Lipid nanoparticles having non-polar lipids for extrahepatic nucleic acid delivery

Abstract

The present disclosure provides a lipid nanoparticle comprising: nucleic acid cargo; ionizable lipid; non-polar, neutral lipid, such as a phospholipid; non-polar lipid, such as a glyceride; and sterol, the lipid nanoparticle being unshielded and wherein each mol% content is relative to total lipid present in the lipid nanoparticle.

Description

Lipid Nanoparticles having Non-Polar Lipids for Extrahepatic Nucleic Acid DeliveryTechnical Field

[0001] The present disclosure relates to lipid nanoparticle formulations for the delivery of cargo such as nucleic acid.Background

[0002] Lipid nanoparticle (LNP) formulations represent a revolution in the field of nucleic acid delivery. An early example of a lipid nanoparticle product approved for clinical use is Onpattro™. Onpattro™ is a lipid nanoparticle -based short interfering RNA (siRNA) drug for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis. The success of this LNP delivery system paved the way for the clinical development of the leading LNP -based COVID-19 mRNA vaccines.

[0003] The Onpattro™ LNP formulation consists of four main lipid components, namely: ionizable amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and polyethylene glycol conjugated lipids (PEG-lipids) at respective molar amounts of 50 / 10 / 38.5 / 1.5. Onpattro™ is often considered the gold standard for comparison in studies of LNP-mediated efficacy and current approaches to LNP design make few deviations from the four-component system.

[0004] Of the four Onpattro™ lipid components, the ionizable cationic lipid is deemed most important for the in vitro and in vivo activity of the LNP system. Accordingly, most work in the field has focused primarily on improving this lipid component. The ionizable cationic lipid is positively charged at low pH, which facilitates association with the negatively charged nucleic acid but is neutral at physiological pH, making it more biocompatible in biological systems. Further, it has been suggested that after the lipid nanoparticles are taken up by a cell by endocytosis, the ability of these lipids to ionize at low pH enables endosomal escape. This in turn allows the nucleic acid to be released into the intracellular compartment.

[0005] The PEG-lipid is also considered an essential component of LNP formulations. While serum stable, large unilamellar vesicles (LUV) (termed “liposomes”) used for small molecule delivery have been prepared without PEG (Semple et al., 1996, Biochemistry, 35, 2521-2525), its inclusion in solid-core type LNPs containing nucleic acid cargo is considered essential as it is thought to prevent aggregation of the particles during the formulation process (Kulkami et al., 2020,Nanoscale, 12:23959-23966). LNPs for nucleic acid delivery are formed by a rapid mixing technique during which an organic solvent (e.g., ethanolic) lipid phase is combined with an acidic aqueous phase containing the nucleic acid. The resulting LNP preparation produced during or after mixing is buffer exchanged to neutral pH and the resulting particles contain an electron dense core (referred to often as “solid core”). As the pH of the formulation is raised, the ionizable lipid becomes uncharged and without any electrostatic repulsion, the particles fuse. In the absence of PEG, the particles fuse to produce large polydisperse particles (e.g., around 1000 nm), which lack clinical utility. Indeed, numerous studies have shown that, without PEG, Onpattro™-type siRNAcontaining LNPs (N / P of 3) formed aggregates and heterogeneous suspensions (high polydispersity), while the same particles spiked with PEG-lipid had a particle size of around 40- 60 nm and a polydispersity in the range of 0.1. It is thought that the inclusion of PEG during neutralization of the formulation provides a steric barrier against further fusion and is essential for the creation of particles of a size range (e.g., between 40 and 120 nm) that are suitable for systemic administration. By contrast, liposomes for small molecule drug delivery, such as large unilamellar vesicles (LUVs) lacking PEG (e.g., DSPC / chol or SM / chol) are prepared by a thin film / hydration method that is not reliant on such fusion events during particle formation.

[0006] Thus, while the inclusion of PEG-lipid during liposome (LUV) formation is not required, the prevailing view is that PEG-lipid is critical for producing the type of solid core-type lipid nanoparticles used for nucleic acid delivery. While some investigators have reported the preparation of PEGless (unshielded) LNPs similar to the Onpattro™-type formulation (containing about 10 mol% DSPC) described above (U.S. Patent No. 11,285,222), the examined LNPs lacking DMG-PEG (l,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) or having low levels thereof (0 to 0.25 mol%) were found to exhibit low B cell uptake and activation relative to the conventional LNPs having 1 .5 mol% DMG-PEG.

[0007] Equally important, the inclusion of PEG-lipid in LNPs improves their circulation lifetime. Researchers are now focusing on delivering nucleic acid cargo beyond the liver to expand the repertoire of diseases that are treatable by LNP -mediated nucleic acid delivery and improving blood stability is crucial to bypassing hepatic tissue. PEG-lipids are known to shield the lipid nanoparticle surface, which protects the particles from opsonins and uptake by the mononuclear phagocyte system, as well as preventing their aggregation after administration (Evers et al., 2018,Small Methods, 2, 1700375). It has been shown that siRNA-LNPs containing PEG-DSG accumulate in distal tumours (Lee et al., 2016, Molecular Therapy-Nucleic Acids, 5, e348).

[0008] Nevertheless, PEG-lipids have certain drawbacks as they can prevent the delivery of nucleic acid into cells by decreasing fusogenicity with target cells. The hydrophilic coating, or steric barrier provided by the PEG-lipid has been reported to inhibit interactions with the endosomal membrane, thereby preventing endosomal escape, which is required for cytosolic delivery. The presence of a PEG coating dramatically reduces gene-silencing potency (Cullis and Hope, 2017, Molecular Therapy, 25(7): 1467-1475). To address this problem, short PEGs (e.g., PEG-DMG) have been examined that diffuse out of the LNP post-administration. The design of such particles involves finding a balance between stabilization in storage or particle formation and shedding of the PEG-lipid post administration. Despite these advances, there are reports of undesirable immune responses being mounted in vivo against PEG molecules.

[0009] Some recent studies have investigated improving in vivo delivery properties by introducing targeting lipid components into LNP formulations. One approach uses Selective Organ Targeting (SORT) LNPs (Wang et al., 2023, Nat. Protoc.18:265-291), which are tuned for tissue-specific mRNA delivery by the introduction of a fifth targeting lipid component, such as a permanently charged anionic or cationic lipid. However, such LNPs are still reliant on maintaining the traditional four lipid components of the clinically approved Onpattro™ formulation. Moreover, the introduction of a fifth “SORT”, permanently charged cationic or anionic lipid may introduce toxicity in vivo.

[0010] Other investigators have examined the use of LNPs with triacylglycerols, such as triolein, to target specific cell types in vivo. WO 2023 / 233042 describes LNPs comprising triacylglycerols for targeting myeloid cells. The particles require an apolipoprotein stabilizing agent that acts as a targeting moiety to direct the particles to the myeloid compartment. In addition, the particles comprise polyvalent molecules with multiple positively charged groups and employ phospholipids with low phase transition temperatures. WO 2018 / 119514 describes triacylglycerol-containing LNPs for neuron targeting. However, such particles incorporate a stabilizing agent, such as PEG- lipid, which is subject to the foregoing drawbacks.

[0011] Despite the foregoing advances in the art, there is a continuing need for LNP formulations that have desirable properties for the delivery of nucleic acids to a target organ or tissue.Summary

[0012] The present disclosure addresses one or more problems in the art and / or provides useful alternatives thereof.

[0013] The present disclosure is based in part on the finding that lipid nanoparticles (LNPs) prepared with non-polar lipids as described herein and without a surface stabilizer (e.g., PEG-lipid and / or apolipoprotein), or low levels thereof, have favourable nucleic acid delivery properties. In particular, the LNPs described herein have been found to exhibit increased nucleic acid delivery to extrahepatic tissues and / or organs relative to benchmark formulations containing a surface stabilizer.

[0014] In one example of the disclosure, the LNPs having a combination of a non-polar glyceride lipid and lacking a surface stabilizer or having low levels thereof, exhibit improved nucleic acid delivery to cells of the bone marrow and / or spleen, (e.g., macrophages), over the liver. In some embodiments, nucleic acid delivery to the bone marrow and / or spleen is significantly improved relative to a benchmark LNP comprising a PEG2000-DMG.

[0015] According to one aspect of the disclosure, there is provided a lipid nanoparticle comprising: (i) nucleic acid cargo; (ii) ionizable lipid present at a content of between 15 mol% and 40 mol%; (iii) neutral lipid having at least two tails and a head group, the neutral lipid present at a content of 10 to 55 mol%; (iii) a non-polar lipid content of between 5 mol% and 40 mol%, wherein the nonpolar lipid is a glyceride; and (iv) sterol at a content of between 4 mol% and 45 mol%, wherein the lipid nanoparticle is unshielded and wherein each mol% content is relative to total lipid present in the lipid nanoparticle.

[0016] According to another aspect of the disclosure, there is provided a lipid nanoparticle comprising: (i)nucleic acid cargo; (ii) ionizable lipid; (iii) neutral lipid having at least two tails and a head group, the neutral lipid having a phase transition temperature of at least 25 degrees as measured by differential scanning calorimetry; (iii) a non-polar lipid; and (iv) sterol, wherein the lipid nanoparticle is unshielded and wherein each mol% content is relative to total lipid present in the lipid nanoparticle.

[0017] In some embodiments of any one of the foregoing aspects, the non-polar lipid content includes a combination of two or more non-polar lipids.

[0018] In some embodiments of any one of the foregoing aspects or embodiments thereof, the non-polar lipid is a triglyceride.

[0019] In some embodiments of any one of the foregoing aspects or embodiments thereof, the triglyceride is selected from triolein, tristearin, trilaurin, trilinoein, trilinolenin, trimyristin, tripalmitin, tricaprylin, triarachidin and oleoyldipalmitin.

[0020] In some embodiments of any one of the foregoing aspects or embodiments thereof, the non-polar lipid is a diglyceride.

[0021] In some embodiments of any one of the foregoing aspects or embodiments thereof, the diglyceride is selected from glycerol dilaurate, glycerol dimyristate, glycerol dipalmitate, glycerol distearate, glycerol diarachidate, glycerol dibehenate, glycerol dipalmitoleate, glycerl dioleate, glycerol dilinoleate, glycerol dilinolenate and glycerol diarachidonate.

[0022] In some embodiments of any one of the foregoing aspects or embodiments thereof, the non-polar lipid is a monoglyceride.

[0023] In some embodiments of any one of the foregoing aspects or embodiments thereof, the monoglyceride is selected from lauroyl -rac-glycerol, glycerol monomyristate, glycerol monopalmitate, glycerol monostearate, glycerol monoarachidate, glycerol monobehenate, glycerol monopalmitoleate, glycerol monooleate, glycerol monolinoleate, glycerol monolinolenate, glycerol monoarachidonate, and glycerol monocaprylate, and / or for example 1 -monomyristoyl - rac glycerol, 1 -mono-palm itoyl -rac-glycerol, 2-monopalm itoylglycerol, 1 -mono-palm itolenyl- rac -glycerol, 1 -monostearoyl -rac-glycerol, 1-monoleoyl-rac -glycerol, 1- monolinoleoyl-rac- glycerol and 1 -mono linolenoyl -rac-glycerol.

[0024] In some embodiments of any one of the foregoing aspects or embodiments thereof, the non-polar lipid is castor oil.

[0025] In some embodiments of any one of the foregoing aspects or embodiments thereof, the non-polar lipid is methyl ricinoleate.

[0026] In some embodiments of any one of the foregoing aspects or embodiments thereof, the non-polar lipid is present at a content between 10 mol% and 40 mol%.

[0027] In some embodiments of any one of the foregoing aspects or embodiments thereof, the non-polar lipid is present at a content between 10 mol% and 35 mol%.

[0028] In some embodiments of any one of the foregoing aspects or embodiments thereof, the non-polar lipid is present at a content between 10 mol% and 32.5 mol%.

[0029] In some embodiments of any one of the foregoing aspects or embodiments thereof, the non-polar lipid is present at a content between 30 mol% and 32 mol%.

[0030] In some embodiments of any one of the foregoing aspects or embodiments thereof, the ionizable lipid is an amino lipid.

[0031] In some embodiments of any one of the foregoing aspects or embodiments thereof, the ionizable lipid is present at a content between 23 mol% and 47 mol%.

[0032] In some embodiments of any one of the foregoing aspects or embodiments thereof, the ionizable lipid is present at a content between 25 mol% and 45 mol%.

[0033] In some embodiments of any one of the foregoing aspects or embodiments thereof, the ionizable lipid is present at a content between 27 mol% and 43 mol%.

[0034] In some embodiments of any one of the foregoing aspects or embodiments thereof, the ionizable lipid is present at a content between 28 mol% and 42 mol%.

[0035] In some embodiments of any one of the foregoing aspects or embodiments thereof, the ionizable lipid is present at a content between 29 mol% and 41.5 mol%.

[0036] In some embodiments of any one of the foregoing aspects or embodiments thereof, the ionizable lipid is present at a content between 29.5 mol% and 41 mol%.

[0037] In some embodiments of any one of the foregoing aspects or embodiments thereof, the ionizable lipid is present at a content between 30 mol% and 40 mol%.

[0038] In some embodiments of any one of the foregoing aspects or embodiments thereof, the neutral lipid content is greater than 12 mol%.

[0039] In some embodiments of any one of the foregoing aspects or embodiments thereof, the neutral lipid content is greater than 15 mol%.

