethyl, l,2-dielaidoyl-sn-glycero-3-phosphoethanolamine, 1 -stearoyl -2-oleoyl-phosphatidy ethanol amine, 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine, distearoylphosphatidylcholine, dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, palmitoyloleyolphosphatidylglycerol, cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, monosialoganglioside GM1, or a combination thereof.

[0015] In some embodiments, the sterol includes cholesterol, beta-sitosterol, 20-alpha-hydroxysterol, phytosterol, or a combination thereof. In some embodiments, the stabilizing agent has a molecular weight of about 500 Da to about 50,000 Da. In some embodiments, the lipid nanoparticle composition also includes a second stabilizing agent, wherein the second stabilizing agent is a polysorbate, N-dodecyl beta-D-maltoside, D-a-Tocopherol polyethylene glycol 1000 succinate, or a combination thereof. In some embodiments, the lipid nanoparticle composition includes about 20 to about 70 mol% ionizable lipid, about 1 to about 75 mol% structural lipid, about 1 to about 75 mol% sterol, and about 0.1 to about 50 mol% stabilizing agent.

[0016] In one aspect, the disclosure provides a lipid nanoparticle including the lipid nanoparticle composition of any preceding embodiment and a nucleic acid. In some embodiments, the nucleic acid is encapsulated by the lipid nanoparticle composition. In someDocket no: 2024-24253-P-WOembodiments, the nucleic acid is an antisense oligonucleotide, a siRNA, a miRNA, a selfamplifying RNA (SAM or saRNA), a circular RNA, a self-replicating DNA, an LNA, a DNA, a replicon, an mRNA, a circRNA, a guide RNA, a transposon, a single gene, a vector, a plasmid, a viral particle, an AAV, a complex of RNA and RNA-binding protein, or a combination thereof. In some embodiments, the nucleic acid is an antigen encoded mRNA for prophylactic or therapeutic vaccine, a nucleic acid for gene therapy, or a nucleic acid for immunogenic cell incorporation, wherein the immunogenic cell is a T cell. In some embodiments, the diameter of the lipid nanoparticle is about 15 nm to about 500 nm. In some embodiments, the lipid nanoparticle has a polydispersity index of about 0.01 to about 0.40. In some embodiments, the lipid nanoparticle has an encapsulation efficiency of about 50% to about 100%.

[0017] In one aspect, the disclosure provides a pharmaceutical composition including the lipid nanoparticle composition of any preceding embodiment and a pharmaceutically acceptable carrier.

[0018] In one aspect, the disclosure provides a method for preparing the lipid nanoparticle of any preceding embodiment or the pharmaceutical composition, the method including: forming the lipid nanoparticle composition by combining the ionizable lipid, the stabilizing agent, and optionally one or more lipids including the structural lipid and / or the sterol; preparing the lipid nanoparticle by combining an organic phase including the lipid nanoparticle composition and an aqueous phase including the nucleic acid using a microfluidic mixer; and optionally purifying the lipid nanoparticle. In some embodiments, the lipid nanoparticle composition and the nucleic acid are combined using a flow ratio of about 1: 1 to about 10:1 by volume (aqueous phase: organic phase) at a N / P ratio of about 2 to about 25, and a combined flow rate of the aqueous phase and the organic phase is about 2 to about 2000 mL / min. In some embodiments, the aqueous phase includes a low pH buffer. In some embodiments, the aqueous phase includes a citrate or acetate buffer. In some embodiments, the organic phase includes 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, alcohols, or a combination thereof. In some embodiments, the organic phase includes an alcohol and the alcohol includes aqueous or anhydrous alcohol, wherein the alcohol includes a primary, secondary, or tertiary alcohol having from 1 to 12 branched or unbranched carbons, or a combination thereof.

[0019] In one aspect, the disclosure provides use of the lipid nanoparticle of any preceding embodiment or the pharmaceutical composition for preventing, treating, or amelioratingDocket no: 2024-24253-P-WOconditions or diseases including administering the lipid nanoparticle as a vaccine or as a treatment to prevent or reduce the severity of a contagion, administering the lipid nanoparticle as a gene therapeutic, or administering the lipid nanoparticle to an immunogenic cell for the treatment of cancer or an infection.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Various aspects of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0021] FIG. 1 is a bar graph illustrating the in vitro potency of various LNPs formulated with sugar-based stabilizing agents.

[0022] FIG. 2 is a dot plot illustrating hEPO expression levels (6 hr and 24 hr post administration) in C57BL / 6 mice following IV administration of 0.25 mg / Kg dose of recombinant human EPO-encoded mRNA-LNPs. FIG. 2 shows the mean [EPO] value at 24 hr is less than the value at 6 hr for all nanoparticles evaluated.

[0023] FIGS. 3A - 3C are a bar graphs showing enhanced expression of eGFP when LNPs comprising TDM was used as a single agent with LNPs in human primary T cells. FIG.3A shows the percent of T cells expressing GFP for each LNP. FIG.3B shows the mean fluorescence intensity (MFI) detected for each LNP. FIG.3C shows the percent viability of the cell population for each LNP.

[0024] FIGS. 4A and 4B are bar graphs illustrating the ex vivo gene editing of CD45 in HSCs using different LNPs formulated with sugar-based stabilizing agents encapsulating CRISPR-Cas9 gene editing mRNA. FIG. 4A shows the percent of CD45 negative cells for each LNP. FIG. 4B shows the percent viability of the cell population for each LNP.

[0025] FIGS. 5A and 5B are plots illustrating the in vitro potency of LNPs (lipid composition: 40% PNI728, 12.5% DSPC, (47.5-x)% Cholesterol (Choi), x% TDM), using a payload of enhanced green fluorescent protein (eGFP) mRNA) in Jurkat cells. FIG. 5A shows the percent of cells expressing GFP. FIG. 5B shows the MFI of GFP in Jurkat cells.

[0026] FIGS. 6A and 6B are plots illustrating the in vitro potency of LNPs (lipid composition: 40% PNI728, 12.5% DSPC, 42.5% Choi, 5% TDM), using a payload of enhanced green fluorescent protein (eGFP) mRNA) in BHK cells. FIG. 6 A shows the percent of cells expressing GFP. FIG. 6B shows the MFI of GFP in BHK cells.Docket no: 2024-24253-P-WO

[0027] FIG. 7 is a dot plot illustrating hEPO expression levels (24 hr post administration) in C57BL / 6 mice following intramuscular (IM) administration of 0.25 mg / Kg dose of recombinant human EPO-encoded mRNA-LNPs using the lipid composition; 40% PNI516, 12.5% DSPC, (47.5-x)% cholesterol, x% stabilizer. The stabilizers tested include PEG-DMG and TDM.DETAILED DESCRIPTIONI. Introduction

[0028] The disclosure provides sugar-based stabilizing agents and nanoparticle (LNP) compositions including the sugar-based stabilizing agents, as well as methods for preparing the lipid nanoparticles. These lipid nanoparticle (LNP) compositions may be configured to encapsulate nucleic acids. The lipid nanoparticle compositions for encapsulating nucleic acids comprise an ionizable lipid, a stabilizing agent, and one or both of a structural lipid, or a sterol, as described herein, where the stabilizing agent has a sugar-based structure such as, for example, tridecyl β-D-maltodside (TDM).

[0029] PEGylated-lipid is an FDA-approved modality and has been widely utilized for the development of lipid nanoparticle-based drug delivery systems. However, PEGylation can also present challenges such as its limited chemistry, inhibiting the ability to add functional groups to the PEG backbone for new applications, and its susceptibility to oxidation during storage and potential immunogenicity upon prolonged use that can cause allergic reactions in some patients. Further, it has been reported that the inclusion of high mol% of PEG-lipid (e.g., > 5%) as a stabilizer in the lipid composition, compromises the critical quality attributes and in vivo efficacy of the resulting LNPs.

[0030] Advantageously, in contrast with LNP compositions including polyethylene glycol (PEG) or PEGylated lipid (polyethylene glycol (PEG)-lipid conjugate) component, LNP and NALNP compositions including a stabilizing agent in accordance with the disclosure are substantially free of PEG and PEGylated lipids. The inventors discovered that the inventive stabilizing agents allow for LNPs that have higher compatibility and safety and lower immunogenicity when compared to PEG or PEGylated lipid based products and outperform the PEG or PEGylated lipid based products both ex vivo and in vivo. Such advantageous characteristics of the LNPs of the instant disclosure also allows for repeated dosing of aDocket no: 2024-24253-P-WOpharmaceutical composition including the LNPs of the instant disclosure, offering improvements over conventional PEG or PEGylated lipid based products.

[0031] In some cases, the PEGylated lipid refers to PEG-R, where R is any atom or molecule. In some cases, R is DMG, DSG, DSPE, DOPE, or DPPE. In some cases, the PEGylated lipid includes DMG-PEG, DSG-PEG, DSPE-PEG, DOPE-PEG, or DPPE-PEG.

[0032] The resulting encapsulated LNP formulations may be used in a variety of applications, including, but not limited to, medical applications such as oligonucleotide-based therapeutics, e.g., vaccines, immunogenic cell incorporation, gene editing, and gene therapy.

[0033] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.II. Definitions

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0035] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0036] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0037] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0038] The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.Docket no: 2024-24253-P-WO

[0039] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the sevenelement set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0040] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0041] Reference throughout this specification to “one aspect (or embodiment),” “an aspect (or embodiment),” “certain aspects (or embodiments),” or “some aspects (or embodiments),” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0042] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with aDocket no: 2024-24253-P-WOmeasured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such an interval of accuracy is + / - 10%. Thus, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1 %, 0.05%, 0.01 %, or 0.001 % greater or less than the stated value. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0043] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0044] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0045] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0046] The term "administering" as used herein refers to the physical introduction of an agent to a subject, such as a lipid nanoparticle disclosed herein, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration forDocket no: 2024-24253-P-WOthe formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intratympanic, intralesional, intracapsular, infraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intratarsal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0047] The term "cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A "cancer" or "cancer tissue" can include a tumor.

[0048] The term "in vitro" refers to events occurring in an artificial environment, e.g., in a test tube, reaction vessel, cell culture, etc., rather than within a multi-cellular organism. The term "in vitro cell" refers to any cell which is cultured ex vivo. In particular, an in vitro cell can include a T cell. The term "in vivo" refers to events that occur within a multi-cellular organism, such as a human or a non -human animal.

[0049] The term "nucleic acid" refers to any polymeric chain of nucleotides. A nucleic acid may be DNA, RNA, or a combination thereof. In some embodiments, a nucleic acid comprises one or more natural nucleic acid residues. In some embodiments, a nucleic acid comprises of one or more nucleic acid analogs. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60,65, 70, 75,80, 85,90,95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000,Docket no: 2024-24253-P-WO1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long (e.g., 20 to 100, 20 to 500, 20 to 1000, 20 to 2000, or 20 to 5000 or more residues). In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.

[0050] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not deleterious to the recipient thereof, or that any deleterious effect is outweighed by a benefit to the recipient thereof. With respect to a carrier, diluent, or excipient used to formulate a composition as disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof, or any deleterious effect must be outweighed by a benefit to the recipient. The term "pharmaceutically acceptable carrier" means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one portion of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient, or any deleterious effect must be outweighed by a benefit to the recipient. Some examples of materials which may serve as pharmaceutically acceptable carriers comprise: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other nontoxic compatible substances employed in pharmaceutical formulations.

[0051] Treatment" or "treating" of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset,Docket no: 2024-24253-P-WOprogression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease. In one embodiment, "treatment" or "treating" includes a partial remission. In another embodiment, "treatment" or "treating" includes a complete remission. In some embodiments, treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition.

[0052] A "disease", as used herein, is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, the subject's health continues to deteriorate. In contrast, a "disorder" is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health. A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.

[0053] As used herein, the terms “subject”, “individual”, and “patient” are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein includes humans. The term "subject" may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).

[0054] As used herein, the term “preventing a disease” in a subject means, for example, to stop the development of one or more clinical symptoms of a disease or disorder in a subject before they occur or are detectable. Preferably, the disease or disorder does not develop at all,Docket no: 2024-24253-P-WOi.e., no symptoms of the disease or disorder are detectable. In some aspects, it can also mean delaying or slowing of the development of one or more symptoms of the disease or disorder. Alternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms.

[0055] As used herein, the term “targeting group” can be in the form of a moiety capable of specifically binding to a molecule on the surface of a target cell, such as a cell within a target tissue of interest. In certain embodiments, the targeting group is a peptide, antibody, sugar, dopamine, oligosaccharide, aminoglycoside, sterol, phenyl boronic acid, or a combination thereof.

[0056] The invention is defined in the claims. However, below is a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.III. Stabilizing Agents

[0057] Lipid nanoparticles including liposomes, cubosomes, hexosomes, solid lipid nanoparticles, and nanostructured lipid carriers may require steric stabilizers to maintain colloidal stability and improve pharmacokinetics and biodistribution profiles. One indication of colloidal stability is the polydispersity index (PDI), which needs to be sufficiently low such that aggregation of the nanoparticles does not occur. A high PDI would indicate a decrease in the stability and viability of the nanoparticles over time.

[0058] Currently, polyethylene glycol (PEG)-lipid conjugates are the most commonly employed stabilizers in lipid nanoparticles. However, this PEGylated class of stabilizers can elicit an undesirable immunogenic response and impede cell interactions with nanoparticles containing a PEG layer. Surprisingly, the class of sugar-based stabilizers as described herein was discovered to have favorable profiles (e.g., size, polydispersity index values, and encapsulation efficiency) without eliciting the same immunogenic response and without impeding cell interactions compared to PEGylated stabilizers.