[0040] In some embodiments of any one of the foregoing aspects or embodiments thereof, the neutral lipid content is between 10 and 40 mol%.

[0041] In some embodiments of any one of the foregoing aspects or embodiments thereof, the neutral lipid is a phospholipid.

[0042] In some embodiments of any one of the foregoing aspects or embodiments thereof, the phospholipid is a phosphocholine selected from a phosphatidylcholine and a sphingolipid.

[0043] In some embodiments of any one of the foregoing aspects or embodiments thereof, the lipid nanoparticle has less than 0.75 mol% of a surface stabilizer based on a total lipid content of the lipid nanoparticle.

[0044] In some embodiments of any one of the foregoing aspects or embodiments thereof, the surface stabilizer is present at a content between 0 mol% and 0.50 mol%.

[0045] In some embodiments of any one of the foregoing aspects or embodiments thereof, the surface stabilizer is present at a content between 0 mol% and 0.40 mol%.

[0046] In some embodiments of any one of the foregoing aspects or embodiments thereof, the surface stabilizer is present at a content between 0 mol% and 0.30 mol%.

[0047] In some embodiments of any one of the foregoing aspects or embodiments thereof, the surface stabilizer is present at a content between 0 mol% and 0.20 mol%.

[0048] In some embodiments of any one of the foregoing aspects or embodiments thereof, the sterol is present at a content of less than 30 mol%.

[0049] In some embodiments of any one of the foregoing aspects or embodiments thereof, the sterol is cholesterol.

[0050] In some embodiments of any one of the foregoing aspects or embodiments thereof, the sterol is present at a content of greater than 15 mol%.

[0051] In some embodiments of any one of the foregoing aspects or embodiments thereof, the sterol is present at a content between 18 mol% and 40 mol%.

[0052] In some embodiments of any one of the foregoing aspects or embodiments thereof, the sterol is present at a content between 20 mol% and 40 mol%.

[0053] In some embodiments of any one of the foregoing aspects or embodiments thereof, the sterol is present at a content between 25 mol% and 38 mol%.

[0054] In some embodiments of any one of the foregoing aspects or embodiments thereof, an N / P charge ratio of the cationic charge (N) of the ionizable lipid to the anionic charge (P) of the nucleic acid cargo is between 1 and 15.

[0055] In some embodiments of any one of the foregoing aspects or embodiments thereof, the N / P charge ratio is between 2 and 9.

[0056] In some embodiments of any one of the foregoing aspects or embodiments thereof, the ratio is between 3 and 9.

[0057] In some embodiments of any one of the foregoing aspects or embodiments thereof, the nucleic acid is selected from an siRNA, mRNA, a vector nucleic acid, an antisense oligonucleotide, a nucleic acid-protein complex, and a nucleic acid-peptide complex.

[0058] In some embodiments of any one of the foregoing aspects or embodiments thereof, the nucleic acid is selected from siRNA, vector nucleic acid, and an antisense oligonucleotide.

[0059] In some embodiments of any one of the foregoing aspects or embodiments thereof, the nucleic acid is mRNA.

[0060] In some embodiments of any one of the foregoing aspects or embodiments thereof, the lipid nanoparticle has a solid core as visualized by cryogenic electron microscopy (cryo-TEM).

[0061] In some embodiments of any one of the foregoing aspects or embodiments thereof, the lipid nanoparticle has a lipid layer surrounding the core as visualized by cryogenic electron microscopy (cryo-TEM).

[0062] In some embodiments of any one of the foregoing aspects or embodiments thereof, the lipid nanoparticle resulting in a ratio of bone marrow / liver expression that is between 1.5 and 30.

[0063] In some embodiments of any one of the foregoing aspects or embodiments thereof, the lipid nanoparticle resulting in a ratio of bone marrow / liver expression that is between 1.5 and 25.

[0064] In some embodiments of any one of the foregoing aspects or embodiments thereof, the ratio of bone marrow / liver expression is between 4 and 20.

[0065] In some embodiments of any one of the foregoing aspects or embodiments thereof, the ratio of bone marrow / liver expression is between 5 and 20.

[0066] In some embodiments of any one of the foregoing aspects or embodiments thereof, the lipid nanoparticle has a ratio of bone marrow / liver expression that is greater than 1.5 or greater than 2.0.

[0067] In some embodiments of any one of the foregoing aspects or embodiments thereof, the neutral lipid is a phosphatidylglycerol or sphingolipid and wherein the phase transition temperature of the neutral lipid is at least 25°C as measured by differential scanning calorimetry (DSC).

[0068] In some embodiments of any one of the foregoing aspects or embodiments thereof, dimyristoylphosphatidylcholine (DMPC) is present at 0 to 10 mol%, 0 to 8 mol%, 0 to 7 mol%, 0 to 6 mol%, 0 to 5 mol%, 0 to 4 mol% or 0 to 3 mol% relative to the total lipid content of the lipid nanoparticle.

[0069] In one embodiment, there is provided a method for delivery of mRNA or vector DNA for in vivo expression of protein or peptide in the bone marrow or spleen, the method comprising administering to a mammal a lipid nanoparticle of any one of the foregoing aspects or embodiments thereof. In some embodiments, a ratio of bone marrow / liver expression is between 2 and 30. In some embodiments, a ratio of bone marrow / liver expression is between 4 and 20. In some embodiments, a ratio of bone marrow / liver expression is between 5 and 20. In some embodiments, a ratio of bone marrow / liver expression is greater than 1.5 or is greater than 2.

[0070] In one embodiment, there is provided a method for delivery of siRNA or antisense oligonucleotide for in vivo silencing of a gene in the bone marrow or spleen, the method comprising administering to a mammal a lipid nanoparticle of any one of the aspects or embodiments thereof, wherein the siRNA or antisense oligonucleotide is encapsulated within the lipid nanoparticle and wherein the administering of the lipid nanoparticle results in an increased bone marrow- or spleen-specific silencing of the gene as compared as compared to a benchmark that is an otherwise identical LNP lacking a surface stabilizer and encapsulating the siRNA or antisense oligonucleotide.

[0071] In one embodiment, there is provided a method for delivering a nucleic acid to a macrophage to treat a disease, disorder or condition, the method comprising contacting a lipid nanoparticle of any one of the aspects or embodiments thereof, with the macrophage in vivo or in vitro.

[0072] In another aspect of the disclosure, there is provided a lipid nanoparticle of any one of the foregoing aspects or embodiments thereof for use to deliver mRNA or vector DNA for in vivo expression of protein or peptide in the bone marrow or spleen to a subject in need of mRNA or vector DNA to treat, ameliorate or prevent a condition or disease by expression of said protein or peptide.

[0073] In one embodiment of the foregoing aspect, ratio of bone marrow / liver expression in the subject is between 2 and 30.

[0074] In one embodiment of the foregoing aspect, the ratio of bone marrow / liver expression is between 4 and 20.

[0075] In one embodiment of the foregoing aspect, the ratio of bone marrow / liver expression is between 5 and 20.

[0076] In a further embodiment, a ratio of bone marrow / liver expression is greater than 1.5 or is greater than 2.

[0077] In another aspect of the disclosure, there is provided a lipid nanoparticle as described in any one of the foregoing aspects or embodiments for use to deliver siRNA or antisense oligonucleotide in a subject to treat, ameliorate or prevent a condition or disease in vivo by silencing of a gene in the bone marrow or spleen, wherein the siRNA or antisense oligonucleotide is encapsulated within the lipid nanoparticle and wherein the administering of the lipid nanoparticle results in an increased bone marrow- or spleen-specific silencing of the gene as compared as compared to a benchmark that is an otherwise identical LNP lacking a surface stabilizer and encapsulating the siRNA or antisense oligonucleotide.

[0078] In another aspect of the disclosure, there is provided a lipid nanoparticle as described in any one of the foregoing aspects or embodiments thereof, to deliver a nucleic acid to a macrophage to treat, ameliorate or prevent a disease, disorder or condition in a subject, the use comprising contacting the lipid nanoparticle with the macrophage in vivo, ex vivo or in vitro.Brief Description of the Figures

[0079] Figure 1 is a graph showing particle size, encapsulation percentage and polydispersity index for triacylglycerol unshielded LNPs (TAG-LNP) having ionizable lipid 1 / DSPC / triolein / cholesterol PEG2000-DMG (29.62 / 19.62 / 29.26 / 19.62 / 1.5 mol / mol) and ionizablelipid 1 / DSPC / triolein / cholesterol (30 / 20 / 30 / 20 mol / mol). The ionizable lipid 1 is set forth in Example 1.

[0080] Figure 2A is a graph showing particle size, encapsulation percentage and polydispersity index (physiochemical properties) for LNPs having ionizable lipid 1 / DSPC / triolein / cholesterol (30 / 20 / 30 / 20 mol / mol) after being subjected to vortexing for 2 minutes at concentrations of 0.5 mg / mL and 0.05 mg / mL.

[0081] Figure 2B is a graph showing the physicochemical properties of PEG-containing LNPs (control LNPs) having 10 / 50 / 38.5 / 1.5 of DSPC / norMC3 ionizable lipid / cholesterol / PEG-DMG after being subjected to vortexing for the time points indicated. The ionizable lipid, norMC3, is set forth in Example 1.

[0082] Figure 3A is a graph showing the physicochemical properties of unshielded triolein LNPs containing 1,2-dibehenoylphosphatidylcholine (22:0 PC), dipalmitoylphosphatidylcholine (DPPC), 1 -stearoyl -2 -oleoylphosphatidylcholine (SOPC), 1 -palmitoyl -2-oleoyl- phosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylglycerol (DOPG) and distearoylphosphatidylcholine (DSPC). The LNPs contained 30 / 20 / 30 / 20 ionizable lipid 1 / phospholipid / triolein / cholesterol (mol / mol). The unshielded triolein LNPs were prepared with an unmodified preparation method set forth in the Materials and Methods.

[0083] Figure 3B is a graph showing the physicochemical properties of unshielded triolein LNPs containing DSPC, DOPC, SOPC, POPC and DMPC diluted with different buffers. The LNPs contained 30 / 20 / 30 / 20 ionizable lipid 1 / phospholipid / triolein / cholesterol (mol / mol). The unshielded triolein LNPs were prepared with a modified preparation method set forth in the Materials and Methods using 25 mM phosphate buffer (PB), Tris-HCl (Tris) and sodium acetate (Ac) as indicated.

[0084] Figure 4 is a graph showing the physicochemical properties of unshielded triolein LNPs composed of ionizable lipid 1 / DSPC / triolein / cholesterol and various amounts of DSPC and cholesterol as set forth in Table 2.

[0085] Figure 5 is a graph showing the physicochemical properties of unshielded triolein LNPs having ionizable lipid 1 / DSPC / triolein / cholesterol at molar ratios of 30 / 20 / 30 / 20 and 30 / 25 / 25 / 20 and encapsulating cargo at nitrogen-to-phosphate ratios (N / P) of 2, 3, 4, 6 and 9 as set forth in Table 3.

[0086] Figure 6 is a graph showing the physicochemical properties of unshielded triolein LNPs having ionizable lipid / DSPC / triolein / chole sterol (30 / 20 / 30 / 20, mol / mol) with ionizable lipid (IL) 2, 3 and 4 as set forth in Table 4.

[0087] Figure 7A is a graph showing luminescence intensity in the liver for PEG-containing IcLNP™ nMC3 LNP having ionizable lipid / DSPC / cholesterol / PEG2ooo-DMG (27.4 / 50 / 21.1 / 1.5 mol / mol), PEG-containing IcLNP™ ionizable lipid 2 / DSPC / cholesterol / PEG20oo-DMG (27.4 / 50 / 21. 1 / 1.5 mol / mol), ionizable lipid 2 / DSPC / cholesterol (20 / 40 / 40 mol / mol), and ionizable lipid 1 / DSPC / triolein / cholesterol (30 / 20 / 30 / 20 mol / mol) at 24 and 4 hours post-injection (see Table 5).

[0088] Figure 7B is a graph showing luminescence intensity in the spleen for the PEG-containing LNPs and the unshielded DSPC and DSPC / triolein LNPs of Figure 7A (see also Table 5).

[0089] Figure 7C is a graph showing luminescence intensity in the bone marrow (BM) for the PEG-containing LNPs and unshielded DSPC and unshielded DSPC / triolein LNPs of Figure 7A (see also Table 5).

[0090] Figure 8A is a graph showing luminescence intensity in the liver for the PEG-containing LNPs and unshielded DSPC / triolein LNPs set forth in Table 6.

[0091] Figure 8B is a graph showing luminescence intensity in the spleen for the PEG-containing LNPs and DSPC / triolein LNPs set forth in Table 6.

[0092] Figure 8C is a graph showing luminescence intensity in the bone marrow for the PEG- containing LNPs and DSPC / triolein LNPs set forth in Table 6.

[0093] Figure 9A is a graph showing physicochemical properties for the PEG-containing and unshielded control LNPs and DSPC / triolein unshielded LNPs of Table 7 used in flow cytometry studies.

[0094] Figure 9B is a graph showing % eGFP+ neutrophils and macrophage cell populations in the bone marrow for PEG-containing and unshielded LNPs of Table 7 as determined by flow cytometry.

[0095] Figure 9C is a graph showing % eGFP+ neutrophils and macrophage cell populations in the spleen for PEG-containing and unshielded LNPs of Table 7 as determined by flow cytometry.