[0059] The sugar-based stabilizers have the general structure of formula (I):Docket no: 2024-24253-P-WOX R2'X'Y / wherein Y is a substituted or unsubstituted monosaccharide, a substituted or unsubstituted disaccharide, a substituted or unsubstituted trisaccharide, a substituted or unsubstituted oligosaccharide, a substituted or unsubstituted galactosamine, a substituted or unsubstituted aminoglycoside, or a derivative thereof; X is null (when Y is directly attached to Ri), a methyl (CH2), an O (ether), a NH (amine), a S (sulfide), a carbonyl (C=O), ester (O-C=O), carbamate, triazole, or a disulfide (S-S); and Ri and R2 are each individually and independently a H, a sterol, a cholesterol, a tocopherol, a substituted or unsubstituted C1-C50 alkyl, a saturated or unsaturated C1-C50 alkyl, a substituted or unsubstituted C1-C50 acyl, and / or a substituted or unsubstituted linear or branched C1-C50 alkyl.

[0060] In some embodiments, the monosaccharide comprises galactose, glucose, ribose, mannose, xylose, or fructose. In some embodiments, the disaccharide is a sucrose, maltose, trehalose, cellobiose, or lactulose. In some embodiments, Ri is directly attached to the monosaccharide, disaccharide, trisaccharide, oligosaccharide, galactosamine, or aminoglycoside. In some embodiments, R2 is H. In some embodiments, R2 is H and Ri is saturated or unsaturated C3- C15 alkyl. In some embodiments, X is C=O, R2 is H, and Ri is saturated or unsaturated C3-C15 alkyl and / or a linear or branched C3-C15 alkyl. In some embodiments, the stabilizing agent is used for stabilizing a lipid nanoparticle or liposome. Non-limiting examples of structures of the sugar- based stabilizers are illustrated in Table 1.Table 1Structure M.w. Stabilizer # Name(Da) P48 OH OH 5250 - 1 0 - ITridecyl-B- maltoside V-AA — kiHOH OHP82 OH OH 510N-Dodecyl- 0 - 1 0 - IBeta- Z OPk / OPkMaltoside \ — — / 0HOH OHDocket no: 2024-24253-P-WOP99 OH OH 566N-Hexadecyl o - 1 o - IB-D- 1 / oX / o\ maltoside \ — 1 / \ / \ — kiHOH OHP100 \0524II 1H°sucrose ° / °\ 0X Rmonolaurate JXL fxoz\ \zlI 1 ^OHOHP102 \ O\ 496II 1H0N-decanoyl O / °x^1 oXsucrose JXL zxoz\ \zlI 1 ^OHP94 875SucrosedistearateL °I 1 ^OHP90 \ 0'S OH 600 TrehalosemonooleateOHP101 \z^ \z^ r / 00H580 trehalose 6- hexadecanoate Q xiOHDocket no: 2024-24253-P-WOIV. Nanoparticle Compositions Including Sugar-based Stabilizing Agents

[0061] Conventionally, lipid nanoparticles include a PEGylated lipid to impart critical quality attribute (CQA) values important for stability of the nanoparticle and delivery of the nucleic acid cargo. Surprisingly, the inventors discovered that CQA values that increase the stability of the nanoparticles and delivery of the nucleic acid cargo may be achieved by using nanoparticles that are substantially free of any PEGylated lipids, which can be represented as PEG-R, where R is any atom or molecule. In some cases, R is DMG, DPG, DSG, DSPE, DMPE, DOPE, or DPPE. In some cases, the PEGylated lipid includes DMG-PEG, DPG-PEG, DSG-PEG, DSPE-PEG, DMPE-PEG, DOPE-PEG, or DPPE-PEG.

[0062] The disclosure provides lipid nanoparticle (LNP) compositions configured to encapsulate nucleic acids. The lipid nanoparticle compositions for encapsulating nucleic acids may include an ionizable lipid. The lipid nanoparticle compositions for encapsulating nucleic acids may also include a stabilizing agent. The lipid nanoparticle compositions for encapsulating nucleic acids may also include a sterol (e.g., cholesterol). The lipid nanoparticle compositions for encapsulating nucleic acids may also include a structural lipid (also referred to as “helper lipid”Docket no: 2024-24253-P-WOin the disclosure). The resulting encapsulated LNP formulations may be used in a variety of applications, particularly medical applications such as oligonucleotide-based therapeutics, e.g., gene therapy, cell therapy.

[0063] In some embodiments, the lipid nanoparticle composition includes an ionizable lipid, two or more lipids, and a stabilizing agent, where the two or more lipids comprise a structural lipid, a sterol, or a combination thereof. In some embodiments, the lipid nanoparticle composition includes an ionizable lipid, one or more lipids, and a stabilizing agent, where the one or more lipids comprise a structural lipid, a sterol, or a combination thereof.

[0064] Any suitable ionizable lipid can be present in the lipid nanoparticle composition and lipid nanoparticle. An ionizable lipid is a lipid that is cationic or becomes ionizable (protonated) as the pH is lowered below the pKa of the ionizable group of the lipid but is more neutral at higher pH values. At pH values below the pKa, the lipid is able to associate with negatively charged nucleic acids / oligonucleotides. Ionizable lipid includes lipids that assume a positive charge on pH lower than physiological pH, or lipids that carry a net positive charge at a selective pH.

[0065] In some embodiments, the lipid nanoparticle composition or lipid nanoparticle comprises one or more ionizable lipids, e.g., two or more ionizable lipids, three or more ionizable lipids, or four or more ionizable lipids. In some embodiments, the ionizable lipid includes, but is not limited to, DODMA (l,2-dioleyloxy-3 -dimethylaminopropane), DLin-MC3-DMA (O-(Z, Z, Z, Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N, N-dimethylamino)), DLin-KC2-DMA (2-dilinoleyl-4-dimethylaminoethyl- [l,3]-dioxolane), BOCHD-C3-DMA (4-(dimethylamino)-,9-(2-octylcyclopropyl)-l-[8-(2 octylcyclopropyl) octyl]nonyl ester), C12-200 (l,l'-[[2-[4-[2-[[2-[Z> A(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l-piperazinyl]ethyl]imino]Z> A-2-dodecanol), or a combination thereof.

[0066] In some embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof includes a cyclopentyl or a tetrahydrofuranyl head group or scaffold.

[0067] In some embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure including a cyclopentyl scaffold, according to the formula (IA):Docket no: 2024-24253-P-WOwherein: Li is a direct bond or C1-C5 alkylene;E1is –O–, –OC(O)O–, –OC(O)–δ1, –OC(O)N(Q)–δ1, –OC(O)S–δ1, –N(Q)C(O)–δ1, –N(Q)C(O)O–δ1, –C(O)O–δ1, or –C(O)N(Q)–δ1; Q is H or C1-C5 alkyl; δ1designates the bond linked to R1;R1is selected from:and wherein;R4and R5are each independently Ci-Ce alkyl, C2-C6 alkenyl or C2-C6 alkynyl; alternatively R4and R5may join to form 4-6 membered heterocyclic ring containing oxygen (O) or up to 2 nitrogen (N),optionally substituted with 1 or 2 substituents each independently a Ci-Ce alkyl, cyclopropyl, OH, or a C1-C3 alkoxy;R6is Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl or a 2 -hydroxy ethyl;R7is H, Ci-Ce alkyl, C2-C6 alkenyl, or a C2-C6 alkynyl;a and c’ are independently 1, 2, 3, 4, or 5;b, c and e are independently 0, 1, or 2;d is 1 or 2;R2is H, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or$ \. / I.5L2is a direct bond or δ2·(CR8R8')k·δ3wherein R8and R8'are each independently H, C1-C12alkyl, C2-C12alkenyl or C2-C12alkynyl; δ2designates the bond linked to E2, and δ3designates the bond linked to the cyclopentyl scaffold described in formula (IA);k is 1, 2, 3, 4, or 5;E2is –O–, –OC(O)O–, –OC(O)–δ4, –OC(O)N(Q)–δ4, –N(Q)C(O)–δ4, –N(Q)C(O)O–δ4, –C(O)N(Q)–δ4or –C(O)O–δ4; Q is H or C1-C5alkyl; where δ4designates the bond linked to R3;Docket no: 2024-24253-P-WOR3is C8-C20 alkyl, C8-C20 alkenyl, C8-C20 alkynyl,wherein: f is 0 or 1;g is 1 or 2;g’ is 1, 2, 3, 4, or 5;h is 0, 1, 2, 3 or 4;R9is a C6-C20 chain having the formula (CH2), [L4-(CH2)]J R12, wherein:L4 is selected fromi is an integer in the range 6-20;j is 0, 1, 2, or 3;R12is H or C4-C8 alkyl;R9is H, C4-C10 alkyl, C4-C10 alkenyl, or C4-C10 alkynyl;R10and RIO’ are each independently C4-C10 alkyl, C4-C10 alkenyl or C4-C10 alkynyl;L3 is –OC(O)–δ5, –O–δ5, or a direct bond; δ5designates the bond linked to R10and R10'; and R11= R9, or has the formula:

[0068] In some embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IA):wherein: Li is a direct bond or C1-C5 alkylene;Docket no: 2024-24253-P-WOE1is –OC(O)O–, –OC(O)–δ1, –OC(O)N(Q)–δ1, or –OC(O)S–δ1; Q is H or C1-C5alkyl; δ1designates the bond linked to the R1;R1is selected from:eNand wherein:R4and R5are each independently Ci-Ce alkyl, C2-C6 alkenyl or C2-C6 alkynyl; alternatively R4and R5may join to form 5-6 membered heterocyclic ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents each independently a Ci-Ce alkyl or a cyclopropyl; R6is Ci-Ce alkyl or C3-C6 cycloalkyl;R7is H or Ci-Ce alkyl;a is 1, 2, or 3;b and c are independently 0, 1, or 2;c’ is 2, 3, or 4;d is 1 or 2;e is 0 or 1;R2is H, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl,orL2is a direct bond or δ2·(CR8R8')k·δ3wherein R8and R8'are each independently H, C1-C12alkyl, C2-C12alkenyl or C2-C12alkynyl; δ2designates the bond linked to E2and δ3designates the bond linked to the cyclopentyl scaffold described in formula (IA);k is 1;E2is –O–, –OC(O)O–, –OC(O)–δ4, –OC(O)N(Q)–δ4, –C(O)N(Q)–δ4or –C(O)O–δ4; Q is H or C1-C5alkyl; where δ4designates the bond linked to R3;R3is selected from C8-C20 alkyl, C8-C20 alkenyl, C8-C20 alkynyl,1 R111 R9'o T 9 |>0 R!>RR9. H hj^R9O 0 wherein: f and h are each 0;g is 1 or 2;R9is a C6-C20 chain having the formula –(CH2)i–[L4-(CH2)]j–R12, wherein:Docket no: 2024-24253-P-WOH H H 'z,.. =. andL4 is selected fromi is an integer in the range 6-20;j is 0, 1, or 2;R12is H or C4-C8 alkyl;R9is H or C4-C10 alkyl;R10and R10are each independently C4-C10 alkyl, C4-C10 alkenyl or C4-C10 alkynyl;L3is –OC(O)–δ5, or a direct bond; δ5designates the bond linked to R10and R10';R11is the same as R9.

[0069] In some embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IIA):wherein: Li is a direct bond;E1 is –OC(O)O–, –OC(O)–δ1, –OC(O)N(Q)–δ1, or –OC(O)S–δ1; Q is H or C1-C5 alkyl; δ1designates the bond linked to R1;R1is selected fromR5N-R4Mewherein:R4and R5are each independently Ci-Ce alkyl; alternatively R4and R5may join to form 5-6 membered heterocyclic ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents each independently a Ci-Ce alkyl;R6is Ci-Ce alkyl or cyclopropyl;R7is H or Ci-Ce alkyl;a is 1, 2, or 3;b is 0 or 1;Docket no: 2024-24253-P-WOc is 0, 1, or 2;c’ is 2, 3, or 4;d is 2;e is 1;4 — \ ^MeR2is H, C1-C5 alkyl, C2-C5 alkenyl, or "' v HR3is selected from:wherein: f and h are each 0;g is 1 or 2;R9is a C6-C20 chain having the formula –(CH2)i–[L4-(CH2)]j–R12, wherein:Hy\HL4is selected from\ ■and■i is an integer in the range 6-20;j is 0, 1, or 2;R12is H or C4-C8 alkyl;R9is H or C4-C10 alkyl;R10and R10'are each independently C4-C10 alkyl;L3 is a direct bond;R13is the same as R11.

[0070] In some embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IIIA):0(IIIA)wherein: R1is selected from:Docket no: 2024-24253-P-WOR5N R4wherein:R4and R5are each independently Ci-Ce alkyl; alternatively, R4and R5may join to form 5-6 membered heterocyclic ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents each independently a Ci-Ce alkyl;R6is Ci-Ce alkyl or cyclopropyl;R7is H or Ci-Ce alkyl;a is 1, 2, or 3;b is 0 or 1;c is 0, 1, or 2;c’ is 2, 3, or 4;d is 2;e is 1;R2is H, C1-C5 alkyl, C2-C5 alkenyl orHR3is selected from:f and h are 0;g is 1 or 2;R9is a C6-C20 chain having the formula –(CH2)i–[L4-(CH2)]j–R12, wherein:i is an integer in the range 6-20;j is 0, 1, or 2;R12is H or C4-C8 alkyl;R9is H or C4-C10 alkyl;R10and R10'are each independently C4-C10 alkyl;L3 is a direct bond;Docket no: 2024-24253-P-WOR11is the same as R9.