[0096] Figure 10A is a graph showing physicochemical properties for the PEG-containing control LNPs and DSPC / triolein unshielded LNPs of Table 8.

[0097] Figure 10B is a graph showing luminescence intensity in the liver for the PEG-containing LNPs and unshielded DSPC / triolein LNPs set forth in Table 8.

[0098] Figure IOC is a graph showing luminescence intensity in the bone marrow for the PEG- containing LNPs and DSPC / triolein LNPs set forth in Table 8.

[0099] Figure 11A is a graph showing physicochemical properties for the PEG-containing control LNPs and DSPC / triolein unshielded LNPs of Table 9.

[0100] Figure 1 IB is a graph showing luminescence intensity in the liver for the PEG-containing LNPs and unshielded DSPC / triolein LNPs set forth in Table 9.

[0101] Figure 11C is a graph showing luminescence intensity in the spleen for the PEG- containing LNPs and unshielded DSPC / triolein LNPs set forth in Table 9.

[0102] Figure 1 ID is a graph showing luminescence intensity in the bone marrow (BM) for the PEG-containing LNPs and unshielded DSPC / triolein LNPs set forth in Table 9.

[0103] Figure HE is agraph showing luminescence intensity in the heart forthe PEG-containing LNPs and unshielded DSPC / triolein LNPs set forth in Table 9.

[0104] Figure HF is a graph showing luminescence intensity in the lungs for the PEG- containing LNPs and unshielded DSPC / triolein LNPs set forth in Table 9.

[0105] Figure 11G is a graph showing luminescence intensity in the abdominal skin for the PEG- containing LNPs and unshielded DSPC / triolein LNPs set forth in Table 9.

[0106] Figure 12A is a graph showing the physicochemical properties of LNPs havingMC3 / DSPC / cholesterol / PEG2ooo-DMG (50 / 10 / 38.5 / 1.5 mol / mol) and ionizablelipid / DSPC / triolein / cholesterol (20 / 40 / 10 / 30, mol / mol) with ionizable lipid (IL) 2 and 6 as set forth in Table 10.6 as set forth in Table 10.

[0107] Figure 12B is a graph showing luminescence intensity in the liver for the PEG-containing control LNP and unshielded DSPC / triolein LNPs set forth in Table 10.

[0108] Figure 12C is a graph showing luminescence intensity in the spleen for the PEG- containing control LNP and unshielded DSPC / triolein LNPs set forth in Table 10.

[0109] Figure 12D is a graph showing luminescence intensity in the bone marrow for the PEG- containing control LNP and unshielded DSPC / triolein LNPs set forth in Table 10.

[0110] Figure 13A is a graph showing the physicochemical properties of LNPs having MC3 / DSPC / cholesterol / PEG2ooo-DMG (50 / 10 / 38.5 / 1.5 mol / mol) and unshielded TAG-LNPs with ionizable lipid / DSPC / triolein / cholesterol with ionizable lipids (IL) and molar ratios as set forth in Table 11.

[0111] Figure 13B is a graph showing luminescence intensity in the liver for the PEG-containing control LNP and unshielded DSPC / triolein LNPs set forth in Table 11.

[0112] Figure 13C is a graph showing luminescence intensity in the spleen for the PEG- containing control LNP and unshielded DSPC / triolein LNPs set forth in Table 11.

[0113] Figure 13D is a graph showing luminescence intensity in the bone marrow for the PEG- containing control LNP and unshielded DSPC / triolein LNPs set forth in Table 11.

[0114] Figure 14 is a graph showing the physicochemical properties of control LNPs having MC3 / DSPC / cholesterol / PEG20oo-DMG at 50 / 10 / 38.5 / 1 / 5 mol / mol (control LNP), ionizable lipid 2 / DSPC / cholesterol / PEG2000-DMG (IcLNP™) and unshielded TAG-LNPs with ionizable lipid / DSPC / triolein / cholesterol with ionizable lipids 5 or 6 as indicated and molar ratios of each lipid component as set forth in Table 12.

[0115] Figure 15A is a graph showing linear negative (Lin"), Sca-1 positive (Sca-1+) and c-Kit positive (c-Kit+) (LSK) cells positive for expression of Thy 1.1 in hematopoietic stem and progenitor cells (HSPC) in the bone marrow for the control LNP, PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0116] Figure 15B is a graph showing CD117+cells positive for expression of Thy 1.1 in HSPC in the bone marrow for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0117] Figure 15C is a graph showing long-term hematopoietic stem cells (LT-HSCs) positive for expression of Thy 1.1 in HSPC in the bone marrow for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0118] Figure 16A is a graph showing monocytes positive for expression of Thy 1 .1 in the bone marrow for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0119] Figure 16B is a graph showing macrophages positive for expression of Thy 1.1 in the bone marrow for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0120] Figure 16C is a graph showing neutrophils positive for expression of Thyl. 1 in the bone marrow for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0121] Figure 16D is a graph showing CDl lb+cells positive for expression of Thyl .1 in the bone marrow for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0122] Figure 16E is a graph showing T cells positive for expression of Thy 1.1 in the bone marrow for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0123] Figure 16F is a graph showing B cells positive for expression of Thy 1.1 in the bone marrow for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0124] Figure 17A is a graph showing monocytes positive for expression of Thy 1.1 in the blood for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0125] Figure 17B is a graph showing other myeloid cells positive for expression of Thyl. l in the blood for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0126] Figure 17C is a graph showing CDl lb+positive for expression of Thy 1.1 in the blood for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0127] Figure 17D is a graph showing neutrophils cells positive for expression of Thy 1.1 in the blood for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0128] Figure 17E is a graph showing T cells positive for expression of Thyl. 1 in the blood for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0129] Figure 17F is a graph showing B cells positive for expression of Thy 1. 1 in the blood for the PEG-containing control IcLNP™ and unshielded DSPC / triolein LNPs set forth in Table 12.

[0130] Figure 18 is a cryogenic transmission electron microscopy (Cryo-TEM) image of was LNPs composed of ionizable lipid 5 / DSPC / triolein / cholesterol (TAG) at molar ratios of 30 / 45 / 5 / 20.Detailed DescriptionSubstantially no surface stabilizer

[0131] The lipid nanoparticle has “substantially no hydrophilic polymer-lipid conjugate” or is “non-sterically stabilized”, “unshielded” or “uncoated”, meaning the lipid nanoparticle has less than 0.8 mol% surface stabilizer content as measured based on the total lipid content of the nanoparticle.

[0132] As used herein, the term “surface stabilizer” is a macromolecule, including a protein, polysaccharide or polymer, including a block copolymer, that is used to stabilize a lipid nanoparticle, and in which at least a portion (e.g., hydrophilic portion) is present on the surface of the lipid nanoparticle. Such molecules are employed by those of skill in the art to prevent aggregation, improve shelflife and / or improve the stability of the particle after administration, so as to increase the circulation lifetime of the lipid nanoparticle. The term includes surfacestabilizers that are known to control the size of lipid nanoparticles, such as amphiphilic polymers (e.g., block co-polymer). As would be appreciated by those of skill in the art, a hydrophobic portion of the surface stabilizer may partition in a lipophilic portion of the lipid nanoparticle.

[0133] In some embodiments, the surface stabilizer is present at less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, or 0.10 mol% as measured based on the total lipid content of the lipid nanoparticle . In further embodiments, the surface stabilizer mol% content is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol% or 0 and 0.10 mol%.

[0134] In some embodiments, the hydrophilic-polymer conjugate (e.g., a hydrophilic-polymer lipid conjugate) content of the LNP is less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15 or 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the hydrophilic-polymer conjugate (e.g., a hydrophilic-polymer lipid conjugate) mol% content is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol%, 0 and 0.10 mol% or 0 and 0.05 mol%.

[0135] The hydrophilic-polymer conjugate (e.g., a lipid conjugate) includes a lipophilic portion that resides in the lipid nanoparticle lipid layer (e.g., bilayer or monolayer) and a polymer chain that is hydrophilic and typically extends into the surrounding solution. Examples of hydrophilic polymers that form part of the conjudate include polyethyleneglycol (PEG), polyvinylpyrrolidone, polyvinylmethylether, polyhydroxypropyl methacrylate, polyhydroxypropylmethacrylamide, polyhydroxyethyl acrylate, polymethacrylamide, polydimethylacrylamide, polymethyloxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polysarcosine and polyaspartamide. In one embodiment, the hydrophilic-polymer conjugate is a PEG-lipid conjugate. In an alternative embodiment, the hydrophilic polymer may be composed of sugar monomer units. For example, the hydrophilic polymer conjugate may be a naturally-occurring or synthesized oligo saccharide -containing molecule, such as, for example, monosialoganglioside (GMI). In further embodiments, the lipid nanoparticle may comprise a hydrophilic polymer-lipid conjugate conjugated to a targeting ligand at its distal end.

[0136] In some embodiments, the amphipathic polymer content in the LNP is less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the amphipathic polymer mol% content is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol%, 0 and 0.10 mol% or 0 and 0.05 mol%. Examples of amphipathic polymers are provided in US 2021 / 0046192, which is incorporated herein by reference.

[0137] In some embodiments, the poloxamer content is less than 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the poloxamer mol% content is between 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol%, 0 and 0.10 mol% or 0 and 0.05 mol%.

[0138] In further embodiments, the LNP lacks a surface stabilizer that is a protein, referred to as a protein stabilizer. This includes an apolipoprotein stabilizer, derivative or mimetic thereof (see e.g., WO 2023 / 233042, which is incorporated herein by reference). Such apolipoprotein may be selected from one or a combination of apo Al, apo Al- Milano, apo A2, apo A4, apo A5, apo B48, apo B100, apo C-l, apo C-l I, apo C-lll, apo C-IV, apo D, apo E, apo F, apo H, apo L and apo M. In some embodiments, the protein stabilizer content is less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15 or 0.10 mol% as measured based on the total lipid content of the nanoparticle. In some embodiments, the protein stabilizer content is less than 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15 or 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the protein stabilizer content mol% content is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol% or 0 and 0.10 mol%. Since lipid nanoparticles can adsorb proteins after administration, the protein content is measured in vitro prior to administration.

[0139] Alternatively, in some embodiments a lipid nanoparticle preparation lacks or has low levels thereof of one or more stabilizing agents, which includes a surface stabilizer as described above and / or a cryoprotectant that functions as a stabilizer. In some embodiments, the lipidnanoparticle preparation has less than 2 w / v, 1.75 w / v, 1.50 w / v, 1.25 w / v, 1.00 w / v, 0.75 w / v, 0.50 w / v, 0.25, 0.10 or 0.05 w / v of one or more cryoprotectants in the preparation. In some embodiments, a lipid nanoparticle preparation having a plurality of LNPs has low levels or lacks glycerol and / or propylene glycol as a cryoprotectant, such as at concentration levels less than 2 w / v, 1.75 w / v, 1.50 w / v, 1.25 w / v, 1.00 w / v, 0.75 w / v, 0.50 w / v, 0.25, 0.10 or 0.05 w / v in the preparation. In some embodiments, the lipid nanoparticle preparation consists essentially of buffer, meaning that a lipid nanoparticle preparation comprises buffer and has less than 2 w / v, 1.75 w / v, 1.50 w / v, 1.25 w / v, 1.00 w / v, 0.75 w / v, 0.50 w / v, 0.25, 0.10 or 0.05 w / v of one or more cryoprotectants in the preparation. In some embodiments, the lipid nanoparticle preparation comprises no cryoprotectant, such as for example, glycerol and / or propylene glycol.

[0140] In another embodiment, the lipid nanoparticle is “PEG-less”, meaning that the lipid nanoparticle has no detectable amounts of polyethyl ene-glycol, such as a PEG-lipid conjugate. In further embodiments, the lipid nanoparticle is “PEGless”, meaning that the LNP lacks a surface stabilizer comprising a hydrophilic polymer with a polyethylene oxide repeating unit or includes low levels thereof, such as less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mol% as measured based on the total lipid content of the lipid nanoparticle. In further embodiments, surface stabilizer comprising a hydrophilic polymer has a mol% content that is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol% or 0 and 0.10 mol%.Neutral lipid

[0141] The LNP includes a neutral lipid. As used herein, the term neutral lipid refers to an amphipathic lipid having a lipophilic portion (typically at least two tails) and a head group. The term includes, without limitation, phospholipids and includes lipids with choline head groups (e.g., phosphatidylcholine), meaning an amphipathic lipid that has a choline head group and that bears no or substantially no net charge at physiological pH. The term also includes phospholipids conjugated to sterols. In some embodiments, the lipid with a choline head group is a phosphatidylcholine lipid, such as a lipid that is selected from distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC) and dipalmitoyl-phosphatidylcholine (DPPC) and sphingomyelin with a phosphocholine head group. In some advantageous embodiments, thelipid with a choline head group is selected from distearoylphosphatidylcholine (DSPC), dipalmitoyl -phosphatidylcholine (DPPC) and sphingomyelin with a phosphocholine head group. In particularly advantageous embodiments, the lipid with a choline head group is selected from distearoylphosphatidylcholine (DSPC) and dipalmitoyl-phosphatidylcholine (DPPC).

[0142] As used herein, “head group” means a moiety of the neutral lipid that imparts polarity to the lipid and comprises one or more electronegative atoms, such as nitrogen, oxygen and / or phosphorus. Generally, the electronegative atom or atoms are part of one or more functional groups. In some embodiments, the head group is zwitterionic. In some embodiments, the head group comprises a phosphate group and a nitrogen atom. In some embodiments, the head group is zwitterionic and has no net charge at physiological pH.

[0143] The neutral lipid (e.g., phospholipid) content in some embodiments (excluding sterol) is greater than 5 mol%, greater than 10 mol%, greater than 15 mol%, greater than 20 mol%, greater than 22 mol%, greater than 22.5 mol% or greater than 25 mol%. In some embodiments, the upper limit of neutral lipid content is 45 mol%, 40 mol% or 35 mol%. The disclosure also encompasses sub-ranges of any combination of the foregoing numerical upper and lower limits.

[0144] The content of neutral lipid (e.g., phospholipid) having a choline head group in some embodiments (excluding sterol) is greater than 5 mol%, greater than 10 mol%, greater than 15 mol%, greater than 20 mol%, greater than 22 mol%, greater than 22.5 mol% or greater than 25 mol%. In some embodiments, the upper limit of neutral lipid having a choline head group is 45 mol%, 40 mol% or 35 mol%. The disclosure also encompasses sub-ranges of any combination of the foregoing numerical upper and lower limits.

[0145] The content of neutral lipid having at least two tails and a polar region (e.g., head group) in some embodiments (excluding sterol) is greater than 5 mol%, greater than 10 mol%, greater than 15 mol%, greater than 20 mol%, greater than 22 mol%, greater than 22.5 mol% or greater than 25 mol%. In some embodiments, the upper limit of neutral lipid having at least two tails and a polar region (e.g., head group) is 45 mol%, 40 mol% or 35 mol%. The disclosure also encompasses sub-ranges of any combination of the foregoing numerical upper and lower limits.

[0146] The phosphatidylcholine lipid content in some embodiments is greater than 5 mol%, greater than 10 mol%, greater than 15 mol%, greater than 20 mol%, greater than 22 mol%, greater than 22.5 mol% or greater than 25 mol%. In some embodiments, the upper limit ofphosphatidylcholine lipid is 45 mol%, 40 mol% or 35 mol%. The disclosure also encompasses sub-ranges of any combination of the foregoing numerical upper and lower limits.

[0147] The neutral lipid (e.g., phospholipid) content may include mixtures of two or more types of different neutral lipids. In one embodiment, the neutral lipid content is a mixture of two or more of the phosphatidylcholine lipids selected from DSPC, DPPC, DMPC, DOPC and POPC. In some embodiments, the phosphatidylcholine lipid content is primarily DSPC. In some embodiments, the DMPC, DOPC or POPC content is less than 5 mol%. In some embodiments, the DMPC content is less than 5 mol%.

[0148] In such embodiments, the lipid nanoparticle may have a DSPC content of at least 10, 15, 16, 18, 20, 22, 30, 35, 40 or 45 mol% based on the total lipid content of the lipid nanoparticle with the balance of the neutral lipid content being another neutral lipid (e.g., other phosphatidylcholine lipid(s)). In another embodiment, the neutral lipid (e.g., phosphatidylcholine) content is made up of at least 40 or 50 mol% DSPC relative to the total neutral lipid (phosphatidylcholine content) of the lipid nanoparticle.

[0149] DMPC has two myristoyl (C14:0) fatty acid chains and has a phase transition temperature of about 23-24°C. The inclusion of greater than 10 mol% of DMPC could negatively impact particle stability. In one embodiment, the lipid nanoparticle has less than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mol% DMPC based on total lipid in the LNP. In some embodiments, the lipid nanoparticle has between 0 and 10 mol% DMPC. In some embodiments, the lipid nanoparticle has between 0 and 9 mol% DMPC. In some embodiments, the lipid nanoparticle has between 0 and 8 mol% DMPC. In some embodiments, the lipid nanoparticle has between 0 and 7 mol% DMPC. In some embodiments, the lipid nanoparticle has between 0 and 6 mol% DMPC. In some embodiments, the lipid nanoparticle has between 0 and 5 mol% DMPC. In some embodiments, the lipid nanoparticle has between 0 and 4 mol% DMPC. In some embodiments, the lipid nanoparticle has between 0 and 3 mol% DMPC. In some embodiments, the lipid nanoparticle has between 0 and 2 mol% DMPC. In some embodiments, the lipid nanoparticle has between 0 and 1 mol% DMPC.

[0150] In another embodiment the phospholipid content of the lipid nanoparticle has less than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mol% of non-phosphatidylcholine lipids, such as DOPE based on total lipid in the LNP.

[0151] Without intending to be limited by any particular theory, it is believed that fusion and agglomeration of lipid nanoparticles with no hydrophilic polymer lipid conjugate (or low levels thereof) during particle formation using the mixing method described herein could be avoided by selecting a neutral lipid (e.g., phospholipid) that is in the gel phase rather than in the disordered liquid crystalline phase at room temperature and above. The inclusion of such neutral lipids (e.g., high Tmphosphatidylcholine lipids) in the lipid nanoparticle may also improve blood stability after injection.

[0152] In some embodiments, the transition temperature (Tm) of the neutral lipid (e.g., phospholipid) is at least 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C. In some embodiments, the transition temperature of the neutral lipid (e.g., phospholipid) is at least 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C. In some embodiments, the transition temperature of the neutral lipid (e.g., phospholipid) is between 20°C and 60°C or between 42°C and 60°C. The phase transition temperature of the lipid as used herein is as published by Avanti™ Research or in Lipid Thermotropic Phase Transition Database (LIPIDAT) - NIST Standard Reference Database or if not published therein is measured using differential scanning calorimetry (DSC) using techniques known to those of skill in the art. As used herein, the transition temperature of the lipid is the main phase transition temperature. The main phase transition is when the lipid undergoes a transition from a low degree of fluidity (e.g., gel or rippled phase) to a liquid disordered phase. The liquid disordered phase is accompanied by an increase in lipid disordering and is associated with a larger enthalpy change than a pre-transition phase.

[0153] In some embodiments, the neutral lipid is a sterol conjugate, such as a phosphatidylcholine-sterol conjugate, including a soy phosphatidylcholine-cholesterol conjugate (SPC-cholesterol), oleoyl-phosphatidylcholine-cholesterol conjugate (OPC-cholesterol) or a palmitoyl-phosphatidylcholine-cholesterol conjugate (PPC-cholesterol conjugate). Additional phospholipid-sterol conjugates are described in US2011 / 0177156, which is incorporated herein by reference. An example of a suitable phospholipid that is an SPC-cholesterol conjugate is set forth below:

[0154] In some embodiments, the neutral lipid (e.g., phospholipid) is a sphingolipid. As used herein the term “sphingolipid”, means a lipid comprising a sphingosine backbone and that is suitable for formulation in the LNPs herein. The sphingolipid includes a ceramide, a sphingomyelin, a cerebroside, a ganglioside, or derivatives, such as but not limited to reduced analogues thereof, that lack a double bond in the sphingosine unit. The sphingolipid has a phosphocholine head group and includes sphingomyelin.Non-polar lipid

[0155] The term “non-polar lipid” as used herein refers to a lipid that is neutral and uncharged at physiological pH, lacks a head group moiety (e.g., phosphate), and that comprises one or more lipophilic chains that are at least 10 carbon atoms in length that impart an overall hydrophobic character to the lipid. The non-polar lipid is typically a glyceride and includes but is not limited to esterified fatty acids and glycerides described below.

[0156] In one example of the disclosure, the non-polar lipid has a structure of F ormula I:Formula I or a tautomer or stereoisomer thereof, wherein Rl, R2 and R3 are at each occurrence, independently, H or C1-C26 alkyl or a C1-C26 alkenyl having 1-6 double bonds, wherein at least one, at least two or all of Rl-X, R2-Y and R3-Z is the C1-C26 alkyl or alkenyl bonded to a heteroatom substituent represented by X, Y and Z, and wherein at least one of Rl, R2 and R3 comprises at least 10 carbon atoms and terminates with a methyl; andwherein X, Y and Z are at each occurrence, a direct bond or are independently, the heteroatom substituent selected from -O-; — (C=O)O-; — O(C=O)-; — C(=O)-; — O(C=O)O-; — S(O)X-; — S— S-; — C(=O)S-; — SC(=O)-; —NR'—; — NR'C(=O) — ; — C(=O)NR'— ; — NR'C(=O)NR'— ; — OC(=O)NR'— ; — NR'C(=O)OR’— ; — NR'S(O)XNR'— ; — NR'S(O)XR’— ;and — S(O)XNR' — , wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2, and wherein a and b are independently 0 to 4, wherein the alkyl or alkenyl of Rl, R2 and R3 is optionally substituted. In some advantageous embodiments, X, Y and Z are at each occurrence, independently, -O-; -(C=O)O-; or -O(C=O)- and wherein b is 1. In some advantageous embodiments, X, Y and Z are at each occurrence, independently, -C=O)O- or -O(C=O)- and wherein b is 1.

[0157] The term “optionally substituted” with reference to an Rl, R2 or R3 alkyl or alkenyl means that at least one hydrogen atom of the alkyl or alkenyl group can be replaced by a nonhydrogen atom or group of atoms (i.e., a “substituent”), and / or the alkyl is interrupted (i.e., a - (CH)2- group replaced) by a non-carbon atom or one or more substituents, provided the non-polar lipid retains sufficient hydrophobicity so as to form the hydrophobic core of the LNP. The substituent that replaces the hydrogen atom may include -OR’, -SR’ or -NR’. Substituents that may replace a methylene -(CH)2- group include but are not limited to -O-, -S- and -NR'-, wherein R' is as defined above. In one embodiment, X, Y and Z are independently selected from — (C=O)O- ; — O(C=O)-; NR'C(=O) — ; — C(=O)NR'— ; — NR'C(=O)NR'— ; — OC(=O)NR'— ; and — NR'C(=O)OR’ — In one embodiment, the X, Y and Z are independently selected from — (C=O)O- and — O(C=O)-. In one embodiment, the non-polar lipid, is a non-polar glyceride lipid or analogues thereof that includes triglycerides, diglycerides, monoglycerides or mixtures thereof.

[0158] In one embodiment, the non-polar glyceride lipid is a tri -acylglycerol (TAG), which refers to a glyceride having three fatty acid chains covalently bonded to a glycerol backbone through respective ester linkages. In one embodiment, all three fatty acids bonded to the glycerol backbone are the same. In alternative embodiments, the three fatty acids of the triglyceride are different, e.g., having different lengths and / or degrees of saturation.

[0159] In some embodiments, the TAG is characterized by the degree of saturation of each fatty acid chain. In some embodiments, TAGs have SSS, SUS, UUU and USU- fatty acid chains and are therefore considered symmetrical triglycerides, where S represents a saturated fatty acid andU represents an unsaturated fatty acid. In other embodiments, asymmetrical TAGS are used in the LNP. In some embodiments, the sn-1 and sn-3 positions of the TAG contain different fatty acids, in which case the central carbon atom is a chiral carbon and the TAG is asymmetrical.

[0160] Non-limiting examples of the triglyceride include triolein, tristearin, trilaurin, trilinoein, trilinolenin, trimyristin, tripalmitin, tricaprylin, triarachidin, triricinolein, tribehenin, tripalmitolein, triarachidonin, glyceryl distearate oleate, glyceryl distearate linoleate, glyceryl palmitate oleate stearate, glyceryl palmitate dioleate, glyceryl stearate dioleate, glyceryl palmitate dilinoleate, glyceryl stearate oleate linoleate. In some embodiments, a mixture of two or more triglycerides is included in the LNP.

[0161] In embodiments of the disclosure, the triglyceride in the LNP is triolein, a symmetrical triglyceride derived from glycerol and three units of the unsaturated fatty acid oleic acid. The IUPAC name for triolein is 2,3-bis[[(Z)-octadec-9-enoyl]oxy]propyl (Z)-octadec- 9-enoate, and synonyms include glycerol trioleate, glycerol trioleyl, trielaidin, trioleoylglycerol, and trioleyl glycerol.

[0162] In some embodiments, the tri-acylglyceride is naturally derived. Examples of natural origin fatty acids used in some embodiments include soy oil, castor oil, sunflower oil, canola oil and palm oil, omega 3 fatty acid and omega 6 fatty acid. Further, examples of omega 3 fatty acids used in some embodiments include, but are not limited to, alpha- linolenic acid and docasahexaenoic acid. Examples of omega 6 fatty acids used in some embodiments include, but are not limited to, linoleic acid and gamma linolenic acid.

[0163] Additional commercially available triglycerides include Captex® and Sterotex®. In some embodiments, the TAG is selected from Captex™ 8000, Captex™ GTO, and Captex™ 1000.

[0164] In some embodiments, the number of carbon atoms on the aliphatic tails of the triglyceride can be used to classify the TAG as a medium chain triglyceride (MCT) having 6 to 12 carbon atoms (6, 7, 8, 9, 10, 11, 12). In some embodiments, the LNP contains one or more MCT, or mixtures thereof. In some embodiments, the medium chain triglyceride comprises a fatty acid selected from one or more of caproic acid, octanoic acid, capric acid, caprylic acid, and / or lauric acid. In some embodiments, the MCT is highly pure. In some embodiments, the MCT has a purity by weight % of equal to or greater than about: 90%, 95%, 97%, 98%, 99%, 100%, or ranges including and / or spanning the aforementioned values. In some embodiments, the MCT (or LCT)is present in the lipid-based particle composition at dry weight % of equal to or greater than about:10%, 20%, 30%, 35%, 40%, 45%, 50%, or ranges including and / or spanning the aforementioned values.