[0071] In some embodiments of the formula (IA), (HA), and (IIIA), R1is one of:Me

[0072] In some embodiments of the formula (IA), (HA), and (IIIA), each R3is independently:ooDocket no: 2024-24253-P-WOoo o ooDocket no: 2024-24253-P-WO

[0073] In some embodiments of the formula (IA), (HA), and (IIIA), R2is selected from:

[0074] In still some embodiments of the formula (IA), (HA), and (IIIA), Ei is selected from:, wherein δ1designates the bond linked to R1; 51designates the bond linked to Li.

[0075] In still some embodiments of the formula (IA), (HA), and (IIIA), E2 is selected from:H MeO, wherein δ4designates the bond linked to R3.Docket no: 2024-24253-P-WO

[0076] In some embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure including a tetrahydrofuranyl scaffold, according to the formula (IB)wherein p is 0 or 1;E1 is –O–δ1, –OC(O)O–δ1, –OC(O)–δ1, –OC(O)N(Q)–δ1, –OC(O)S–δ1, –C(O)N(Q)–δ1, –C(O)O–δ1, –N(Q)C(O)–δ1, –N(Q)C(O)O–δ1, –N(Q)C(O)S–δ1, or –N(Q)C(O)N(Q)–δ1; wherein Q is H or C1-C5 alkyl; δ1designates the bond linked to R1; R1is selected from:wherein: R3and R4are each independently Ci-Ce alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; alternatively R3and R4may join to form 4-6 membered ring containing oxygen (O) or up to 2 nitrogen (N), optionally substituted with 1-2 substituents, each independently a Ci-Ce alkyl, cyclopropyl, OH, or a C1-C3 alkoxy;R5is Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, or a 2-hydroxyethyl;R6is H or Ci-Ce alkyl;a is 1, 2, 3, 4 or 5;b and c are independently 0, 1, or 2;c’ is 1, 2, 3, 4, or 5;d is 1 or 2;e is 0, 1, or 2;E2is -OC(O)-52, -OC(O)O-52, -OC(O)N(Q)-52, -O-52, -OCH2CH2O-δ2, or -OC(O)(CH2)6C(O)O-52; Q is H or C1-C5 alkyl; 52designates the bond linked to R2;Docket no: 2024-24253-P-WOLI-R'R2isf L2~R® or has the formula (CH2)g[L3-(CH2)]h R9, wherein:L1 and L2 are each independently a direct bond, –O–δ3, –CH2OC(O)–δ3, or –CH2O–δ3; δ3designates the bond linked to R7and R8;R7and R8are each independently C4-C10 alkyl, C4-C10 alkenyl or C4-C10 alkynyl;f is 0, 1, 2, 3, 4, or 5;H H H A HL3 is selected from'X H •;R9is H or C4-C8 alkyl;g is an integer in the range of 1-18;h is 0, 1, 2, or 3.

[0077] In some embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IIB):(HB)wherein Ei is OC(O)O 51, OC(O) 51, OC(O)N(Q) 51, or OC(O)S 51; Q is H or C1-C5 alkyl; and δ1designates the bond linked to R1;R1is selected from:R4N-R3’wherein:R3and R4are each independently a Ci-Ce alkyl; alternatively R3and R4may join to form 5-6 membered ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents selected from a Ci-Ce alkyl;R5is a Ci-Ce alkyl or C3-C6 cycloalkyl;R6is an H or Ci-Ce alkyl;a is 1, 2, 3, or 4;Docket no: 2024-24253-P-WOb and c are independently 0, 1, or 2;c’ is 2, 3, or 4;d is 2;e is 0 or 1;E2is -O-δ2, -OC(O)-δ2, -OCH2CH2O-δ2, or -OC(O)(CH2)6C(O)O-δ2; where δ2designates the bond linked to R2;R2isf L2~R8or has the formula -(CH2)g-[L3-(CH2)]h R9, wherein:L1 and L2are each independently a direct bond, –O–δ3, –CH2OC(O)–δ3, or –CH2O–δ3; δ3designates the bond linked to R7and R8;R7and R8are each independently C4-C10 alkyl, C4-C10 alkenyl or C4-C10 alkynyl;f is 0, 1, 2, 3, 4, or 5;_ H H A HL3 is selected fromX Z ■ X HR9is H or a C4-C8 alkyl;g is an integer in the range of 1-18;h is 0, 1, or 2.

[0078] In some embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IIB):(HB)Wherein Ei is OC(O)O 51, OC(O) 51, OC(O)N(Q) 51, or OC(O)S 51; Q is H or C1-C5 alkyl; and δ1designates the bond linked to R1;R1is selected from:wherein:Docket no: 2024-24253-P-WOR3and R4are each independently a Ci-Ce alkyl; alternatively R3and R4may join to form 5-6 membered ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents selected from a Ci-Ce alkyl;R5is Ci-Ce alkyl or cyclopropyl;R6is H or Ci-Ce alkyl;a is 1, 2, 3, or 4;b is 0 or 1;c is 0, 1, or 2;c’ is 2, 3, or 4;d is 2;e is 1;E2is –O–δ2, –OC(O)–δ2, –OCH2CH2O–δ2, or –OC(O)(CH2)6C(O)O–δ2; where δ2designates the bond linked to R2;R2is f L2~R8or has the formula –(CH2)g-[L3-(CH2)]h–R9, wherein:Li and L2are each a direct bond;R7and R8are each independently C4-C10 alkyl;f is 0 or 1;R9is H or a C4-C8 alkyl;g is an integer in the range of 1-18;h is 0, 1, or 2.

[0079] In some embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IIIB):(IIIB)Docket no: 2024-24253-P-WOR1is selected from:N-R3’wherein:R3and R4are each independently a Ci-Ce alkyl group; alternatively R3and R4may join to form 5-6 membered ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents selected from a Ci-Ce alkyl;R5is Ci-Ce alkyl or cyclopropyl;R6is H or Ci-Ce alkyl;a is 1, 2, 3, or 4;b is 0 or 1;c is 0, 1, or 2;c’ is 2, 3, or 4;d is 2;e is 1;E2is –O–δ2, –OC(O)–δ2, –OCH2CH2O–δ2, or –OC(O)(CH2)6C(O)O–δ2; where δ2designates the bond linked to R2;R2isor has the formula –(CH2)g–[L3-(CH2)]h–R9, wherein:Li and L2are each a direct bond;R7and R8are each independently a C4-C10 alkyl;f is 0 or 1;H HW T L3 i •s se 1lecte dd f from X i X r* ’andz,R9is H or a C4-C8 alkyl;g is an integer in the range of 1-18;h is 0, 1, or 2.

[0080] According to embodiments of the formula (IB), (IIB), and (IIIB), R1is one of:Docket no: 2024-24253-P-WO

[0081] In some embodiments of the formula (IB), (IIB), and (IIIB), each R2isindependently:Docket no: 2024-24253-P-WO0Me, wherein δ1designates the bond linked to R1.

[0083] In still some embodiments of the formula (IB), (IIB), and (IIIB), E2 is selected from:linked to R2or a pharmaceutically acceptable salt thereof.Docket no: 2024-24253-P-WO

[0084] In the instant disclosure, “alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon. Representative saturated straight chain alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, or the like; while saturated branched alkyls include, but are not limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, or the like. The number of carbons may depend on the context in which the term alkyl is used. For example, in some cases, an ionizable lipid may contain from 1 to 24 carbon atoms. In some cases, a stabilizing agent may contain from 1 to 50 carbon atoms. In certain cases, alkyl may also include heteroalkyl. In some embodiments, alkyl group may be substituted or unsubstituted. In some embodiments, alkyl group may be saturated or unsaturated. In some embodiments, alkyl may include, for example, a substituted or unsubstituted C1-C50 alkyl, a saturated or unsaturated C1-C50 alkyl, a substituted or unsubstituted C1-C50 heteroalkyl, and / or linear or branched C1-C50 alkyl. In some embodiments, alkyl may include a substituted or unsubstituted C1-C40 alkyl, a saturated or unsaturated C1-C40 alkyl, a substituted or unsubstituted C1-C40 heteroalkyl, and / or linear or branched C1-C40 alkyl. In some embodiments, alkyl may include a substituted or unsubstituted C1-C30 alkyl, a saturated or unsaturated C1-C30 alkyl, a substituted or unsubstituted C1-C30 heteroalkyl, and / or linear or branched C1-C30 alkyl. In some embodiments, alkyl may include a substituted or unsubstituted C1-C20 alkyl, a saturated or unsaturated C1-C20 alkyl, a substituted or unsubstituted C1-C20 heteroalkyl, and / or linear or branched C1-C20 alkyl. In some embodiments, alkyl may include a substituted or unsubstituted C1-C15 alkyl, a saturated or unsaturated C1-C15 alkyl, a substituted or unsubstituted C1-C15 heteroalkyl, and / or linear or branched C1-C15 alkyl.

[0085] As used herein, the term “cycloalkyl group” or “cycloalkyl” is a subset of “alkyl” and may be in the form of a saturated or partially saturated cyclic group of from 3 to about 10 carbon atoms with no ring heteroatoms. Representative saturated cyclic alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like; while unsaturated cyclic alkyls include cyclopentenyl or cyclohexenyl, or the like.

[0086] “Alkenyl” means an alkyl, as defined above, which additionally contains at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers.Representative straight chain and branched alkenyls include, but are not limited to, ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-l-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, or the like.Docket no: 2024-24253-P-WO

[0087] “Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1 -pentynyl, 2-pentynyl, 3 -methyl- 1 butynyl, or the like.

[0088] The term “acyl” refers to hydrogen, alkyl, partially saturated or fully saturated cycloalkyl, partially saturated or fully saturated heterocycle, aryl, or heteroaryl substituted carbonyl groups. For example, non-limiting examples of acyl include groups such as (C1-C50)alkanoyl (e.g., formyl, acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.), (C3-C50)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl, pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl) or heteroaroyl (e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl, furanyl-3 -carbonyl, 1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl, benzo[b]thiophenyl-2-carbonyl, etc.). In some embodiments, acyl includes C1-C45 alkanoyl, C1-C40 alkanoyl, C1-C35 alkanoyl, C1-C30 alkanoyl, C1-C25 alkanoyl, C1-C20 alkanoyl, or C1-C15 alkanoyl.

[0089] The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom can be substituted. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, anthracenyl, or pyrenyl.

[0090] “Heterocycle” means a 3- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen (N), oxygen (O) and sulfur (S), and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quatemized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include, but are not limited to, heteroaryls as defined below.Heterocycles include, but are not limited to, morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, or the like.Docket no: 2024-24253-P-WO

[0091] The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom can be substituted. The heteroaryl groups herein described may also contain fused rings that share a common carboncarbon bond. The term “alkylheterocyle” refers to a heteroaryl wherein at least one of the ring atoms is substituted with alkyl, alkenyl or alkynyl.

[0092] The term “substituted” refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, oxo, thioxy, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, or aliphatic. In some embodiments, non-limiting examples of substituents include alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, amino, alkylamino, acyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid. It is understood that the substituent defined in the term “substituted” may be further substituted. Exemplary substituents include, but are not limited to, amino, alkylamino, dialkylamino, or cyclic amino compounds.

[0093] “Halogen” means fluoro-, chloro-, bromo- or iodo- substituents.

[0094] The terms “alkylamine” and “dialkylamine” refer to -NH(alkyl) and -N(alkyl)2 radicals respectively. The term "hydroxyalkyl" means -alkyl-OH radical. The term “alkylheterocycle” refers to an alkyl where at least one methylene has been replaced by a heterocycle.

[0095] Representative ionizable lipids of the instant disclosure include, but are not limited to, DODMA (l,2-dioleyloxy-3-dimethylaminopropane), DLin-MC3-DMA (O-(Z, Z, Z, Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N, N-dimethylamino)), DLin-KC2-DMA (2-dilinoleyl-4-dimethylaminoethyl- [l,3]-dioxolane), BOCHD-C3-DMA (4-(dimethylamino)-,9-(2-octylcyclopropyl)-l-[8-(2 octylcyclopropyl) octyl]nonyl ester), C12-200 (1, 1 '-[[2-[4-[2-[[2-Docket no: 2024-24253-P-WO[Z>z5(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l-piperazinyl]ethyl]imino]Z>z'5-2-dodecanol), PNI 127 ((2R,3S,4S)-2-(((l,4-dimethylpiperidine-4-carbonyl)oxy)methyl)tetrahydrofuran-3,4-diyl (9Z,9'Z, 12Z, 12'Z)-bis(octadeca-9, 12-di enoate)), PNI 516 ((Z)-3-(2-((l, 17-bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-l-yl)cyclopentyl 4-(dimethylamino)butanoate), PNI 550 (3-(2-((l,17-bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)cyclopentyl 4-(dimethylamino)butanoate), PNI 560 ((Z)-3-(2-((l, 17-bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-l-yl)cyclopentyl l,4-dimethylpiperidine-4-carboxylate), PNI 580 ((2R,3S,4S)-2-(((4-(dimethylamino)butanoyl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-hexyldecanoate)), PNI 659 ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl 4-(dimethylamino)butanoate), PNI 660 (((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)m ethyl l,4-dimethylpiperidine-4-carboxylate), PNI 714 ((Z)-l-(2-(3-(2-(l-methylpyrrolidin-3-yl)acetoxy)-2-(pent-2-en- 1 -yl)cyclopentyl)acetoxy)- 11 -(2-octylcyclopropyl)undecan-3 -yl 2-hexyldecanoate), PNI 721 ((2R,3S,4S)-2-((((2-(dimethylamino)ethyl)carbamoyl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-hexyldecanoate)), PNI 722 (2-(((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methoxy)-N, N-dimethylethan-1 -amine), PNI 723 (((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl 4-(diethylamino)butanoate), PNI 726 ((2R,3S,4S)-2-((3-(dimethylamino)propoxy)methyl)tetrahydrofuran-3,4-diyl bis(2 -hexyldecanoate)), PNI 728 (((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl (2-(dimethylamino)ethyl)carbamate), PNI 730 ((2R,3S,4S)-2-((2-(dimethylamino)ethoxy)methyl)tetrahydrofuran-3,4-diyl bis(2-hexyldecanoate)), PNI 761 ((2R,3S,4S)-2-((((3-(diethylamino)propyl)carbamoyl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-hexyldecanoate)), PNI 762 (((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl (2-(diethylamino)ethyl)carbamate), PNI 768 ((2R,3S,4S)-2-((((2-(dimethylamino)ethyl)carbamoyl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-octyldodecanoate), PNI 769 (((2R,3R,4S)-3,4-bis((2-octyldodecyl)oxy)tetrahydrofuran-2-yl)methyl (2-(dimethylamino)ethyl)carbamate), PNI 771 (((2R,3S,4S)-2-((3-(dimethylamino)propoxy)methyl)tetrahydrofuran-3,4-diyl bis(2-octyldodecanoate)), PNI 825 (((2R,3R,4S)-3,4-bis((2-octyldodecyl)oxy)tetrahydrofuran-2-yl)methyl 1,4-dimethylpiperidine-4-carboxylate), or any combinations thereof. Additional ionizable lipids that can be used in theDocket no: 2024-24253-P-WOinstant disclosure are disclosed in PCT Publication Nos. WO20252589 and W02021000041, each of which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipids include stereoisomers. In some embodiments, the ionizable lipids include mixtures of enantiomers and / or diastereomers.

[0096] The ionizable lipid may be present in the lipid nanoparticle composition or lipid nanoparticle in any suitable amount or concentration. In some embodiments, the ionizable lipid is present at a concentration of about 10 to about 90 mol% or about 20 to about 70 mol%, e.g., about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, or about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, or a concentration within a range defined by any two of the foregoing values. In some cases, the ionizable lipid is present at a concentration of more than about 10 mol%, more than about 12 mol%, more than about 14 mol%, more than about 16 mol%, more than about 18 mol%, more than about 20 mol%, more than about 22 mol%, more than about 24 mol%, more than about 26 mol%, more than about 28 mol%, or more than about 30 mol%. In some cases, the ionizable lipid is present at a concentration of less than about 90 mol%, less than about 88 mol%, less than about 86 mol%, less than about 84 mol%, less than about 82 mol%, less than about 80 mol%, less than about 78 mol%, less than about 76 mol%, less than about 74 mol%, less than about 72 mol%, less than about 70 mol%, less than about 68 mol%, less than about 66 mol%, less than about 64 mol%, less than about 62 mol%, less than about 60 mol%, less than about 58 mol%, less than about 56 mol%, less than about 54 mol%, less than about 52 mol%, or less than about 50 mol%.

[0097] In some embodiments, the lipid nanoparticle composition comprises a structural lipid. Any suitable structural lipid can be present in the lipid nanoparticle composition and lipid nanoparticle. A structural lipid supports the formation of particles during manufacture. In some embodiments, the structural lipid includes a phospholipid. In various embodiments, the structural lipid includes one or more neutrally charged, positively charged, or negatively charged molecules. In some embodiments, the structural lipid has a net negative charge. In some embodiments, the structural lipid has a net neutral charge. In some embodiments, the structural lipid has a net positive charge.Docket no: 2024-24253-P-WO

[0098] In some embodiments, the lipid nanoparticle composition or lipid nanoparticle comprise one or more structural lipids, e.g., two or more structural lipids, three or more structural lipids, or four or more structural lipids. In some embodiments, the structural lipid comprises diacylphosphatidylcholines, diacylphosphatidylethanolamines, diacylphosphatidylglycerols, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, cerebrosides, or a combination thereof. In some embodiments, the structural lipid is distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylethanolamine, palmitoyloleoylphosphatidylcholine, 1 -stearoyl -2-oleoyl-sn-gly cero-3 -phosphocholine, palmitoyloleoyl-phosphatidylethanolamine, di oleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate, dipalmitoyl phosphatidyl ethanolamine, dimyristoylphosphoethanolamine, distearoylphosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine-N-m ethyl, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N, N-dimethyl, l,2-dielaidoyl-sn-glycero-3-phosphoethanolamine, 1 -stearoyl -2-oleoyl -phosphatidy ethanol amine, 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine, distearoylphosphatidylcholine, or a combination thereof.

[0099] In some embodiments, the structural lipid comprises any suitable lipid that is negatively charged (anionic) at physiological pH. In certain embodiments, the structural lipid comprises dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, palmitoyloleyolphosphatidylglycerol, cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, monosialoganglioside GM1, or a combination thereof. In some embodiments, the structural lipid is distearoylphosphatidylcholine.

[0100] The structural lipid may be present in the lipid nanoparticle composition in any suitable amount. In some embodiments the structural lipid is present in the lipid nanoparticle composition at a concentration of about 1 to about 75 mol% or about 5 to about 60 mol%, e.g., about 1 mol%, about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, or about 70 mol%, about 75 mol%, or a concentration within a range defined by any two of the aforementioned values. In some cases, the structural lipid is present in the lipid nanoparticle composition at a concentration of less than about 1%. In some cases, the structural lipid is present in the lipid nanoparticle composition at a concentration of more than about 20%, more than about 25%, more than about 30%, more than about 35%,Docket no: 2024-24253-P-WOmore than about 40%, more than about 45%, more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, or more than about 75%.

[0101] In some embodiments, the lipid nanoparticle composition comprises a sterol. Any suitable sterol can be present in the lipid nanoparticle composition. In some embodiments, the lipid nanoparticle composition comprises one or more sterols, e.g., two or more sterols, three or more sterols, or four or more sterols. In some embodiments, the sterol includes cholesterol, betasitosterol, 20-alpha-hydroxysterol, phytosterol, or a combination thereof. In some embodiments, the sterol is cholesterol.

[0102] The sterol may be present in the lipid nanoparticle composition in any suitable amount. In some embodiments the sterol is present in the lipid nanoparticle composition a concentration of about 1 to about 75 mol% or about 5 to about 60 mol%, e.g., about 1 mol%, about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, or about 70 mol%, about 75 mol%, or a concentration within a range defined by any two of the aforementioned values. In some cases, the sterol is present at a concentration of more than about 20 mol%, more than about 25 mol %, more than about 30 mol %, more than about 35 mol %, more than about 40 mol %, more than about 45 mol %, more than about 50 mol %, more than about 55 mol %, more than about 60 mol %, more than about 65 mol %, more than about 70 mol %, or more than about 75 mol %.

[0103] In some embodiments, the lipid nanoparticle composition comprises one or more stabilizing agents, e.g., two or more stabilizing agents, three or more stabilizing agents, or four or more stabilizing agents.

[0104] In some embodiments, the stabilizing agent includes the sugar-based stabilizing agent, tridecyl β-D-maltodside (TDM), having the structure:OHHO"'*1OH

[0105] In some embodiments, the stabilizing agent includes two or more sugar-based stabilizing agents. In some embodiments, the stabilizing agent includes one, two, three, four, or five sugar-based stabilizing agents.Docket no: 2024-24253-P-WO

[0106] The stabilizing agent can have any suitable molecular weight. In some embodiments, the stabilizing agent has a molecular weight of about 200 to about 4000 Da, e.g., about 200 Da, about 300 Da, about 400 Da, about 500 Da, about 600 Da, about 700 Da, about 800 Da, about 900 Da, about 1000 Da, about 1200 Da, about 1400 Da, about 1600 Da, about 1800 Da, about 2000 Da, about 2200 Da, about 2400 Da, about 2600 Da, about 2800 Da, about 3000 Da, about 3200 Da, about 3400 Da, about 3600 Da, about 3800 Da, or about 4000 Da, or a molecular weight defined by the range of any two of the foregoing values. In some cases, the stabilizing agent has a molecular weight of less than about 200 Da. In some cases, the stabilizing agent has a molecular weight of more than about 4000 Da, about 5000 Da, about 6000 Da, about 7000 Da, about 8000 Da, or about 10000 Da.

[0107] The stabilizing agent can be present in any suitable concentration. In some cases, the stabilizing agent has a concentration from about 0.1 mol% to about 50 mol%, e.g., about 0.1 mol%, about 0.2 mol%, about 0.3 mol%, about 0.4 mol%, about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, about 1 mol%, about 1.2 mol%, about 1.4 mol%, about 1.6 mol%, about 1.8 mol%, about 2 mol%, about 2.5 mol%, about 3 mol%, about 3.5 mol%, about 4 mol%, about 4.5 mol%, about 5 mol%, about 5.5 mol%, about 6 mol%, about 6.5 mol%, about 7 mol%, about 7.5 mol%, about 8 mol%, about 8.5 mol%, about 9 mol%, about 9.5 mol%, about 10 mol%, about 10.5 mol%, about 11 mol%, about 11.5 mol%, about 12 mol%, about 12.5 mol%, about 13 mol%, about 13.5 mol%, about 14 mol%, about 14.5 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 22 mol%, about 24 mol%, about 26 mol%, about 28 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, or a concentration defined by a range of any two of the foregoing values. In some cases, the stabilizing agent has a concentration of more than about 0.02 mol%, more than about 0.04 mol%, more than about 0.06 mol%, more than about 0.08 mol%, more than about 0.1 mol%, more than about 0.2 mol%, more than about 0.3 mol%, more than about 0.4 mol%, more than about 0.5 mol%, more than about 0.6 mol%, more than about 0.7 mol%, more than about 0.8 mol%, more than about 0.9 mol%, more than about 1.0 mol%, more than about 5 mol%, more than about 10 mol%, more than about 15 mol%, more than about 20 mol%, more than about 25 mol%, more than about 30 mol%, more than about 35 mol%, more than about 40 mol%, more than about 45 mol%, or more than about 50 mol%. In some cases, the stabilizing agent has a concentration of less than about 30 mol%, less than aboutDocket no: 2024-24253-P-WO25 mol%, less than about 20 mol%, less than about 18 mol%, less than about 16 mol%, less than about 14 mol%, less than about 12 mol%, less than about 10 mol%, less than about 9 mol%, less than about 8 mol%, less than about 7 mol%, less than about 6 mol%, less than about 5 mol%, less than about 4 mol%, or less than about 3 mol%.

[0108] In some embodiments, the lipid nanoparticle composition comprises about 10 to about 90 mol% ionizable lipid, about 1 to about 75 mol% structural lipid, about 1 to about 75 mol% sterol, and about 0.1 to about 50 mol% stabilizing agent. In certain embodiments, the lipid nanoparticle composition comprises about 18 mol% ionizable lipid, about 54 mol % structural lipid, about 27 mol % sterol, and about 1 mol % stabilizing agent. In certain embodiments, the lipid nanoparticle composition comprises about 29 mol% ionizable lipid, about 50 mol% structural lipid, about 20 mol% sterol, and about 1.5 mol% stabilizing agent. In some embodiments, the lipid nanoparticle composition comprises about 30% mol ionizable lipid, about 35% mol structural lipid, about 30% sterol, and about 5% stabilizing agent. In some embodiments, the lipid nanoparticle composition comprises about 40 mol% ionizable lipid, about 12.5 mol% structural lipid, about 37.5 mol% sterol, and about 10 mol% stabilizing agent. In certain embodiments, the lipid nanoparticle composition comprises about 47.5 mol% ionizable lipid, about 12.5 mol% structural lipid, about 38.5 mol% sterol, and about 1.5 mol% stabilizing agent. In certain embodiments, the lipid nanoparticle composition comprises about 75 mol% ionizable lipid, about 19.1 mol% structural lipid, about 4.4 mol% sterol, and about 1.5 mol% stabilizing agent.

[0109] In some embodiments, the stabilizing agent includes one or more sugar-based stabilizing agents and additional one or more stabilizing agents with backbones based on structures other than a sugar-based backbone. Non-limiting examples of additional stabilizing agents with non sugar-based backbone include the ones described in PCT applications PCT / EP2024 / 075129, PCT / EP2024 / 075124, and PCT / EP2024 / 075128, each of which is incorporated herein by reference in its entirety.

[0110] In some embodiments, the lipid nanoparticle composition comprises about 10 to about 80 mol% ionizable lipid, about 20 to about 70 mol% sterol, and about 0.1 to about 10 mol% stabilizing agent. In certain embodiments, the lipid nanoparticle composition comprises about 47.5 mol% ionizable lipid, about 51 mol% sterol, and about 1.5 mol% stabilizing agent. In some embodiments, the lipid nanoparticle composition comprises about 40 mol% ionizable lipid,Docket no: 2024-24253-P-WOabout 57.5 mol% sterol, and about 2.5 mol% stabilizing agent. In some embodiments, the lipid nanoparticle composition comprises about 40 mol% ionizable lipid, about 58.5 mol% sterol, and about 1.5 mol% stabilizing agent.