[0165] In some embodiments, the triglyceride comprises a fatty acid greater than 12 carbons in length. In some embodiments, the lipid component comprises a long chain triglyceride (LCT) greater than or equal to 13, 14, 15, 16, 17, 18, 19, or 20 carbons in length or ranges including and / or spanning the aforementioned values.

[0166] The term “diacylglycerol lipid” or “diacylglyceride” (DAG) herein refers to a glyceride having two fatty acid chains covalently bonded to a glycerol backbone through respective ester linkages. The DAG includes in some embodiments rac-1,3 or sn-1,2 lipids.

[0167] Exemplary diacylglycerides include, but are not limited to, glycerol dilaurate, glycerol dimyristate, glycerol dipalmitate, glycerol distearate, glycerol diarachidate, glycerol dibehenate, glycerol dipalmitoleate, glycerol dioleate, glycerol dilinoleate, glycerol dilinolenate, glycerol diarachidonate, glycerol dicaprylate, l-stearoyl-3-oleyl-glycerol, 1 -stearoyl -2 -oleyl-sn-glycerol or combinations thereof.

[0168] The term "monoacylglycerol lipid" or "monoacylglyceride" (MAG) as used herein refers to a glyceride having one fatty acid chain covalently bonded to a glycerol backbone through an ester linkage. In one embodiment, the monoacylglycerol lipid is a 1 -monoacylglycerol or 2- monoacylglycerol, depending on the position of the ester bond on the glycerol backbone.

[0169] Exemplary monoacylglycerol lipids for incorporation in the LNP include, but are not limited to, glycerol monolaurate, glycerol monomyristate, glycerol monopalmitate, glycerol monostearate, glycerol monoarachidate, glycerol monobehenate, glycerol monopalmitoleate, glycerol monooleate, glycerol monolinoleate, glycerol monolinolenate, glycerol monoarachidonate, and glycerol monocaprylate, and / or for example 1 -monomyristoyl -rac glycerol, 1 -mono-palm itoyl-rac -glycerol, 2-monopahnitoylglycerol, 1-mono-palmitolenyl-rac- glycerol, 1 -monostearoyl -rac-glycerol, 1 -monoleoyl -rac-glycerol, 1- monolinoleoyl-rac-glycerol, 1-monolinolenoyl-rac-glycerol or combinations thereof.Sterol

[0170] The lipid nanoparticles include “sterol”, which refers to steroids that are naturally- occurring or synthetic and encompasses derivatives thereof. The term includes phytosterols, zoosterols and derivatives thereof. The term “sterol derivatives” refers to modified sterols or precursors thereof, including triterpenes.

[0171] In one embodiment, the sterol is cholesterol. The term “cholesterol” refers to a naturally- occurring or synthetic compound having a gonane skeleton, and derivatives thereof, and that has a hydroxyl bonded to one of its rings, typically the A-ring. The cholesterol derivative may be naturally-occurring or synthetic and includes but is not limited to a cholesterol molecule having a gonane structure and one or more additional functional groups, including derivatization of the terminal hydroxyl group.

[0172] In another embodiment, the LNP may comprise a triterpene. Non-limiting examples include squalene, achilleol A, polypodatetraene, malabaricane, lanostane, cucuribitacin, hopane, oleanane and urosolic acid.

[0173] In certain embodiments, the cholesterol derivative is a phytosterol. The phytosterol may be [3-sitosterol, 3-sitosterol, campesterol, stigmasterol, fucosterol, or stigmastanol or a salt or ester thereof.

[0174] In certain embodiments, the cholesterol derivative is selected from [3-sitosterol, [3- sitosterol acetate, 3-sitosterol, campesterol, stigmasterol, fucosterol, or stigmastanol, dihydrocholesterol, ent-cholesterol, epi-cholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 3P[N-(N'N'- dimethylaminoethyl)carbamoyl cholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25- hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23 -oxacholesterol, 24- oxacholesterol, cycloartenol, 22 -ketosterol, 20-hydroxysterol, 7 -hydroxy cholesterol, 19- hydroxycholesterol, 22-hydroxycholesterol, 25 -hydroxycholesterol, 7-dehydrocholesterol, 5a- cholest-7-en-3p-ol, 3,6,9-trioxaoctan-l-ol-cholesteryl-3e-ol, dehydroergosterol, 9, 11 - dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, sitocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22- dihydroegocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fucosterol, fecosterol, daucosterol or a salt or ester thereof.

[0175] The sterol or cholesterol derivative may be conjugated to another moiety, such as an amino acid or an alkyl group.

[0176] In one embodiment, the sterol is present at from 0 mol% to 50 mol%, 10 mol% to 50 mol%, 15 mol% to 45 mol% or 20 mol% to 35 mol%, based on the total lipid present in the lipid nanoparticle.

[0177] In one embodiment, the cholesterol is present at from 0 mol% to 50 mol%, 10 mol% to 50 mol%, 15 mol% to 45 mol% or 20 mol% to 35 mol%, based on the total lipid present in the lipid nanoparticle.Ionizable lipid

[0178] The LNP of the disclosure has an ionizable lipid. The ionizable lipid may be charged at low pH and have substantially no net charge at physiological pH. This allows for electrostatic interactions between the lipid and the negatively charged nucleic acid cargo during initial formulation. Since the ionizable lipid is near neutral at physiological pH, toxicity and renal clearance is reduced. After cellular uptake by endocytosis, the acidic environment of the endosome leads to an increase in the net positive charge of the ionizable amino lipids, which promotes fusion with the anionic lipids of the endosomal membrane and subsequent membrane destabilization and release of the nucleic acid-based therapeutics into the cytoplasm to exert their effects.

[0179] In some embodiments, it is desirable to include less than 50 mol% ionizable lipid. That is, the ionizable lipid content may be less than 50 mol%, less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol% or less than 5 mol%.

[0180] In certain embodiments, the ionizable lipid content is from 5 mol%to 50 mol%or 8 mol% to 47 mol% or 10 mol%to 50 mol% or 15 mol% to 45 mol% or 15 mol%to 35 mol% of total lipid present in the lipid nanoparticle.

[0181] As used herein, the term "cationic lipid" refers to a lipid that, at a given pH, such as physiological pH, is in an electrostatically neutral form and that may either accept or donate protons, thereby becoming electrostatically positively charged, and for which the electrostatically neutral form has a calculated logarithm of the partition coefficient between water and 1 -octanol(i.e a cLogP) greater than 8. In some embodiments, the cationic lipid has an apparent pKa that is between 5.0 and 8.0 when formulated in an LNP.

[0182] In some embodiments, the LNP has an apparent pKa of between 5.0 and 7.5, between 6.5 and 7.5 or between 6.8 and 7.3. The apparent pKa is measured using a 6-(p-Toluidino)-2- naphthalenesulfonic acid (TNS) assay adapted from previous studies from other groups (Shobaki et al., 2018, International Journal of Nanomedicine, 13:8395-8410; and Jayaraman et al., 2012, Angew. Chem Int. Ed., 51:8529-8533, which are incorporated herein by reference for the purposes of determining apparent pKa). According to the method, a series of buffers are prepared spanning a pH range of 2-11 in 0.5 pH unit increments consisting of 130 mM NaCl, 10 mM ammonium acetate, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), and 10 mM HEPES. 0.15-0.2 mM ofthe LNP. A solution of 0.06 mM of TNS is subsequently mixed with 175 pL of the LNP at each buffered pH in triplicate in a black, polysterene 96-well plate, to yield a final concentration of 6.25 and 6 pM of lipid and TNS in each well, respectively. Fluorescence is subsequently measured using an SpectraMax™ M5 microplate reader at ex 321 nm, em 445 nm. The fluorescence is then plotted against pH using a sigmoidal curve fit through Prism™, in which the pKa is determined to be the pH value with 50% of maximal fluorescent intensity.

[0183] In some embodiments, the cationic lipid has an amino group. In some embodiments, the cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group, C16 to C 18 alkyl chains, optionally ether linkages between its head group and alkyl chains, and 0 to 3 double bonds. Such lipids include, but are not limited to sulfur lipids, such as MF019 described in WO 2022 / 155728A1 and DODMA. Other lipids that may be used in the practice of the disclosure include MC3- and KC2-type lipids, which are well-known to those of skill in the art. In further embodiments, the ionizable lipid is selected from one or more lipids set forth in WO 2022 / 246555; WO 2022 / 246568; WO 2022 / 246571; WO 2023 / 147657; WO2022 / 155728; WO 2023 / 215989; WO 2024 / 065041; WO 2024 / 065042; PCT / CA2023 / 051727 filed on December 21, 2023; WO 2024 / 065043; and U.S. patent application No. 18 / 442,431 filed on February 15, 2024, each incorporated herein by reference.

[0184] In one embodiment, the ionizable cationic lipid comprises an ionizable amino head group and at least two lipophilic groups, at least one of which comprises a heteroatom or a moiety comprising one or more heteroatoms, such as an ester or one or more sulfur atoms. In someembodiments, at least one lipophilic group comprises distal branching and / or one or more cyclic groups. Examples of ionizable cationic lipids comprising an ionizable amino head group and two lipophilic chains, at least one chain comprising one or more sulfur atoms and / or ester groups are described in co-owned and co-pending WO 2023 / 215989; WO 2024 / 065041; WO 2024 / 065042; PCT / CA2023 / 051727 fded on December 21, 2023; and WO 2024 / 065043. Functional groups comprising one or more heteroatoms may be biodegradable in vivo.

[0185] In one embodiment, the ionizable cationic lipid has a protonatable amino head group; at least two lipophilic moieties, wherein the amino head group has a central nitrogen atom or carbon atom to which each of the two lipophilic moieties are directly bonded; each lipophilic chain has between 15 and 40 carbon atoms in total; and wherein the lipid has (i) an apparent pKaof between 6 and 7.5 (measured as per the TNS assay); and (ii) a ClogP of at least 11.

[0186] Optionally, at least one of the lipophilic moieties bonded to the head group has a biodegradable group, including but not limited to an ester group or a moiety comprising an ester (in any orientation). In one embodiment, at least one of the lipophilic moieties comprises a sulfur atom. In another embodiment, at least one of the lipophilic moieties comprises an ester or a moiety comprising an ester, one or more sulfur atoms or any combination thereof.

[0187] In one embodiment, the lipid nanoparticle comprises a “sulfur-containing amino lipid”, which is an ionizable lipid having at least one ionizable nitrogen (tertiary nitrogen) and one or more sulfur atoms in at least one of its lipophilic chains. In one embodiment, the sulfur-containing amino lipid further comprises a biodegradable group such as an ester moiety in one or more of its lipophilic chains. Non-limiting examples of sulfur-containing lipids are provided in Table 3 and in the foregoing co-owned PCT applications. The incorporation of the biodegradable group(s) into the lipid increases metabolism post-administration and improves clearance of the lipid from the body following delivery of the active agent to a target area. Generally, such lipids have decreased toxicity when compared to similar lipids without the biodegradable groups.

[0188] Sulfur-containing ionizable lipids and / or ionizable lipids with one or more biodegradable groups (e.g., esters, groups comprising an ester or a disulfide) in one or more of their lipophilic chains have been shown to be particularly efficacious when formulated in LNPs, including unshielded LNPs, as described in the Example section herein.

[0189] Non-limiting examples of atoms or substituents that may replace a carbon atom (interrupt the alkyl) in the lipophilic chains of the ionizable cationic lipid include cycloalkyl groups (mono or polycyclic); — O— ; — (C=O)O— ; — O(C=O)-; — C(=O); — O(C=O)O-; — S(O)X-; — S— ; — S— S— ; — C(=O)S-; — SC(=O)-; —NR'—; — NR'C(=O) — ; — C(=O)NR'— ; — NR'C(=O)NR'— ; — OC(=O)NR'— ; — NR'C(=O)OR’— ; — NR'S(O)XNR'— ; — NR'S(O)XR’— ; and — S(O)XNR' — , wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0190] By the term “biodegradable group”, with reference to a group in a lipophilic chain of an ionizable lipid, includes a functional group with one or more electronegative atoms (e.g., O, N, S or P) that is metabolized in vivo by an enzyme, thereby increasing its metabolism relative to an otherwise identical ionizable lipid that does not contain such group. Non-limiting examples include — O— ; — (C=O)O— ; — O(C=O)-; — C(=O); — O(C=O)O-; — S(O)X-; — S— ; — S— S— ; — C(=O)S-; — SC(=O)-; —NR'—; — NR'C(=O) — ; — C(=O)NR'— ; — NR'C(=O)NR'— ; — OC(=O)NR'— ; — NR'C(=O)OR’— ; — NR'S(O)XNR'— ; — NR'S(O)XR’— ; and — S(O)XNR'— , wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0191] Non-limiting examples of atoms or substituents that may replace a hydrogen atom in the ionizable cationic lipid include halogen; deuterium, an alkyl group; a cycloalkyl group (mono or polycyclic); an oxo group (=0); a hydroxyl group (-OH); — (C=O)OR'; — O(C=O)R'; — C(=O)R'; O(C=O)OR'-; —OR'; — S(O)XR'; —SR', — S— SR'; — C(=O)SR'; — SC(=O)R'; — NR'R'; — NR'C(=O)R'; — C(=O)NR'R'; — NR'C(=O)NR'R'; — OC(=O)NR'R'; — NR'C(=O)OR'; — NR'S(O)XNR'R'; — NR'S(O)XR'; and — S(O)XNR'R', wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0192] In one embodiment, the ionizable amino lipid is a “non-furan ring-containing ionizable lipid”, which is an ionizable lipid that lacks a furan ring, such as an oxolane in the head group moiety. In one embodiment, the ionizable lipid is a “non-MC3 type lipid”, which is an ionizable lipid that lacks in its head group a terminal ionizable amino group that is a methyl 4- (dimethylamino)butanoate. In another embodiment, the ionizable lipid is a non-furan ringcontaining ionizable lipid that is also a non-MC3 type lipid. In one embodiment, the ionizableamino lipid is a “non-cyclopentane ring-containing ionizable lipid”, which is an ionizable lipid that lacks a cyclopentane moiety in the head group moiety.

[0193] In one non-limiting example, at least one of the lipophilic groups or moieties of the ionizable lipid has the formula:or is an amino acid or a sarcosine group. The ester (which may be denoted as “E”) is present in either orientation (i.e., the carbonyl (C=O) is bonded to R1or the -O- of the ester is bonded to R1).

[0194] In one embodiment, R1and R2are, independently, linear, cyclic and / or branched optionally substituted C3-C20 alkyl and optionally with varying degrees of unsaturation; and n is 4 to 8. In an alternative embodiment, R2is C2-C 12 alkyl (including alkenyl).

[0195] In some embodiments, it is desirable to include less than 50 mol% ionizable cationic lipid in the LNP. That is, the ionizable cationic lipid content may be less than 50 mol%, less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol% or less than 5 mol%.

[0196] In certain embodiments, the ionizable cationic lipid content is from 5 mol% to 50 mol% or 8 mol% to 47 mol% or 10 mol% to 50 mol% or 15 mol% to 45 mol% or 15 mol% to 35 mol% of total lipid present in the lipid nanoparticle.

[0197] The ionizable lipid component may include an ionizable anionic lipid as part of the ionizable lipid content. An example of such a lipid is cholesteryl hemisuccinate (CHEMS). Further examples of ionizable anionic lipids are described in co-pending and co-owned U.S. PCT / CA2024 / 050347 filed on March 22, 2024, which is incorporated herein by reference in its entirety.Additional components

[0198] The LNP may comprise additional lipid components or modifications to the sterol (e.g., cholesterol), sterol derivative (e.g., cholesterol derivative) and / or other lipid components.

[0199] For example, the surface of the LNP may be grafted to comprise a targeting ligand. The targeting ligand may be conjugated to cholesterol. The targeting ligand may be conjugated to the distal end of a hydrophilic polymer-lipid conjugate when present (typically at less than 0.5 mol%). In some embodiments, the targeting ligand may be used to target receptors on cells in vivo. In some embodiments, the targeting ligand may be conjugated to a phospholipid.

[0200] The ligand includes peptides, polypeptides or proteins and includes antibodies or fragments thereof. In one embodiment, the ligand may be a single-chain antibody fragment. Examples of ligands are described in PCT / CA2023 / 051632 filed on December 8, 2023, which is incorporated herein by reference.

[0201] The LNP may further comprise a tocopherol as an additional component. The tocopherol includes an a-tocopherol, [3-tocopherol, y-tocopherol, 5-tocopherol or a salt or ester thereof. The tocopherol may be present at 0.5 mol% to 20 mol%, 1 mol% to 15 mol% or 2 mol% to 10 mol%.Nanoparticle preparation and morphology

[0202] Lipid nanoparticles can be prepared using any of a variety of suitable methods, such as a rapid mixing / ethanol dilution process. Examples of preparation methods are described in Jeffs, L.B., et al., Pharm Res, 2005, 22(3):362-72; and Leung, A.K., et al., The Journal of Physical Chemistry. C, Nanomaterials and Interfaces, 2012, 116(34): 18440-18450, each of which is incorporated herein by reference in its entirety.

[0203] For example, the method of preparing the lipid nanoparticles may comprise dissolving lipid components (e.g., ionizable lipid, phosphatidylcholine and a sterol or derivative thereof) at appropriate ratios in an organic solvent (e.g., ethanol). An aqueous buffer comprising nucleic acid is prepared separately at a suitable pH to ensure that the head group (e.g., amino group) of an ionizable lipid is protonated to facilitate electrostatic interaction with the negatively charged cargo, and the positively charged ionizable lipid. Such charge interaction improves nucleic acid encapsulation.

[0204] In some embodiments, the aqueous phase comprising the nucleic acid is subsequently combined with the organic solvent-lipid mixture comprising the lipids. Combining of the aqueous phase and the organic-solvent-lipid mixture may be carried out in a mixing device (e.g., in-line mixer), such as a T-junction mixer with pumps (e.g., a T-tube mixer), a herringbone micromixer,a toroidal mixer, a multi -inlet vortex mixer or other suitable mixing devices known to those of skill in the art. In some embodiments, the mixing device refers to a device comprising two or more inlets meeting in a central mixing region and an outlet through which the mixture exits the device. The LNP formation may occur upon mixture of the aqueous phase and organic solvent-lipid mixture and / or subsequent to such mixing. (Kulkami et al., 2019, Nanoscale, 11(18):9023-9031, which is incorporated herein by reference).

[0205] The aqueous phase typically comprises a buffer. Non-limiting examples of suitable buffers include one or more of sodium acetate, phosphate buffered saline (PBS), sodium formate and sodium succinate. Examples of suitable solvents to prepare the organic solvent-lipid mixture are organic solvents including ethanol, isopropanol, methanol and acetone. In one embodiment, the organic solvent-lipid mixture comprises ethanol.

[0206] The aqueous phase and organic-solvent lipid mixture may be introduced to the mixer as two separate respective streams via pumps. The volumetric flow rate of each stream may be the same or different and the respective flow rates of each stream may be adjusted to achieve optimal mixing and / or LNP formation.

[0207] In one embodiment, the lipid nanoparticle is prepared using a modified process in which the amount of organic solvent in the lipid nanoparticle preparation formed in the mixing device is reduced relative to the conventional process. The modified process allows for the formulation of ionizable cationic lipids in PEG-less lipid nanoparticles that may not otherwise be capable of being formulated in such particles (see Materials and Methods).

[0208] The inventive process involves producing unshielded lipid nanoparticles (e.g., PEG-less lipid nanoparticles or lipid nanoparticles with substantially no hydrophilic polymer-lipid conjugate) with lower amounts of organic solvent relative to the conventional rapid mixing process. In one exemplary embodiment, this is achieved by adjusting volumetric flow rates of the two streams combined in the mixer. In one embodiment, the volumetric flow rate of the aqueous buffer solution comprising the nucleic acid is greater than the flow rate of the organic solvent lipid containing mixture, thereby resulting in a lipid nanoparticle solution having reduced levels of the organic solvent. A non-limiting example of a suitable flow rate ratio is 3: 1 aqueous: organic solutions v / v. A person of ordinary skill in the art can select a suitable flow rate to the mixer or this could be determined empirically to best suit the mixer used. The flow rate of the aqueousphase may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% greater than the volumetric flow rate of the organic solvent lipid mixture.

[0209] Alternatively or additionally, the lipid nanoparticle solution may be diluted with a buffer solution after exiting the mixing device to reduce the organic solvent concentration. In some embodiments, the lipid nanoparticle so produced may be diluted at a dilution ratio above 0.5:1 (v:v) of buffer solution: lipid nanoparticle solution after existing the mixing device. In some embodiments, the dilution ratio of bufferlipid nanoparticle solution is at least 0.5: 1, 0.55: 1, 0.60: 1, 0.65:1, 0.70:1, 0.75:1, 0.80:1, 0.85:1, 0.90:1, 0.95:1, 1:1, 1.05:1, 1.10:1, 1.15:1, 1.20:1, 1.25:1, 1.30:1, 1.35:1, 1.40:1, 1.45:1, 1.50:1, 1.55:1, 1.60:1, 1.65:1, 1.70:1, 1.75:1, 1.80:1, 1.85:1, 1.90:1, 1.95 : 1 or 2.0: 1. In some advantageous embodiments, the dilution ratio of buffer: lipid nanoparticle solution is at least 0.90: 1,0.95:1, 1:1, 1.05:1, 1.10:1, 1.15:1, 1.20:1, 1.25:1, 1.30:1, 1.35:1, 1.40:1, 1.45:1, 1.50:1, 1.55:1, 1.60:1, 1.65:1, 1.70:1, 1.75:1, 1.80:1, 1.85:1, 1.90:1, 1.95:1 or 2.0:1. In some embodiments, the upper limit of the dilution ratio of buffer: I ipid nanoparticle solution is 5 : 1 , 4.5:1, 4.0:1, 3.5:1, 3.0:1 or 2.5:1.

[0210] In some examples, the buffer used to dilute the lipid nanoparticle solution after exiting the mixing device may be sodium acetate, phosphate buffered saline (PBS), sodium formate and / or sodium succinate. The buffer may be the same or different from the buffer used in the aqueous buffer solution comprising the nucleic acid.

[0211] The lipid nanoparticles may have an average size of between 40 and 200 nm or between 40 and 170 nm or between 45 and 130 nm or any range therebetween. In another embodiment, the lipid nanoparticle has a PDI of less than 0.30, or less than 0.20 or less than 0.15 or less than 0.12 or less than 0.10.

[0212] The nitrogen-to-phosphate ratio of the lipid nanoparticle may be between 1 and 12. In another embodiment, the nitrogen-to-phosphate ratio of the lipid nanoparticle may be between 1 and 9.

[0213] The LNP generally comprises a “core” region, which may be characterized as being electron dense as visualized by cryo-TEM microscopy or having an electron dense region and an aqueous region within the core. The morphology is assessed visually by cryo-TEM microscopy as set forth in the Materials and Methods herein. Figure 18 shows particles that display the latter morphology.

[0214] Without being limiting, the electron dense region within the core may be partially surrounded by an aqueous portion within the enclosed space, entirely surrounded or enveloped by an aqueous portion within the core or may have a solid core without an aqueous portion as observed by cryo-TEM. The core may comprise nucleic acid and ionizable lipid. In one embodiment, the phosphatidylcholine and sterol or derivative thereof is present primarily in an outer lipid layer and the ionizable cationic lipid and nucleic acid cargo is present in the core of the LNP.

[0215] In one embodiment, at least one about fifth of the core (trapped volume) contains the aqueous portion or compartment, and in which the electron dense region within the core is partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM. In another embodiment, at least one about quarter of the core contains the aqueous portion or compartment, and in which the electron dense core is either partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM. In a further embodiment, at least one about one third of the core contains the aqueous portion or compartment, and in which the electron dense region is either partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM. In another embodiment, at least one about one half of the core contains the aqueous portion or compartment, and in which the electron dense core is either partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM.

[0216] In another embodiment, the electron dense region of the LNP surprisingly appears to be completely surrounded by the aqueous portion of the core as visualized by cryo-TEM microscopy. This morphology is observed in a single plane and a portion of the electron dense region as observed is contiguous with the lipid layer (e.g., bilayer) but cannot be seen since this portion is not within the plane that can be visualized.

[0217] In some embodiments, the LNP comprises at least a bilayer surrounding the core. In some embodiments, the LNP comprises a continuous or discontinuous bilayer surrounding the aqueous region of the core. In some embodiments, the LNP comprises a bilayer surrounding at least the aqueous region of the core. In some examples, the electron dense region is surrounded by a monolayer and the aqueous region is surrounded by a bilayer as visualized by cryo-TEM (see Figure 18).

[0218] In one embodiment, the electron dense region is generally spherical in shape. In another embodiment, the electron dense region is hydrophobic.

[0219] In some embodiments, the density of the electron dense region has an intensity as measured by imaging software that is at least 10%, at least 20%, at least 30%, at least 40% greater or at least 50% greater than that of the aqueous region. The density of the electron dense region relative to the aqueous region of the core is assessed by analysis of the cryo-TEM image of the LNP using Image J™ software available at https: / / imagej .net / ij / (incorporated herein by reference), which is a Java-based image processing program developed by the National Institute of Health to analyze images. The cryo-TEM image is first focused and subsequently defocused to enhance the contrast for ease of visualization. The level of defocusing can be determined by a person of ordinary skill in the art. A suitable amount of defocus is 0.5-2 pm. After the desired level of contrast is achieved, a line is drawn through the LNP particle image bisecting the aqueous region and electron dense region. The image intensity of the line is measured along its length using Image J™ software available at https: / / imagej.net / ij / , which is a Java-based image processing program developed by the National Institute of Health to analyze images. The image intensity of two equal areas is measured, one of which is measured at the aqueous region and the other at the electron dense region. After the image intensities are quantified, the increase in intensity of the electron dense region relative to the aqueous portion is determined and expressed as a percentage.

[0220] In some embodiments, the electron dense region in the core has an image intensity that is similar to that of the periphery of the particle, which in this case is a lipid layer (bilayer or monolayer). For example, in some embodiments, the lipid layer image intensity as measured by hnageJ™ does not deviate by more than 20% or 10% relative to that of the electron dense region within the core. The relative intensities are measured in equal areas as described earlier and expressed as a percentage.