[0111] In some embodiments, the lipid nanoparticle composition comprises about 10 to about 80 mol% ionizable lipid, about 20 to about 70 mol% structural lipid, and about 0.1 to about 10 mol% stabilizing agent. In certain embodiments, the lipid nanoparticle composition comprises about 47.5 mol% ionizable lipid, about 51 mol% structural lipid, and about 1.5 mol% stabilizing agent. In some embodiments, the lipid nanoparticle composition comprises about 40 mol% ionizable lipid, about 57.5 mol% structural lipid, and about 2.5 mol% stabilizing agent. In some embodiments, the lipid nanoparticle composition comprises about 40 mol% ionizable lipid, about 58.5 mol% structural lipid, and about 1.5 mol% stabilizing agent.

[0112] In some embodiments, the lipid nanoparticle composition is used in the formation of a lipid nanoparticle in embodiments of the methods described herein. In some embodiments, the diameter of the lipid nanoparticle is about 15 nm to about 500 nm, e.g., about 15 nm, about 25 nm, about 50 nm, about 75 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, or about 500 nm, or a diameter defined by a range of any two of the foregoing values. Such diameters can be useful for improving the tissue targeting and biodistribution of the lipid nanoparticles. In some cases, the diameter of the lipid nanoparticle is more than about 10 nm, more than about 15 nm, more than about 20 nm, more than about 25 nm, more than about 30 nm, more than about 35 nm, more than about 40 nm, or more than about 45 nm. In some cases, the diameter of the lipid nanoparticle is less than about 700 nm, less than about 675 nm, less than about 650 nm, less than about 625 nm, less than about 600 nm, less than about 575 nm, less than about 550 nm, less than about 525 nm, less than about 500 nm, less than about 475 nm, less than about 450 nm, less than about 425 nm, less than about 400 nm, less than about 375 nm, less than about 350 nm, less than about 325 nm, or less than about 300 nm.

[0113] Embodiments of the lipid nanoparticle described herein can have any suitable polydispersity index. In some embodiments, the lipid nanoparticle has a polydispersity index of from about 0.01 to about 0.40, e.g., about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13,Docket no: 2024-24253-P-WOabout 0.14, about 0.15 about 0.16, about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, or about 0.40, or polydispersity index defined by a range of any two of the foregoing values. Embodiments of the lipid nanoparticle described herein can have any suitable encapsulation efficiency. Encapsulation efficiency refers to the percentage of nucleic acid that is successfully entrapped into the lipid nanoparticle. In some embodiments, the lipid nanoparticle has an encapsulation efficiency from about 50% to about 100%, e.g., about 50%, about 52%, about 54%, about 56%, about 58%, about 60%, about 62%, about 64%, about 66%, about 68%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, about 94%, about 96%, about 98%, or about 100%, or an encapsulation efficiency defined by a range of any two of the foregoing values.

[0114] The disclosure also provides a method for preparing embodiments of the lipid nanoparticle as described herein comprising (a) forming the lipid nanoparticle composition by combining two or more of prescribed amounts of ionizable lipid, structural lipid, sterol, or stabilizing agent; (b) preparing the lipid nanoparticle by combining the lipid nanoparticle composition and the nucleic acid using a microfluidic mixer; and (c) optionally, purifying the lipid nanoparticle. In some embodiment, the method for preparing embodiments of the lipid nanoparticle as described herein comprising (a) forming the lipid nanoparticle composition by combining prescribed amounts of ionizable lipid, stabilizing agent, and one or more lipids including a structural lipid or a sterol; (b) preparing the lipid nanoparticle by combining the lipid nanoparticle composition and the nucleic acid using a microfluidic mixer; and (c) optionally, purifying the lipid nanoparticle.

[0115] Any suitable method of mixing can be used to combine the lipid nanoparticle composition and nucleic acid. Any suitable method can be used to combine two or more of the prescribed amounts of ionizable lipid, structural lipid, sterol, or stabilizing agent. In some embodiments, the ionizable lipid, structural lipid, sterol, and / or stabilizing agent are combined by mixing. In some embodiments, the mixing is done using a microfluidic mixer. In some embodiments, the microfluidic mixer comprises a first and second stream of reagents, which feed into the microfluidic mixer, and lipid nanoparticles are collected from the outlet. In someDocket no: 2024-24253-P-WOembodiments, the prescribed amounts of ionizable lipid, structural lipid, sterol, and / or stabilizing agent are as described herein. In some embodiments, the ionizable lipid, structural lipid, sterol, stabilizing agent, and nucleic acid are combined by standard T-tube mixing techniques, turbulent mixing, titration mixing, agitation promoting ordered self-assembly, or passive mixing of all the elements with self-assembly of elements into nanoparticles. A variety of methods have been developed to formulate lipid nanoparticles containing genetic drugs.

[0116] In some embodiments, microfluidic mixing devices, which can involve mixing two or more types of fluids together uniformly in a microfluidic chip, such as the NanoAssemblr® mixers including NanoAssemblr® Spark™, NanoAssemblr® Ignite™, NanoAssemblr® Blaze™, NanoAssemblr® GMP system, and NanoAssemblr® commercial formulation system are used. In some embodiments, the lipid nanoparticles formed by using a microfluidic mixing device has an encapsulation efficiency from about 90 to about 100%.

[0117] Any suitable method may be used to combine the sugar-based nanoparticle composition and the nucleic acid. In some embodiments, the sugar-based nanoparticle composition and the nucleic acid are combined by mixing. In some embodiments, the mixing is done using a microfluidic mixer. In some embodiments, the microfluidic mixer comprises a first and second stream of reagents, which feed into the microfluidic mixer, and sugar-based nanoparticles are collected from an outlet of the microfluidic mixer.

[0118] In some embodiments, the first stream includes a payload in a first solvent. In some embodiments, the payload may include a nucleic acid. In some cases, the payload may include a therapeutic agent. The combination of the payload in the first solvent may be described as the aqueous phase. Any suitable first solvent may be used. Suitable first solvents include solvents in which the payload is soluble and that are miscible with the second solvent. In some embodiments, the first solvent comprises aqueous buffers. In some embodiments, the aqueous buffer includes a low pH buffer. In some embodiments, the low pH buffer includes a citrate or acetate buffer.

[0119] In some embodiments, the second stream includes embodiments of the lipid nanoparticle composition as described herein in a second solvent. The combination of the lipid nanoparticle composition and the second solvent may be described as the organic phase. Any suitable second solvent may be used. Suitable second solvents include solvents in which the ionizable lipids according to embodiments of the invention are soluble, and that are miscibleDocket no: 2024-24253-P-WOwith the first solvent. In some embodiments, the second solvent comprises one or more solvents, two or more solvents, three or more solvents, or four or more solvents. In some embodiments, the second solvent includes, but is not limited to, 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, alcohols, or a combination thereof. In some embodiments, the second solvent comprises aqueous or anhydrous alcohols. In some cases, the alcohol includes a primary, secondary, or tertiary alcohol having from 1 to 12 branched or unbranched carbons (e.g., methanol, ethanol, n-propanol, isopropanol, n-butanol, 2-methyl 1 -propanol, 2-butanol, 2-methylpropan-2-ol), or a combination thereof.

[0120] In some embodiments, a suitable device for mixing includes one or more microchannels (i.e., a channel having its greatest dimension less than 1 millimeter). In some embodiments, the microchannel has a diameter from about 20 pm to about 300 pm. In some embodiments, at least one region of the microchannel has a principal flow direction and one or more surfaces having at least one groove or protrusion defined therein, the groove or protrusion having an orientation that forms an angle with the principal direction (e.g., a staggered herringbone mixer) or a bifurcating toroidal flow mixer. To achieve maximal mixing rates, it is advantageous to avoid undue fluidic resistance prior to the mixing region. In some embodiments, a device has non-microfluidic channels having dimensions greater than 1000 pm, to deliver the fluids to a single mixing channel.

[0121] Any suitable flow ratio may be used to combine the sugar-based nanoparticle composition and the nucleic acid. In some embodiments, the sugar-based nanoparticle composition and the nucleic acid are combined using a flow ratio of about 1: 1 (or 1) to about 20:1 (or 20) (aqueous phase: organic phase) by volume, e.g., about 1, about 2, about 3, about 4, about 5, about 6, or about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, or about 17, about 18, about 19, about 20, or a flow ratio defined by a range of any two of the aforementioned values. In some cases, the flow ratio is more than about 0.5. In some cases, the flow ratio is less than about 30, less than about 28, less than about 26, less than about 24, less than about 22, less than about 20, or less than about 18. Any suitable N / P ratio may be used to combine the sugar-based nanoparticle composition and the nucleic acid. The N / P ratio is the ratio of positively-charged polymer amine (N = nitrogen) groups to negatively-charged nucleic acid phosphate (P) groups. In some embodiments, the sugar-based nanoparticle composition and the nucleic acid are combined at a N / P ratio from about 2 to about 20, e.g.,Docket no: 2024-24253-P-WOabout 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20, or at an N / P ratio defined by a range of any two of the aforementioned values. In some case, the N / P ratio is more than about 1, more than about 2, more than about 3, more than about 4, more than about 5, more than about 6, more than about 7, more than about 8, or more than about 9. In some case, the N / P ratio is less than about 40, less than about 38, less than about 36, less than about 34, less than about 32, less than about 30, less than about 28, less than about 26, less than about 24, less than about 22, less than about 20, less than about 18, less than about 16, less than about 14, less than about 12, or less than about 10. Any suitable total flow rate can be used to combine the sugar-based nanoparticle composition and the nucleic acid. In some embodiments, the sugar-based nanoparticle composition and the nucleic acid are combined with a total flow rate of the organic phase and aqueous phase from about 2 to about 2600 mL / min, e.g., about 2 mL / min, about 4 mL / min, about 6 mL / min, about 8 mL / min, about 10 mL / min, about 20 mL / min, about 40 mL / min, about 60 mL / min, about 80 mL / min, or about 100 mL / min, about 120 mL / min, about 140 mL / min, about 160 mL / min, about 180 mL / min, about 200 mL / min, about 220 mL / min, about 240 mL / min, about 260 mL / min, about 280 mL / min, about 300 mL / min, about 350 mL / min, about 400 mL / min, about 450 mL / min, or about 500 mL / min, about 550 mL / min, about 600 mL / min, about 650 mL / min, about 700 mL / min, about 750 mL / min, about 800 mL / min, about 850 mL / min, or about 900 mL / min, about 950 mL / min, about 1000 mL / min, about 1100 mL / min, about 1200 mL / min, about 1300 mL / min, about 1400 mL / min, about 1500 mL / min, about 1600 mL / min, about 1700 mL / min, about 1800 mL / min, about 1900 mL / min, about 2000 mL / min, about 2100 mL / min, about 2200 mL / min, about 2300 mL / min, about 2400 mL / min, about 2500 mL / min, about 2600 mL / min, or a total flow rate defined by a range of any two of the foregoing values. In some cases, the total flow rate is more than about 1 mL / min, 2 mL / min, 4 mL / min, 6 mL / min, 8 mL / min, 10 mL / min, 20 mL / min, or 40 mL / min. In some case, the total flow rate is less than about 3000 mL / min, less than about 2800 mL / min, less than about 2600 mL / min, less than about 2400 mL / min, less than about 2200 mL / min, less than about 2100 mL / min, less than about 2000 mL / min, less than about 1800 mL / min, less than about 1600 mL / min, less than about 1500 mL / min, less than about 1400 mL / min, less than about 1200 mL / min, less than about 1000 mL / min, or less than about 800 mL / min, In some embodiments, the sugar-based nanoparticle composition and the nucleic acid are combined using a flow ratioDocket no: 2024-24253-P-WOfrom about 1:1 (or 1) to about 20: 1 (or 20) by volume (aqueous phase: organic phase) at a N / P ratio from about 2 to about 20, and a total flow rate from about 2 to about 2600 mL / min. In some embodiments, the flow rate is 3 (aqueous phase: organic phase) to optimize for a particular payload or molar ratio of lipid components.

[0122] Any suitable method of purifying the sugar-based nanoparticles may be used. In some embodiments, the purifying is done using dialysis in a buffer (e.g., PBS, pH 7), a filter or a centrifuge (e.g., Amicon™ centrifugal filters, Millipore, USA), or a tangential flow filtration system. In some embodiments, the method includes concentrating the sugar-based nanoparticles to a predetermined target dose.V. Methods of Use

[0123] In some embodiments, a payload is encapsulated by an exemplary sugar-based nanoparticle composition. Any suitable payload may be encapsulated in the sugar-based nanoparticle composition. The payload may include a nucleic acid. In some cases, the payload may include a therapeutic agent. The nucleic acid may be a substance intended to have a direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have direct effect in restoring, correcting or modifying physiological functions, or to act as a research reagent. Exemplary nucleic acids include, but are not limited to, any oligonucleotide or polynucleotide whose delivery into a cell causes a desirable effect. The nucleic acid may be single-stranded DNA or RNA, double-stranded DNA or RNA, DNA-RNA hybrids, or combinations thereof. In some embodiments, the sugar-based nanoparticle comprises one or more nucleic acids, two or more nucleic acids, three or more nucleic acids, or four or more nucleic acids. Including more than one nucleic acid may be beneficial in some embodiments e.g., gene editing). In some embodiments, the nucleic acid includes antisense oligonucleotide, a siRNA, a miRNA, a self-amplifying RNA (SAM or saRNA), a circular RNA, a self-replicating DNA, an LNA, a DNA, a replicon, an mRNA, a guide RNA, a transposon, a single gene, a vector, a plasmid, a viral particle, an AAV, a complex of RNA and RNA-binding protein, or a combination thereof. In some embodiments, the nucleic acid is an antigen encoded mRNA.