[0221] In some embodiments, the LNP is not a lipoplex. Lipoplexes are prepared by mixing preformed cationic liposomes with nucleic acid in an aqueous solution and may exhibit undesirable properties such as localization of the cargo on the particle surface. Lipoplexes lack the abovedescribed core of the LNP particle. Further, LNPs have a defined size, shape and morphology whereas lipoplexes lack such defined physical characteristics. (See Kubota et al., 2017, Int. J. Nanomedicine, 12:5121-5133 and Kulkami et al., 2018, Nucleic Acid Therapeutics, 28(3): 146- 157, which are each incorporated herein by reference).

[0222] Further, it should be understood that the morphology observed results when particles are prepared with the molar ratios of lipids as described and using T-junction mixing or similar methods. This morphology is not observed with standard methods used to prepare liposomes, such as thin layer evaporation and extrusion.

[0223] As used herein, the term “encapsulation,” with reference to incorporating the nucleic acid cargo within an LNP refers to any association of the nucleic acid with any lipid component or compartment of the lipid nanoparticle. However, this excludes localization of the nucleic acid on the particle surface as in lipoplexes.Nucleic acid cargo

[0224] In one embodiment, the cargo is a nucleic acid. The nucleic acid includes, without limitation, RNA, including small interfering RNA (siRNA), small nuclear RNA (snRNA), micro RNA (miRNA), messenger RNA (mRNA) or DNA such as vector DNA or linear DNA. The nucleic acid length can vary and can include nucleic acid of 1-50,000 nucleotides in length. The nucleic acid can be in any form, including single stranded DNA or RNA, double stranded DNA or RNA, or hybrids thereof. Single stranded nucleic acid includes antisense oligonucleotides. The nucleic acid may be conjugated to another molecule, including a targeting moiety. An example of such a nucleic acid conjugate is an antibody-nucleic acid conjugate, or an oligosaccharide -nucleic acid conjugate, such as a GalNAc-nucleic acid conjugate.

[0225] In one embodiment, the cargo is an mRNA, which includes a polynucleotide that encodes at least one peptide, polypeptide or protein. The mRNA includes, but is not limited to, small activating RNA (saRNA) and trans-amplifying RNA (taRNA), as described in co-pending WO 2023 / 184038, which is incorporated herein by reference.

[0226] The mRNA as used herein encompasses both modified and unmodified mRNA. In one embodiment, the mRNA comprises one or more coding and non-coding regions. The mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, or may be chemically synthesized.

[0227] In those embodiments in which an mRNA is a chemically synthesized molecule, the mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and / or backbone modifications. In some embodiments, an mRNA is or comprises naturalnucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5 -fluorouridine, C5 -iodouridine, C5 -propynyl-uridine, C5-propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5 -methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2 '-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0228] The mRNAs of the disclosure may be synthesized according to any of a variety of known methods. For example, mRNAs in certain embodiments may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.

[0229] In some embodiments, in vitro synthesized mRNA may be purified before encapsulation to remove undesirable impurities including various enzymes and other reagents used during mRNA synthesis.

[0230] The present disclosure may be used to encapsulate mRNAs of a variety of lengths. In some embodiments, the present disclosure may be used to encapsulate in vitro synthesized mRNA ranging from about 1-20 kb, about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5- 15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb in length.

[0231] Typically, mRNA synthesis includes the addition of a “cap” on the 5' end, and a “tail” on the 3' end. The presence of the cap is advantageous in that it may provide resistance to nucleases found in eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

[0232] In some embodiments, mRNAs include a 5' and / or 3' untranslated region. In some embodiments, a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5'untranslated region may be between about 1 and 500 nucleotides in length or 50 and 500 nucleotides in length or longer.

[0233] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between 1 and 500 nucleotides in length or 50 and 500 nucleotides in length or longer.

[0234] In a further embodiment, the mRNA is circular. Advantageously, such mRNA lacks 5’ and 3’ ends and thus may be more stable in vivo due to its resistance to degradation by exonucleases. The circular mRNA may be prepared by any known method, including any one of the methods described in Deviatkin et al., 2023, “Cap-Independent Circular mRNA Translation Efficiency”, Vaccines, 11(2), 238, which is incorporated herein by reference. Translation of the circular mRNA is carried out by a cap-independent initiation mechanism.

[0235] While mRNA provided from in vitro transcription reactions may be desirable in certain embodiments, other sources of mRNA are contemplated, such as mRNA produced from bacteria, fungi, plants, and / or animals.

[0236] The mRNA sequence may comprise a reporter gene sequence, although the inclusion of a reporter gene sequence in pharmaceutical formulations for administration is optional. Such sequences may be incorporated into mRNA for in vitro studies or for in vivo studies in animal models to assess expression and biodistribution.

[0237] In another embodiment, the cargo is an siRNA. An siRNA becomes incorporated into endogenous cellular machineries to result in mRNA breakdown, thereby preventing transcription. Since RNA is easily degraded, its incorporation into a delivery vehicle can reduce or prevent such degradation, thereby facilitating delivery to a target site.

[0238] The siRNA encompassed by embodiments of the disclosure may be used to specifically inhibit expression of a wide variety of target polynucleotides. The siRNA molecules targeting specific polynucleotides for any therapeutic, prophylactic or diagnostic application may be readily prepared according to procedures known in the art. An siRNA target site may be selected and corresponding siRNAs may be chemically synthesized, created by in vitro transcription, or expressed from a vector or PCR product. A wide variety of different siRNA molecules may beused to target a specific gene or transcript. The siRNA may be double-stranded RNA, or a hybrid molecule comprising both RNA and DNA, e.g., one RNA strand and one DNA strand. The siRNA may be of a variety of lengths, such as 1 to 30 nucleotides in length or 15 to 30 nucleotides in length or 20 to 25 nucleotides in length. In certain embodiments, the siRNA is double-stranded and has 3' overhangs or 5' overhangs. In certain embodiments, the overhangs are UU or dTdT 3'. In particular embodiments, the siRNA comprises a stem loop structure.

[0239] In a further embodiment, the cargo molecule is a microRNA or small nuclear RNA. Micro RNAs (miRNAs) are short, noncoding RNA molecules that are transcribed from genomic DNA, but are not translated into protein. These RNA molecules are believed to play a role in regulation of gene expression by binding to regions of target mRNA. Binding of miRNA to target mRNA may downregulate gene expression, such as by inducing translational repression, deadenylation or degradation of target mRNA. Small nuclear RNA (snRNA) are typically longer noncoding RNA molecules that are involved in gene splicing. The snRNA molecules may have therapeutic or diagnostic importance in diseases that are an outcome of splicing defects.

[0240] Examples of the nucleic acid cargo include but are not limited to antisense oligonucleotides, ribozymes, microRNA, mRNA, ribozyme, tRNA, tracrRNA, sgRNA, snRNA, siRNA, shRNA, ncRNA, miRNA, mRNA, pre-condensed DNA, pDNA or an aptamer.

[0241] In another embodiment, the cargo is a DNA vector. The encapsulated DNA vectors may be administered to a subject for the purpose of repairing, enhancing or blocking or reducing the expression of a cellular protein or peptide. In another embodiment, the encapsulated DNA vectors may be administered to a subject for diagnosis of disease. The DNA vector may localize in target cells (e.g., rapidly dividing cells) and expression of encoded DNA may be used to provide a measurable signal. Accordingly, the nucleotide polymers can be nucleotide sequences including genomic DNA, cDNA, or RNA.

[0242] As will be appreciated by those of skill in the art, the vectors may encode promoter regions, operator regions or structural regions. The DNA vectors may contain double-stranded DNA or may be composed of a DNA-RNA hybrid. Non-limiting examples of double-stranded DNA include structural genes, genes including operator control and termination regions, and selfreplicating systems such as vector DNA.

[0243] Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes and triplex-forming oligonucleotides. In order to have prolonged activity, the single-stranded nucleic acids will most advantageously have some or all of the nucleotide linkages substituted with stable, non-phosphodiester linkages, including, for example, phosphorothioate, phosphorodithioate, phophoroselenate, or O-alkyl phosphotriester linkages.

[0244] The DNA vectors may include nucleic acids in which modifications have been made in one or more sugar moieties and / or in one or more of the pyrimidine or purine bases. Such sugar modifications may include replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, azido groups or functionalized as ethers or esters. In another embodiment, the entire sugar may be replaced with sterically and electronically similar structures, including azasugars and carbocyclic sugar analogs. Modifications in the purine or pyrimidine base moiety include, for example, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substitutes known to those of skill in the art.

[0245] The DNA vector may be modified in certain embodiments with a modifier molecule such as a peptide, protein, steroid or sugar moiety. Modification of a DNA vector with such molecule may facilitate delivery to a target site of interest. In some embodiments, such modification translocates the DNA vector across a nucleus of a target cell. By way of example, a modifier may be able to bind to a specific part of the DNA vector (typically not encoding of the gene-of-interest), but also has a peptide or other modifier that has nucleus-homing effects, such as a nuclear localization signal. A non-limiting example of a modifier is a steroid-peptide nucleic acid conjugate as described by Rebuffat et al., 2002, Faseb J. 16(11): 1426-8, which is incorporated herein by reference. The DNA vector may contain sequences encoding different proteins or peptides. Promoter, enhancer, stress or chemically-regulated promoters, antibiotic-sensitive or nutrient-sensitive regions, as well as therapeutic protein encoding sequences, may be included as required. Non-encoding sequences may be present as well in the DNA vector.

[0246] The nucleic acids used in the present disclosure can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries or prepared by synthetic methods. Synthetic nucleic acids can be prepared by a variety of solution or solid phase methods. Generally, solid phase synthesis is preferred. Known procedures for solid phase synthesis of nucleic acids by phosphite-triester, phosphotriester, and H-phosphonate chemistries are widely available.In one embodiment, the DNA vector is double stranded DNA and comprises more than 700 base pairs, more than 800 base pairs or more than 900 base pairs or more than 1000 base pairs.Editing of genetic material

[0247] The LNP may comprise a nucleic acid that encodes for a protein or peptide that forms part of a “genetic editor” or “editor”, which includes without limitation products or compositions that edit genetic material, e.g., nucleic acid is inserted, deleted, modified (e.g., epigenetic editing) or replaced in the genetic material of an organism at a site-specific location.

[0248] The editor may be used for ex vivo or in vivo genetic modification of a hepatic cell and includes post-translational modifications.

[0249] The genetic editor includes, without limitation, Cas-based (e.g., CRISPR or non- CRISPR), transcription activator-like effector nuclease (TALEN), megaTALs, zinc finger nuclease (ZFN), Adenosine Deaminase Acting on RNA (ADAR), prime editors, base editors, epigenetic, transposase, meganuclease, ARCUS gene editing systems or any variant or combination thereof. These editors are exemplary, however, and the disclosure includes any product or composition that can modify genetic material (including RNA transcripts and noncoding regions) of a hepatic cell to treat, prevent or ameliorate a disorder or disease. Without limitation, the editor may include those that are designed by a process referred to as Directed Nuclease Editor (DNE), which is known to those of skill in the art.

[0250] Cas-based editors comprise CRISPR and non-CRISPR gene editing systems. In addition, the editor includes those that cut DNA as well as epigenetic editing systems that modify nucleic acid markers, as discussed below.

[0251] The CRISPR nucleic acid editor most advantageously comprises nucleic acid (e.g., mRNA) encoding for one or more of a Class II Cas nuclease family of proteins and a guide RNA. The nucleases encoded by the nucleic acid are enzymes with DNA endonuclease activity and can be directed to cleave a desired nucleic acid target by an appropriate guide RNA. The nuclease and guide RNA form a complex referred to as a ribonucleoprotein (RNP). In some embodiments, the nuclease is a Class II CRISPR enzyme, which is further subdivided into Types II, V and VI. According to one embodiment, the mRNA encodes for a Cas protein that is part of a Type II CRISPR / Cas system, such as a Cas9 protein or a Cpfl protein.

[0252] In another embodiment, the mRNA encodes for a Cas protein that is part of a Type V CRISPR / Cas system, such as Cas 12a. In another embodiment, the mRNA encodes for a Cas protein that is a Cas 13a, which is an RNA endonuclease and cleaves single-stranded RNA.

[0253] The guide RNA can direct the Cas nuclease to the target sequence on a target nucleic acid molecule, where the guide RNA hybridizes to the target sequence and the Cas nuclease cleaves or modulates the sequence. In some embodiments, the guide RNA binds to a class 2 nuclease, thereby providing specificity of cleavage.

[0254] Guide RNAs for the CRISPR / Cas9 nuclease system include CRISPR RNA (crRNA) or tracr RNA (tracr). In some embodiments, the crRNA can include a targeting sequence that is complementary to and hybridizes to a target sequence on a target nucleic acid molecule. The crRNA can also include a flagpole that is complementary to, and hybridize to, a portion of tracrRNA. In some embodiments, the crRNA can correspond to the structure of a naturally- occurring crRNA transcribed from a bacterial CRISPR locus, wherein the targeting sequence acts as a spacer for the CRISPR / Cas9 system. The flagpole corresponds to the part of the repetitive sequence adjacent to the spacer above the CRISPR locus.

[0255] The guide RNA of the RNP can target any sequence of interest through the targeting sequence of a crRNA. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may be 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule can comprise at least one mismatch.

[0256] The length of the targeting sequence may depend on the RNP system and components used. For example, different Cas proteins from different bacterial species have various optimal targeting sequence lengths. Thus, the targeting sequences are: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more than 50 nucleotides in length can be included. In some embodiments, the targeting sequence can comprise a length of 18 to 24 nucleotides. In some embodiments, the targeting sequence can comprise 19-21 nucleotides in length. In some embodiments, the targeting sequence can comprise a length of 20 nucleotides.