[0124] In some embodiments, the sugar-based nanoparticle composition is a therapeutic composition, such as an mRNA-based therapeutic composition. The therapeutic composition may optionally include one or more therapeutically acceptable carriers, diluents, or excipientsDocket no: 2024-24253-P-WOsuch as salts, buffering agents, preservatives, anti adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film formers or coatings, flavours, fragrances, glidants, lubricants, sorbents, suspending or dispersing agents, sweeteners, waters of hydration, and / or other therapeutic agents. As used herein, the term “excipient” means any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API) (and typically in addition to components of the delivery vehicle compositions), suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices. The disclosed compounds can be administered to a subject or patient in a therapeutically effective amount. The complexes can be administered alone or as part of a pharmaceutically acceptable composition or formulation. In addition, the compositions can be administered all at once, as for example, by a bolus injection, multiple times, or delivered substantially uniformly over a period of time. It is also noted that the dose of the compound can be varied over time.

[0125] In some embodiments, the sugar-based nanoparticle composition may encapsulate an antigen encoded mRNA and be used as a vaccine. In some embodiments, the antigen encoded mRNA is for a prophylactic or therapeutic vaccine. A vaccine may be referred to as a substance used to stimulate the production of antibodies and provide immunity against one or several diseases, prepared from the causative agent of a disease, its products, or a synthetic substitute. The vaccine may further comprise one or more immunologic adjuvants. As used herein, the term "immunologic adjuvant" refers to a compound or a mixture of compounds that acts to accelerate, prolong, enhance or modify immune responses when used in conjugation with an immunogen (e.g., neoantigens). Adjuvant may be non-immunogenic when administered to a host alone, but that augments the host's immune response to another antigen when administered conjointly with that antigen. Specifically, the terms "adjuvant" and "immunologic adjuvant" are used interchangeably in the present disclosure. Adjuvant-mediated enhancement and / or extension of the duration of the immune response can be assessed by any method currently known in the art or later developed including without limitation one or more of the following: (i) an increase in the number of antibodies produced in response to immunization with the adjuvant / antigen combination versus those produced in response to immunization with the antigen alone; (ii) an increase in the number of T cells recognizing the antigen or the adjuvant; and (iii) an increase in the level of one or more cytokines. Adjuvants may be aluminium based adjuvants including butDocket no: 2024-24253-P-WOnot limiting to aluminium hydroxide and aluminium phosphate; saponins such as steroid saponins and triterpenoid saponins; bacterial flagellin and some cytokines such as GM-CSF. Adjuvants selection may depend on antigens, vaccines, and routes of administrations.

[0126] In some aspects, adjuvants improve the adaptive immune response to a vaccine antigen by modulating innate immunity or facilitating transport and presentation. Adjuvants act directly or indirectly on antigen presenting cells (APCs) including dendritic cells (DCs).Adjuvants may be ligands for toll-like receptors (TLRs) and can directly affect DCs to alter the strength, potency, speed, duration, bias, breadth, and scope of adaptive immunity. In other instances, adjuvants may signal via proinflammatory pathways and promote immune cell infiltration, antigen presentation, and effector cell maturation. This class of adjuvants includes mineral salts, oil emulsions, nanoparticles, and polyelectrolytes and comprises colloids and molecular assemblies exhibiting complex, heterogeneous structures. In one example, the composition further comprises pidotimod as an adjuvant. In another example, the composition further comprises CpG as an adjuvant.

[0127] In some cases, the sugar-based nanoparticle composition is used in gene therapy. Gene therapy is a medical technique that produces a therapeutic effect through the manipulation of gene expression or through altering the biological properties of cells. In some cases, a gene encoding a therapeutic protein for incorporation into the host’s DNA or a mRNA encoding the therapeutic protein is administered to treat a disease, where the disease is the result of a missing protein and / or missing activity of the protein. In some cases, a new gene or mRNA is supplied, which may enhance a cell’s function without modifying the genes that cause the disease. In other cases, an antisense oligonucleotide (ASO) or small interfering RNA (siRNA) is used as a therapeutic to silence the activity of a variant protein causing a disease.

[0128] Gene therapy can be performed on a somatic cell level or a germline cell level. Gene therapy can be performed ex vivo or in vivo. Gene therapy can be employed by various gene editing techniques (e.g., CRISPR, homologous recombination, zinc finger nucleases, TALEN). In some cases, the sugar-based nanoparticle composition used in gene therapy includes the elements necessary to perform gene editing (e.g., includes a CRISPR protein or CRISPR protein encoding mRNA, sgRNA, template RNA). In some cases, the elements are provided together in the same LNP. In other cases, the elements are provided separately in one or more LNPs.Docket no: 2024-24253-P-WO

[0129] In some cases, the nucleic acid is for incorporation into an immunogenic cell. In some cases, the immunogenic cell includes a T cell, natural killer cell, dendritic cell, or tumourinfiltrating leukocyte. In some embodiments, the immunogenic cell can be engineered to express a receptor to a specific antigen or neoantigen, engineered to enhance the immunogenic response or the immunogenic cell, and engineered to decrease proteins associated with an adverse response such as neurotoxicity (e.g., reduction of cytokines to ameliorate the effects of cytokine release syndrome). In some embodiments, the sugar-based nanoparticle is in an anhydrous form. In some embodiments, the sugar-based nanoparticle is in an anhydrous form consisting of a lyophilized cake. In some embodiments, the sugar-based nanoparticle is in a reconstituted form. In a reconstituted form, a lyophilized sugar-based nanoparticle may have a pharmaceutically acceptable carrier added to the lyophilized sugar-based nanoparticle.

[0130] In some embodiments, a pharmaceutical composition includes the sugar-based nanoparticle and the pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carrier are provided herein.

[0131] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.EXAMPLESExample 1

[0132] Components of the lipid nanoparticle (LNP) composition including ionizable lipid, one or more lipids, and stabilizing agents were mixed in predetermined molar ratios. Lipid nanoparticle compositions were prepared in an organic solvent (e.g., ethanol) by combining prescribed amounts of individual LNP components. The LNP components can be combined from individual component stocks in the organic solvent or combined by adding individual components in powder to the organic solvent. Lipid nanoparticles (LNPs), also referred to as LNP formulations, were then prepared by running the lipid nanoparticle composition and nucleicDocket no: 2024-24253-P-WOacid through a mixer. Any mixer currently known or future developed may be used for mixing the lipid nanoparticle composition and nucleic acid. Non-limiting examples of the mixer include NanoAssemblr® microfluidic mixers (Cytiva, Marlborough, MA).

[0133] In this non-limiting example, the mixing of nucleic acids and lipid nanoparticle compositions occurred as follows. Ionizable lipids, stabilizing agent, and one or both of structural lipid and sterol were mixed in 100% ethanol at a molar ratio according to various lipid nanoparticle compositions (e.g., lipid nanoparticle compositions listed in Table 1). The aqueous phase was prepared by diluting nucleic acids in a sodium acetate buffer (pH 4). The solutions were combined using the NanoAssemblr® Ignite™ with an Ignite™ NxGen (DVBM) cartridge (Cytiva, Marlborough, MA) at a predetermined flow ratio of (aqueous phase: organic phase). The resulting LNP formulations were diluted with lx PBS (pH 7.4) and the mixture was subjected to an optional step of downstream processing. Downstream processing included ethanol removal through dialysis in PBS (pH 7), or using Amicon™ centrifugal filters (Millipore, USA) at 2500 RPM, or using tangential flow filtration systems. The processed LNP formulations were mixed with a proper cryopreservation buffer and kept at -80 °C until further use.Table 1. Exemplary Lipid Nanoparticle (LNP) CompositionsLNP Description of the LNP compositionComposition #1 10 mol% iL / 18.5 mol% structural lipid / 70 mol% sterol / 1.5 mol%stabilizer2 18 mol% iL / 54 mol % structural lipid / 27 mol % sterol / I mol %stabilizer3 24.66 mol% iL / 36.99 mol % structural lipid / 36.99 mol % sterol / 1.37mol % stabilizer4 29 mol% iL / 50 mol% structural lipid / 20 mol% sterol / 1.5 mol%stabilizer5 39.1 mol% iL / 58.7 mol % structural lipid / 0 mol % sterol / 2.2 mol %stabilizer6 40 mol % iL / 20 mol% structural lipid / 37.5 mol% sterol / 2.5 mol%stabilizer7 40 mol % iL / 20 mol% structural lipid / 39.25 mol% sterol / 0.75 mol %stabilizer8 40 mol % iL / 12.5 mol% structural lipid / 46 mol% sterol / 1.5 mol%stabilizer9 40 mol % iL / 12.5 mol% structural lipid / 44.5 mol% sterol / 3 mol%stabilizer10 40 mol % iL / 12.5 mol% structural lipid / 42.5 mol% sterol / 5 mol%stabilizerDocket no: 2024-24253-P-WO11 40 mol % iL / 12.5 mol% structural lipid / 37.5 mol% sterol / 10 mol%stabilizer12 40 mol % iL / 12.5 mol% structural lipid / 46 mol% sterol / 1.5 mol%stabilizer13 40.0 mol% iL / 25 mol% structural lipid 1 / 12.5 mol% structural lipid 2 / 21 mol% sterol / 1.5 mol% stabilizer14 42mol% IL / 56.5 mol% structural lipid / 0 mol% sterol / 1.5 mol%stabilizer15 47.5 mol% iL / 12.5 mol% structural lipid / 38.5 mol% sterol / 1.5 mol%stabilizer16 54 mol % iL / 10 mol% structural lipid / 35 mol% sterol / 1.0 mol%stabilizer17 60.6 mol % iL / 0 mol% structural lipid / 37.9 mol% sterol / 1.5 mol%stabilizer18 75 mol% iL / 19.1 mol% structural lipid / 4.4 mol% sterol / 1.5 mol%stabilizer*iL = ionizable lipidExample 2

[0134] Size and PDI of the LNPs was measured by Dynamic Light Scattering (DLS) using a ZetaSizer™ Nano ZS™ (Malvern Instruments). He / Ne laser of 633 nm wavelength was used as the light source. Data were measured from the scattered intensity data conducted in backscattering detection mode (measurement angle = 173°). Measurements were an average of 10 runs of two cycles each per sample. Z - Average size was reported as the particle size and is defined as the harmonic intensity averaged particle diameter. Encapsulation efficiency (EE) of the LNPs was measured by Quant-iT™ RiboGreen® RNA reagent. Non-limiting examples of critical quality attributes of LNPs formulated based on different stabilizers are illustrated in Table 2 and Table 3. It is to be understood that while results of specific LNP compositions are listed in Table 2 and Table 3, other LNP compositions, for example, ones listed in Table 1, could also work with the stabilizing agents. As illustrated in Table 2 and Table 3, increasing stabilizer concentration up to 10% retained CQAs in the desired range while LNPs formulated with PEG-DMG mol% higher than 5% showed poor CQAs (e.g., decreased EE).Table 2. Exemplary Lipid Nanoparticle (LNP) CompositionsMW of SizeLipid composition Payload PDI EE stabilizing (nm)ag (%)ent (Da)Docket no: 2024-24253-P-WO40% PNI 516 / 12.5% DSPC / 46% Choi / 1.5%524 EPO 125.1 0.073 99.7 Tridecyl β-D- maltoside (TDM)mRNA40% PNI 516 / 12.5% DSPC / 44.5% Choi / 3% 2509 EPO 57.4 0.136 97.8 PEG-DMG mRNA40% PNI 516 / 12.5% DSPC / 44.5% Choi / 3%524 EPO 115.1 0.082 99.3 Tridecyl β-D- maltosidemRNA40% PNI 516 / 12.5% DSPC / 42.5% Choi / 5% 2509 EPO 54.2 0.195 92.5 PEG-DMG mRNA40% PNI 516 / 12.5% DSPC / 42.5% Choi / 5%524 EPO 118.3 0.225 98.8 Tridecyl β-D- maltosidemRNA40% PNI 516 / 12.5% DSPC / 37.5% Choi / 524 EPO 95.9 0.091 99.8 10% Tridecyl β- D-maltosidemRNA40% PNI 516 / 12.5% DSPC / 42.5% Cholesterol2509 Flue mRNA 62.8 0.18 86.5 / 5% PEG- DMG40% PNI 516 / 12.5% DSPC / 42.5%524 Flue mRNA 107.9 0.25 98.9 Cholesterol / 5% Tridecyl β-D-maltoside40% PNI 516 / 12.5% DSPC / 37.5% Cholesterol2509 Flue mRNA 45.8 0.25 43.2 / 10% PEG- DMG40% PNI 516 / 12.5% DSPC / 37.5%524 Flue mRNA 85.4 0.26 98.2Cholesterol / 10% Tridecyl β-D-maltoseTable 3Stabilizer MW of Lipid composition Payload Size PDI EE stabiliz Rep Re Repl Rep2 Repl Rep2 er (Da) 1 p2P48 525 40% PNI550, 20% GFP 92 91 0.168 0.169 98.58 98.54DSPC, 37.5% Choi, mRNA2.5% TDM [37.5 mM]P48 525 40% PNI728, 12.5% GFP 101 101 0.154 0.037 99.43 99.17DSPC, 46% Choi, 1.5% mRNATDM [25 mM]P48 525 28.7% PNI728, 49.8% GFP 101 102 0.175 0.193 89.92 88.77DSPC, 20% Choi, 1.5% mRNATDM [25 mM]P48 525 40% PNI550, 20% GFP 103 103 0.244 0.251 97.89 98.06DSPC, 35% Choi, 5% mRNATDM [37.5 mM]P48 525 40% PNI728, 12.5% GFP 82 79 0.043 0.048 98.93 99.03DSPC, 42.5% Choi, 5% mRNATDM [25 mM]P48 525 28.7% PNI728, 49.8% GFP 98 97 0.188 0.187 89.1 87.65DSPC, 16.5% Choi, 5% mRNATDM [25 mM]Docket no: 2024-24253-P-WOP48 525 40% PNI728, 12.5% Flue 319 323 0.186 0.092 97.07 97.03DSPC, 46% Choi, 1.5% mRNATDM [25 mM]P48 525 40% PNI769, 12.5% TTR 142 148 0.108 0.104 98.5 98.7 DSPC, 37.5% Choi, gRNA / C10% TDM [25 mM] as9mRNAP48 525 40% PNI769, 12.5% TTR 144 140 0.286 0.301 95.9 97.5 DSPC, 32.5% Choi, gRNA / C15% TDM [25 mM] as9mRNAP102 496 40% PNI728, 12.5% Flue 207 208 0.061 0.043 77 77.3 DSPC, 35% Choi, 10% mRNATDM [25 mM]P48 525 40% PNI516, 12.5% EPO 139. 141 0.085 0.054 99.74 99.74DSPC, 45% Choi, 2.5% mRNA 5.1TDMP48 525 40% PNI516, 12.5% EPO 83.9 83. 0.066 0.077 99.24 99.24DSPC, 37.5% Choi, mRNA 1310% TDMExample 3

[0135] FIG. 1 showed the in vitro potency in HEK cells of LNPs formulated with the sugar-based stabilizing agent, TDM compared to PEG-DMG. The general LNP composition used was 40% PNI 516 / 12.5% DSPC / (47.5)- x% Chol / x% stabilizer, where x is the mol% of the stabilizer used. There was a downward trend for LNP with PEG-DMG stabilizer when the mol% of PEG-DMG was increased from 5 mol% to 10 mol%. Surprisingly, a significant upward trend of luciferase expression was observed for LNP with a TDM stabilizer. TDM outperformed PEG-DMG at concentrations of 5% and > 5% using the same lipid composition in vitro. (UT = untreated cells).Example 4

[0136] This study describes the procedure used for the in vivo erythropoietin (EPO) expression evaluation of LNP encapsulating EPO-encoded mRNA. LNPs were intravenously injected into mice (6-week-old female C57 BL6 mice) at a single dose of 0.25 mg / kg. The sera samples were collected 6 h and 24 h post-injection via the tail nick method. For serum preparation, after collection of the whole blood, the blood was allowed to clot by leaving the collection tube at room temperature for 15-30 minutes. The clot was removed by centrifuging the tubes at 1000-2000 x g for 10 min at 4 °C. The clear golden-yellow color supernatant was carefully removed and transferred to sterile a screw-capped clear polypropylene tube on ice. TheDocket no: 2024-24253-P-WOserum was then stored at -80 °C until further use. The terminal blood collection was performed 24 h post injection. The Erythropoietin (EPO) protein level in sera samples was determined using the Ella kit (ProteinSimple, Catalog # SPCKB-PS-000487).

[0137] FIG. 2 showed hEPO expression levels (6 hr and 24 hr post administration) in C57BL / 6 mice following IV administration of 0.25 mg / Kg dose of recombinant human EPO-encoded mRNA-LNPs using the lipid composition: 40% PNI 516 / 12.5% DSPC / (47.5)-x% Chol / x% stabilizer, where x is the mol% of the stabilizer used. There was a downward trend for LNP including PEG-DMG stabilizer when the mol% of PEG-DMG is increased from 1.5 mol% to 10 mol%. Surprisingly, a significant upward trend of EPO protein production was observed with LNP including TDM stabilizer. In addition, TDM stabilizers outperformed PEG-DMG at high concentration (10%) using the same lipid composition in vivo.Example 5

[0138] FIGS. 3A-3C showed enhanced expression of eGFP when LNPs comprising TDM was used as a single agent with LNPs in human primary T cells. The Ionizable lipid used was PNI550. In this experiment TDM has been used as a single agent stabilizer or a mixture of components of stabilizers (Tween 20 / Tween80 / TDM at 0.5 / 0.5 / 1.5 mol%). The compositions tested in this study include TDM LNP (40 mol% PNI550, 20 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% TDM), 0.5%PEGLNP (40 mol% PNI550, 20 mol% DSPC, 39.5 mol% cholesterol and 0.5 mol% PEG-DMG), 1.5%PEG LNP (47.5 mol% PNI550, 20 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% PEG-DMG) and Triple LNP (40 mol% PNI550, 20 mol% DSPC, 37 mol% cholesterol, 0.5 mol% Tween 20, 0.5 mol% Tween80 and 1.5 mol% TDM). FIG. 3A showed the percent of cells expressing eGFP. FIG. 3B showed the mean fluorescence intensity (MFI) produced by GFP (normalized to untreated cells (UT). FIG. 3C showed the viability of human primary T cells after treatment with the LNPs (normalized to UT).Example 6

[0139] FIGS. 4A and 4B showed LNP -mediated CD45 targeted CRISPR-Cas9 gene editing of HSCs. HSCs were treated with 6.4 ug RNA dose (1: 1 wt ratio of Cas9 mRNA and CD45 targeted sgRNA). FIG.4A showed a knock down of CD45 using LNPs comprising TDM and Brij S10 (Polyoxyethylene (10) stearyl ether) measured using flow cytometry via CD45 surfaceDocket no: 2024-24253-P-WOexpression analysis. FIG. 4B showed cell viability. PNI550 was used as the ionizable lipid. The general LNP composition used was 40% PNI 550, 20% DSPC, (30-x)% Choi, x% stabilizer, where x is the mol% of the stabilizer used.Example 7

[0140] Cells (BHK and Jurkat) were cultured and seeded (96 well-plate, 0.1 mL / well, 12,000 cell / well). Afterwards, cells were treated with the GFP mRNA loaded LNPs for 24 h followed by analysis by fluorescence microscopy (BHK) and flow cytometry (Jurkat).

[0141] FIGS. 5A and 5B are plots illustrating the in vitro potency of LNPs (lipid composition: 40% PNI728, 12.5% DSPC, (47.5-x)% Choi, x% TDM), using a payload of enhanced green fluorescent protein (eGFP) mRNA) in Jurkat cells. FIG. 5 A shows the percent of cells expressing GFP. FIG. 5B shows the MFI of GFP in Jurkat cells.

[0142] FIGS. 6A and 6B are plots illustrating the in vitro potency of LNPs (lipid composition: 40% PNI728, 12.5% DSPC, 42.5% Choi, 5% TDM), using a payload of enhanced green fluorescent protein (eGFP) mRNA) in BHK cells. FIG. 6 A shows the percent of cells expressing GFP. FIG. 6B shows the MFI of GFP in BHK cells.Example 8

[0143] Male C57BL / 6 mice (6-8 weeks; n = 100, 4 / group across TA1-TA25) received single intramuscular injections of ready -to-inject RNA-LNP formulations (2.5 g RNA in 50 pL) on Day 0. Tail-nick blood (~75 pL) was collected at 24 h into serum tubes, allowed to clot (15-30 min, RT), centrifuged (1,000–2,000 × g, 10 min, 4 °C), and the supernatant transferred to labeled polypropylene tubes and stored at £ -70 °C; hemolyzed samples were excluded.Following the 24 h collection, animals were euthanized and briefly necropsied. Serum erythropoietin (EPO) was quantified on the Ella Simple Plex instrument using an EPO cartridge per manufacturer instructions: thawed serum was diluted to fit the calibration range, run with calibrators and quality controls, concentrations calculated via a four-parameter logistic standard curve, and reported as the mean of valid technical replicates (pg / mL). The study was conducted under institutional SOPs in an AAAL AC -accredited vivarium.

[0144] FIG. 7 is a dot plot illustrating hEPO expression levels (24 hr post administration) in C57BL / 6 mice following intramuscular (IM) administration of 0.25 mg / Kg dose of recombinantDocket no: 2024-24253-P-WOhuman EPO-encoded mRNA-LNPs using the lipid composition; 40% PNI516, 12.5% DSPC, (47.5-x)% cholesterol, x% stabilizer. The stabilizers tested include PEG-DMG and TDM. It was clearly shown that increasing TDM mol% from 2.5% to 10% significantly improved EPO protein expression.

[0145] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0146] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Docket no: 2024-24253-P-WOClaims:

1. A stabilizing agent comprising a structure of formula (I):X R2wherein Y is a substituted or unsubstituted monosaccharide, a substituted or unsubstituted disaccharide, a substituted or unsubstituted trisaccharide, a substituted or unsubstituted oligosaccharide, a substituted or unsubstituted galactosamine, a substituted or unsubstituted aminoglycoside, or a derivative thereof; X is null (when Y is directly attached to Ri), a methyl (CH2), an O (ether), a NH (amine), a S (sulfide), a carbonyl (C=O), ester (O-C=O), carbamate, triazole, or a disulfide (S-S); and Ri and R2 are each individually and independently a H, a sterol, a cholesterol, a tocopherol, a substituted or unsubstituted C1-50 alkyl, a saturated or unsaturated Ci-50 alkyl, a substituted or unsubstituted C1-50 acyl, and / or a linear or branched C1-50 alkyl.

2. The stabilizing agent of claim 1, wherein the monosaccharide comprises a galactose, glucose, ribose, mannose, xylose, or fructose.

3. The stabilizing agent of claim 1, wherein the disaccharide is a sucrose, maltose, trehalose, cellobiose, or lactulose.

4. The stabilizing agent of any one of claims 1 to 3, wherein X is O, R2 is H, and Ri is saturated or unsaturated C3-C15 alkyl and / or a linear or branched C3-C15 alkyl.

5. The stabilizing agent of any one of claims 1 to 4, wherein the stabilizing agent is used for stabilizing a lipid nanoparticle or liposome.

6. A lipid nanoparticle composition comprising: (a) an ionizable lipid; (b) one or more lipids; (c) and the stabilizing agent of any one of claims 1 to 5.

7. The lipid nanoparticle composition of claim 6, wherein the one or more lipids comprises a structural lipid, a sterol, or a combination thereof.Docket no: 2024-24253-P-WO8. The lipid nanoparticle composition of claim 6 consisting essentially of: (a) an ionizable lipid; (b) two lipids; (c) and the stabilizing agent of any one of claims 1 to 5.

9. The lipid nanoparticle composition of claim 6 consisting essentially of: (a) an ionizable lipid; (b) a sterol; and (c) and the stabilizing agent of any one of claims 1 to 5.

10. The lipid nanoparticle composition of claim 6 consisting essentially of: (a) an ionizable lipid; (b) a structural lipid; and (c) and the stabilizing agent of any one of claims 1 to 5.

11. The lipid nanoparticle composition of any one of claims 6 to 10, wherein the lipid nanoparticle composition is substantially free of PEG or PEG-R, wherein R is any atom or molecule covalently attached to PEG.

12. The lipid nanoparticle composition of any one of claims 6 to 8, wherein the structural lipid is neutrally charged, positively charged, or negatively charged.

13. The lipid nanoparticle composition of any one of claims 6 to 12, wherein the ionizable lipid is DODMA, DLin-MC3-DMA, DLin-KC2-DMA, BOCHD-C3-DMA, C 12-200, PNI 127, PNI 516, PNI 550, PNI 580, PNI 659, PNI 660, PNI 714, PNI 721, PNI 723, PNI 728, PNI 730, PNI 761, PNI 762, PNI 769, PNI 771, or a combination thereof.

14. The lipid nanoparticle composition of any one of claims 6 to 12, wherein the ionizable lipid includes a cyclopentyl or a tetrahydrofuranyl head group or scaffold.

15. The lipid nanoparticle composition of claim 14, wherein the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure including a cyclopentyl scaffold, according to the formula (IA):wherein: Li is a direct bond or C1-C5 alkylene;Docket no: 2024-24253-P-WOE1 is –O–, –OC(O)O–, –OC(O)–δ1, –OC(O)N(Q)–δ1, –OC(O)S–δ1, –N(Q)C(O)–δ1, –N(Q)C(O)O–δ1, –C(O)O–δ1, or –C(O)N(Q)–δ1; Q is H or C1-C5 alkyl; δ1designates the bond linked to R1;R1is selected from:and wherein;R4and R5are each independently Ci-Ce alkyl, C2-C6 alkenyl or C2-C6 alkynyl; alternatively R4and R5may join to form 4-6 membered heterocyclic ring containing oxygen (O) or up to 2 nitrogen (N),optionally substituted with 1 or 2 substituents each independently a Ci-Ce alkyl, cyclopropyl, OH, or a C1-C3 alkoxy;R6is Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl or a 2-hydroxy ethyl;R7is H, Ci-Ce alkyl, C2-C6 alkenyl, or a C2-C6 alkynyl;a and c’ are independently 1, 2, 3, 4, or 5;b, c and e are independently 0, 1, or 2;d is 1 or 2;R2is H, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, orL2is a direct bond or δ2–(CR8R8')k–δ3wherein R8and R8'are each independently H, C1-C12alkyl, C2-C12alkenyl or C2-C12alkynyl; δ2designates the bond linked to E2, and δ3designates the bond linked to the cyclopentyl scaffold described in formula (IA);k is 1, 2, 3, 4, or 5;E2is O, OC(O)O, OC(O) δ4, OC(O)N(Q) δ4, N(Q)C(O) δ4, N(Q)C(O)O δ4, C(O)N(Q) δ4or C(O)O δ4; Q is H or C1-C5alkyl; where δ4designates the bond linked to R3;R3is C8-C20 alkyl, C8-C20 alkenyl, C8-C20 alkynyl,wherein: f is 0 or 1;Docket no: 2024-24253-P-WOg is 1 or 2;g’ is 1, 2, 3, 4, or 5;h is 0, 1, 2, 3 or 4;R9is a C6-C20chain having the formula–(CH2)i[L4-(CH2)]jR12, wherein:L4is selected fromi is an integer in the range 6-20;j is 0, 1, 2, or 3;R12is H or C4-C8 alkyl;R9’ is H, C4-C10 alkyl, C4-C10 alkenyl, or C4-C10 alkynyl;R10and RIO’ are each independently C4-C10 alkyl, C4-C10 alkenyl or C4-C10 alkynyl;L3 is OC(O) 55, O S5, or a direct bond; S5designates the bond linked to R10and R10’; and R11= R9, or has the formula:

16. The lipid nanoparticle composition of claim 14, wherein the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IIA):(HA)wherein: Li is a direct bond;Ei is OC(O)O, OC(O) 61, OC(O)N(Q) 61, or OC(O)S 51; Q is H or C1-C5 alkyl; δ1designates the bond linked to R1;R1is selected fromDocket no: 2024-24253-P-WOR5N-R4Me'awherein:R4and R5are each independently Ci-Ce alkyl; alternatively R4and R5may join to form 5-6 membered heterocyclic ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents each independently a Ci-Ce alkyl;R6is Ci-Ce alkyl or cyclopropyl;R7is H or Ci-Ce alkyl;a is 1, 2, or 3;b is 0 or 1;c is 0, 1, or 2;c’ is 2, 3, or 4;d is 2;e is 1;MeR2is H, C1-C5 alkyl, C2-C5 alkenyl, or;R3is selected from:wherein: f and h are each 0;g is 1 or 2;R9is a C6-C20 chain having the formula –(CH2)i–[L4-(CH2)]j–R12, wherein:L4is selected from, and ;i is an integer in the range 6-20;j is 0, 1, or 2;R12is H or C4-C8 alkyl;R9is H or C4-C10 alkyl;R10and R10'are each independently C4-C10 alkyl;L3 is a direct bond;Docket no: 2024-24253-P-WOR13is the same as R11.

17. The lipid nanoparticle composition of claim 14, wherein the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IIIA)(IIIA)wherein: R1is selected from:b dR' e Nwherein:R4and R5are each independently Ci-Ce alkyl; alternatively, R4and R5may join to form 5-6 membered heterocyclic ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents each independently a Ci-Ce alkyl;R6is Ci-Ce alkyl or cyclopropyl;R7is H or Ci-Ce alkyl;a is 1, 2, or 3;b is 0 or 1;c is 0, 1, or 2;c’ is 2, 3, or 4;d is 2;e is 1;R2is H, C1-C5 alkyl, C2-C5 alkenyl orR3is selected from:Docket no: 2024-24253-P-WOf and h are 0;g is 1 or 2;R9is a C6-C20chain having the formula –(CH2)i–[L4-(CH2)]j–R12, wherein:L4 is selected fromi is an integer in the range 6-20;j is 0, 1, or 2;R12is H or C4-C8 alkyl;R9is H or C4-C10 alkyl;R10and R10'are each independently C4-C10 alkyl;L3 is a direct bond;R11is the same as R9.

18. The lipid nanoparticle composition of claim 14, wherein the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IB):wherein p is 0 or 1;E1 is –O–δ1, –OC(O)O–δ1, –OC(O)–δ1, –OC(O)N(Q)–δ1, –OC(O)S–δ1, –C(O)N(Q)–δ1, –C(O)O–δ1, –N(Q)C(O)–δ1, –N(Q)C(O)O–δ1, –N(Q)C(O)S–δ1, or –N(Q)C(O)N(Q)–δ1; wherein Q is H or C1-C5 alkyl; δ1designates the bond linked to R1; R1is selected from:Docket no: 2024-24253-P-WOwherein: R3and R4are each independently Ci-Ce alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; alternatively R3and R4may join to form 4-6 membered ring containing oxygen (O) or up to 2 nitrogen (N), optionally substituted with 1-2 substituents, each independently a Ci-Ce alkyl, cyclopropyl, OH, or a C1-C3 alkoxy;R5is Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, or a 2-hydroxyethyl;R6is H or Ci-Ce alkyl;a is 1, 2, 3, 4 or 5;b and c are independently 0, 1, or 2;c’ is 1, 2, 3, 4, or 5;d is 1 or 2;e is 0, 1, or 2;E2is -OC(O)-52, -OC(O)O-52, -OC(O)N(Q)-52, -O-52, -OCH2CH2O-δ2, or -OC(O)(CH2)6C(O)O-52; Q is H or C1-C5 alkyl; 52designates the bond linked to R2;R2isf L2 r8or has the formula (CH2)g[L3-(CH2)]h R9, wherein:L1 and L2 are each independently a direct bond, –O–δ3, –CH2OC(O)–δ3, or –CH2O–δ3; δ3designates the bond linked to R7and R8;R7and R8are each independently C4-C10 alkyl, C4-C10 alkenyl or C4-C10 alkynyl;f is 0, 1, 2, 3, 4, or 5;L3 is selected fromR9is H or C4-C8 alkyl;g is an integer in the range of 1-18;h is 0, 1, 2, or 3.

19. The lipid nanoparticle composition of claim 14, wherein the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IIB):Docket no: 2024-24253-P-WO(IIB)wherein E1 is –OC(O)O–δ1, –OC(O)–δ1, –OC(O)N(Q)–δ1, or –OC(O)S–δ1; Q is H or C1-C5 alkyl; and δ1designates the bond linked to R1;R1is selected from:R4N-R3'wherein:R3and R4are each independently a Ci-Ce alkyl; alternatively R3and R4may join to form 5-6 membered ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents selected from a Ci-Ce alkyl;R5is a Ci-Ce alkyl or C3-C6 cycloalkyl;R6is an H or Ci-Ce alkyl;a is 1, 2, 3, or 4;b and c are independently 0, 1, or 2;c’ is 2, 3, or 4;d is 2;e is 0 or 1;E2is –O–δ2, –OC(O)–δ2, –OCH2CH2O–δ2, or –OC(O)(CH2)6C(O)O–δ2; where δ2designates the bond linked to R2;R2isf L2~R8or has the formula (CH2)g[L3-(CH2)]h R9, wherein:L1 and L2are each independently a direct bond, –O–δ3, –CH2OC(O)–δ3, or –CH2O–δ3; δ3designates the bond linked to R7and R8;R7and R8are each independently C4-C10 alkyl, C4-C10 alkenyl or C4-C10 alkynyl;f is 0, 1, 2, 3, 4, or 5;H HandL3 is selected fromDocket no: 2024-24253-P-WOR9is H or a C4-C8 alkyl;g is an integer in the range of 1-18;h is 0, 1, or 2.

20. The lipid nanoparticle composition of claim 14, wherein the ionizable lipid or a pharmaceutically acceptable salt thereof has the general structure according to the formula (IIIB):R2–E2E2–R2(IIIB) R1is selected from:R3and R4are each independently a Ci-Ce alkyl group; alternatively R3and R4may join to form 5-6 membered ring containing up to 2 nitrogen (N), optionally substituted with 1-2 substituents selected from a Ci-Ce alkyl;R5is Ci-Ce alkyl or cyclopropyl;R6is H or Ci-Ce alkyl;a is 1, 2, 3, or 4;b is 0 or 1;c is 0, 1, or 2;c’ is 2, 3, or 4;d is 2;e is 1;E2is –O–δ2, –OC(O)–δ2, –OCH2CH2O–δ2, or –OC(O)(CH2)6C(O)O–δ2; where δ2designates the bond linked to R2;Docket no: 2024-24253-P-WO>^<L1'R7R2isfL2~R8or has the formula –(CH2)g–[L3-(CH2)]h–R9, wherein:Li and L2 are each a direct bond;R7and R8are each independently a C4-C10 alkyl;f is 0 or 1;L3 is selected fromR9is H or a C4-C8 alkyl;g is an integer in the range of 1-18;h is 0, 1, or 2.

21. The lipid nanoparticle composition of any one of claims 6 to 8, wherein the structural lipid comprises diacylphosphatidylcholines, diacylphosphatidylethanolamines, diacylphosphatidylglycerols, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, cerebrosides, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoyl-phosphatidylethanolamine, palmitoyloleoylphosphatidylcholine, l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, palmitoyloleoyl-phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate, dipalmitoyl phosphatidyl ethanolamine, dimyristoylphosphoethanolamine, distearoyl-phosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-methyl, l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N, N-dimethyl, l,2-dielaidoyl-sn-glycero-3 -phosphoethanolamine, 1 -stearoyl -2-oleoyl-phosphatidy ethanol amine, l,2-dielaidoyl-sn-glycero-3-phophoethanolamine, distearoylphosphatidylcholine, dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, palmitoyloleyolphosphatidylglycerol, cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, monosialoganglioside GM1, or a combination thereof.Docket no: 2024-24253-P-WO22. The lipid nanoparticle composition of any one of claims 6 to 9, wherein the sterol comprises cholesterol, beta-sitosterol, 20-alpha-hydroxysterol, phytosterol, or a combination thereof.

23. The lipid nanoparticle composition of any one of claims 6 to 22, wherein the stabilizing agent has a molecular weight of about 500 Da to about 50,000 Da.

24. The lipid nanoparticle composition of any one of claims 6 to 23, further comprising a second stabilizing agent, wherein the second stabilizing agent is a polysorbate, N-dodecyl beta-D-maltoside, D-a- Tocopherol polyethylene glycol 1000 succinate, or a combination thereof.

25. The lipid nanoparticle composition of any one of claims 6 to 24, wherein the lipid nanoparticle composition comprises about 10 to about 90 mol% ionizable lipid, about 1 to about 75 mol% structural lipid, about 1 to about 75 mol% sterol, and about 0.1 to about 50 mol% stabilizing agent.

26. A lipid nanoparticle comprising the lipid nanoparticle composition of any one of claims 6 to 25 and a nucleic acid.

27. The lipid nanoparticle of claim 26, wherein the nucleic acid is encapsulated by the lipid nanoparticle composition.

28. The lipid nanoparticle of claim 26 or 27, wherein the nucleic acid is an antisense oligonucleotide, a siRNA, a miRNA, a self-amplifying RNA (SAM or saRNA), a self-replicating DNA, an LNA, a DNA, a replicon, an mRNA, a circRNA, a guide RNA, a transposon, a single gene, a vector, a plasmid, a viral particle, an AAV, a complex of RNA and RNA-binding protein, or a combination thereof.

29. The lipid nanoparticle of any one of claims 26 to 28, wherein the nucleic acid is an antigen encoded mRNA for prophylactic or therapeutic vaccine, a nucleic acid for gene therapy, or a nucleic acid for immunogenic cell incorporation, wherein the immunogenic cell is a T cell.Docket no: 2024-24253-P-WO30. The lipid nanoparticle of any one of claims 6 to 29, wherein the diameter of the lipid nanoparticle is about 15 nm to about 500 nm.

31. The lipid nanoparticle of any one of claims 6 to 30, wherein the lipid nanoparticle has a polydispersity index of about 0.01 to about 0.40.

32. The lipid nanoparticle of any one of claims 6 to 31, wherein the lipid nanoparticle has an encapsulation efficiency of about 50% to about 100%.

33. A pharmaceutical composition comprising the lipid nanoparticle composition of any one of claims 6 to 32 and a pharmaceutically acceptable carrier.

34. A method for preparing the lipid nanoparticle of any one of claims 22 to 28 or the pharmaceutical composition of claim 29, the method comprising:(i) forming the lipid nanoparticle composition by combining the ionizable lipid, the stabilizing agent, and one or more lipids including the structural lipid or the sterol;(ii) preparing the lipid nanoparticle by combining an organic phase including the lipid nanoparticle composition and an aqueous phase including the nucleic acid using a microfluidic mixer; and(iii) optionally purifying the lipid nanoparticle.

35. The method of claim 34, wherein the lipid nanoparticle composition and the nucleic acid are combined using a flow ratio of about 1: 1 to about 10:1 by volume (aqueous phase: organic phase) at a N / P ratio of about 2 to about 25, and a combined flow rate of the aqueous phase and the organic phase is about 2 to about 2600 mL / min.

36. The method of claim 34 or 35, wherein the aqueous phase comprises a low pH buffer.

37. The method of any one of claims 34 to 36, wherein the aqueous phase comprises a citrate or acetate buffer.Docket no: 2024-24253-P-WO38. The method of any one of claims 34 to 37, wherein the organic phase comprises 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, alcohols, or a combination thereof.

39. The method of any one of claims 34 to 38, wherein the organic phase includes an alcohol and the alcohol comprises aqueous or anhydrous alcohol, wherein the alcohol includes a primary, secondary, or tertiary alcohol having from 1 to 12 branched or unbranched carbons, or a combination thereof.

40. Use of the lipid nanoparticle of any one of claims 26 to 32 or the pharmaceutical composition of claim 33 for preventing, treating, or ameliorating conditions or diseases including administering the lipid nanoparticle as a vaccine or as a treatment to prevent or reduce the severity of a contagion, administering the lipid nanoparticle as a gene therapeutic, or administering the lipid nanoparticle to an immunogenic cell for the treatment of cancer or an infection.

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