[0257] In some embodiments, the nucleic acid editor includes Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbll l, Csb2, Csb3, Csxl7, Csxl4,CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.

[0258] As noted, non-CRISPR, Cas-based nucleic acid editors are encompassed by embodiments of the disclosure as well. A Cas-based editor may include a Cas enzyme fused to deaminase (Luo et al., 2020, Microbial Cell Factories, 19(93), incorporated herein by reference). An example is a cytosine base editor or an adenine base editor produced by fusing endonuclease Cas to cytosine deaminase pmCDAl or heterodimer adenine deaminase TadA-TadA. A further non-limiting example is Cas fused to reverse transcriptase (Mohr et al., 2018, Mol Cell., 72(4):700-714, incorporated herein by reference).

[0259] Fanzor is a eukaryotic RNA-guided endonuclease that could function as a nucleic acid editor. (See Saito et al., 2023, Nature 620:660-668, which is incorporated herein by reference). In some embodiments, Fanzor proteins use RNA as a guide to target DNA precisely and can be modified to edit a hepatic cell using the LNPs described herein. In some examples, the compact Fanzor systems may have the ability to facilitate more improved delivery than CRISPR-Cas systems.

[0260] In those embodiments in which the editor is a TALEN, the LNP comprises a nucleic acid encoding a peptide having a Transcription Activator-Like (TAL) effector DNA binding domain, a fragment or a variant thereof. In an embodiment, the editor comprises a nucleic acid encoding a peptide having nuclease activity, e.g., endonuclease activity. In an embodiment, the peptide having nuclease activity is atype-II restriction 1-like endonuclease, e.g., a Fokl endonuclease.

[0261] In those embodiments in which the nucleic acid editor is a zinc finger nuclease (ZFN), the nucleic acid may encode a peptide having: a Zinc finger DNA binding domain, a fragment or a variant thereof; and / or nuclease activity, e.g., endonuclease activity. In an embodiment, the Zn finger binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8 or more Zinc fingers. In an embodiment, the peptide having nuclease activity is a type-II restriction 1-like endonuclease, e.g., a Fokl endonuclease.

[0262] Adenosine Deaminase Acting on RNA (ADAR) is another editor encompassed by embodiments of the disclosure that may be used for post-transcriptional modification of RNA. Examples include AD ARI and ADAR2. ADAR1 may catalyze posttranscriptional deaminationof C6 of adenosines in dsRNA, converting them to inosines (see Song et al., 2022, PMC, 13( 1): e 1665, incorporated herein by reference).

[0263] Meganucleases are enzymes in the endonuclease family that may induce homologous recombination, generate mutations and alter reading frames. The meganuclease includes homing endonucleases that are intron or intein endonucleases. In one embodiment, the meganuclease is from the LAGLIDADG family, a GIY -YIG endonuclease, an HNH endonuclease, a His-Cys box endonuclease or a PD-(D / E)XK endonuclease. Meganucleases may be combined with components of other editors. In one embodiment, a DNA binding domain from a transcription activator-like (TAL) effector is combined with a meganuclease to produce a “megaTAL”. In another embodiment, a meganuclease may be fused to a DNA end-processing enzyme to promote an error-prone non-homologous end joining.

[0264] ARCUS nuclease is an editor based on I-Crel, which is a kind of homing endonuclease that evolved in the algae Chlamydomonas reinhardtii . In some embodiments, the nuclease can deactivate itself after gene editing, thereby reducing off-targeting. ARCUS nucleases in some embodiments can generate a unique cleavage site that is a four-base-pair, 3’ overhang and may be able to carry out gene insertion, gene excision, gene repair or a combination thereof.

[0265] Epigenetic editing is also encompassed by examples of the disclosure. Such editing of genetic material does not cut nucleic acid but rather alters epigenomic marks “adorning” DNA. Changing the epigenic signature of a hepatic cell can serve to modify an epigenetic signature of the cell and change its transcriptional profile. In some embodiments, the epigenetic editing system may target and edit one or more methylation sites of a nucleic acid sequence. In some embodiments, genome homing proteins with engineered or naturally occurring nuclease functions for gene editing, can be mutated and adapted to function as only delivery systems. In one embodiment, an epigenetic modifying enzyme or domain can be fused to the homing protein and local epigenetic modifications can be altered upon protein recruitment. A targeting protein that recognizes DNA sequences may be linked to an effector protein that alters epigenomic marks, such as methylation. Examples of targeting proteins include Transcription Activator-Like Effector (TALE), zinc finger proteins, and Cas systems, including but not limited to CRISPR-Cas. Nonlimiting examples of effector proteins include TET1, which induces demethylation of cytosine at CpG sites; LSD1, which induces demethylation of H3K4mel / 2, which also causes an indirecteffect of deacetylation on H3K27; and CIB1 / CRY2, which is a cryptochrome / blue light activated complex allowing chromatin to be modified upon illumination.

[0266] Further examples of effector proteins include DNA methyltransferase, a fragment (e.g., a biologically active fragment) or variant thereof (e.g., DNMT1, DNMT2 DNMT3A, DNMT3B, DNMT3L, or CpG methyltransferase (M. Sssl)); or a poly comb repressive complex or a component thereof, e.g., PRC1 or PRC2, or PR-DUB, or a fragment (e.g., biologically active fragment) or a variant thereof.

[0267] In an embodiment, the epigenetic editor comprises a molecule that modifies chromatin architecture and / or modifies a histone. In an embodiment, the epigenetic modulator is a molecule that modifies chromatin architecture, e.g., a SWI / SNF remodeling complex or a component thereof. In an embodiment, the epigenetic modulator is a molecule that modifies a histone, e.g., methylates and / or acetylates a histone, e.g., a histone modifying enzyme or a fragment (e.g., biologically active fragment) or a variant thereof, e.g., HMT, HDM, HAT, or HD AC.Clinical and non-clinical uses of the unshielded LNP herein

[0268] The unshielded lipid nanoparticles of certain embodiments described herein resulted in high ratios of spleen or bone marrow / liver expression. In some embodiments, the bone marrow- to-liver ratio or spleen-to-liver ratio is between 1.5 and 30, between 4 and 20 or between 5 and 20. The bone marrow-to-liver or spleen-to-liver ratios are measured at 24 hours as set forth in Example 7 and the Materials and Methods herein. In some embodiments, the unshielded LNPs exhibiting bone marrow-to-liver ratios within these ranges comprise at least 20 mol% or 25 mol% of the nonpolar glyceride lipid. In some embodiments, the unshielded LNPs exhibiting bone marrow-to- liver ratios comprise 25 mol% to 35 mol% of the non-polar glyceride lipid. In some embodiments, the unshielded LNPs exhibiting bone marrow-to-liver ratios within these ranges comprise at least 20 mol% or 25 mol% of a triacylglycerol lipid. In some embodiments, the unshielded LNPs exhibiting bone marrow-to-liver ratios comprise 25 mol% to 35 mol% of the triacylglycerol lipid.

[0269] In some embodiments, the unshielded lipid nanoparticles exhibit high rates of transfection of macrophages in the bone marrow. In some embodiments, macrophage transfection is assessed in an animal model using flow cytometry as described in Example 8 and the Materials and Methods herein. In some embodiments, the lipid nanoparticles exhibit a bone marrow macrophagetransfection of at least 20%, 25%, 30% or 35% eGFP+cells at 24 hours post-injection to a mouse model.

[0270] In some embodiments, the unshielded lipid nanoparticles exhibit high rates of transfection of macrophages in the spleen. In some embodiments, the lipid nanoparticles exhibit a spleen macrophage transfection of at least 20%, 25%, 30% or 35% eGFP+cells at 24 hours post-injection to a mouse model using the methods set forth in Example 8 with reference to the Materials and Methods.

[0271] In some embodiments, the unshielded or non-sterically stabilized lipid nanoparticle comprising nucleic acid cargo is part of a pharmaceutical composition. The treatment may provide a prophylactic (preventative), ameliorative or a therapeutic benefit to treat any undesirable condition, such as a disease condition or disorder. In other embodiments, the unshielded lipid nanoparticle encapsulates a diagnostic agent. The pharmaceutical composition will be administered at any suitable dosage.

[0272] The LNPs described herein may be used to treat and / or prevent any disease, disorder or condition in a mammalian subject. This includes a disease, disorder or condition, such as cancer, infectious diseases such as bacterial, viral, fungal or parasitic infections, inflammatory and / or autoimmune disorders, including treatments that induce immune tolerance and cardiovascular diseases such as hypertension, cardiac arrhythmia and restenosis.

[0273] Examples of cancers include lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, stomach (gastric) cancer, esophageal cancer; gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer, renal cancer (e.g., renal cell carcinoma), cancer of the central nervous system, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head and neck cancers, osteogenic sarcomas, and blood cancers. Non-limiting examples of specific types of liver cancer include hepatocellular carcinoma (HCC), secondary liver cancer (e.g., caused by metastasis of some other non-liver cancer cell type), and hepatoblastoma.

[0274] Non-limiting examples of other diseases, disorders or conditions that may be treated by the nucleic acid-LNPs herein and that may be attributed at least in part to an immunological disorder include colitis, Crohn’s disease, allergic encephalitis, allograft transplant / graft vs. host disease (GVHD), diabetes and multiple sclerosis.

[0275] The LNP may be part of a vaccine pharmaceutical formulation.

[0276] In some embodiments, the nucleic acid in the LNP encodes for a protein, peptide or polypeptide that is non-antigenic for infectious disease, such as a virus. In some embodiments, the LNP is a non-vaccine composition for infectious disease.

[0277] The LNPs herein may also be used in other applications besides the treatment and / or prevention of a disease or disorder. The LNPs may be used to treat conditions such as aging, preventative medicine and / or as part of a personalized medicine regime. In further embodiments, the LNP is used in a diagnostic application.

[0278] In one embodiment, the LNP is part of a pharmaceutical composition administered parenterally, i.e., intra-arterially, intravenously, subcutaneously or intramuscularly. In yet a further embodiment, the pharmaceutical compositions are for intra- tumoral administration. In another embodiment, the pharmaceutical compositions are administered intranasally, intravitreally, subretinally, intrathecally or via other local routes.

[0279] The pharmaceutical composition comprises pharmaceutically acceptable salts and / or excipients.

[0280] The compositions described herein may be administered to a patient. The term patient as used herein includes a human or a non-human subject.

[0281] The examples below are intended to illustrate the preparation of specific lipid nanoparticle preparations and properties thereof but are in no way intended to limit the scope of the invention.

[0282] The article “a” or “an” as used herein is meant to include both singular and plural, unless otherwise indicated.ExamplesMaterials and MethodsLNP preparation

[0283] The LNPs were prepared by dissolving mRNA in 25 mM sodium acetate, pH 4.0 (or other buffer as indicated), while the lipid components at the mole % specified were dissolved in absolute ethanol. The lipids in ethanol and the luciferase mRNA (Examples 1-7, 9 and 10) or eGFP mRNA(Example 8) in buffer were combined in a 1 : 3 volume by volume ratio using a t-junction with dualsyringe pumps. The solutions were pushed through the t-junction at a combined flow rate of 20 mL / min (5 mL / minute for the lipid-containing syringe, 15 mL / minute for the mRNA-containing syringe). The mixture was subsequently dialyzed overnight against at least -100 volumes of 1 x phosphate buffered saline, pH 7.4 using Spectro / Por dialysis membranes (molecular weight cutoff 12 000-14 000 Da). The LNPs were concentrated as required with an Amicon Ultra™ 100 000 MWCO (molecular weight cut-off), regenerated cellulose concentrator.Modified LNP preparation method

[0284] The above LNP preparation method was modified in some instances to facilitate formulation of LNPs having low levels ofthe non-polar glyceride orto produce particles of smaller size (e.g., 65 nm). After T-mixing, IX phosphate buffered saline (PBS) or other buffer as indicated, was added to the formulation mixture in increasing ratios of LNP:buffer prior to dialysis and gently mixed, followed by transfer to dialysis in 1 X PBS (or other suitable buffer as indicated) followed by processing as usual.

[0285] Encapsulation efficiency was calculated by determining unencapsulated mRNA content by measuring the fluorescence upon the addition of RiboGreen™ to the mRNA-LNP (F) and comparing this value to the total mRNA content that is obtained upon lysis of the LNP by 2% Triton X-100 (Ft): % encapsulation = (Ft - F) / Ft x 100.

[0286] The particle size and polydispersity index (PDI) were characterized using a Zetasizer Nano ZS™.Determination of N / P ratio of lipid nanoparticles

[0287] N / P values describe the ratio of anionic charge (P) to cationic charge (N) within an oligonucleotide-containing lipid nanoparticle. To calculate the total lipid weight required to obtain a specific N / P ratio, the average molecular weight per anionic charge (P) of a single nucleic acid base / phosphate / ribose monomer (-300 g / mol) versus the molecular weight per cationic charge of the ionizable lipid (N) was first calculated. Prom this molar ratio, required ionizable lipid weights were calculated by required N / P*calculated molar ratio* ionizable lipid molecular weight (g / mol)* required oligonucleotide weight (g). From this, total lipid weights (including non-charged lipid components) were calculated based on the desired lipid composition (i.e., mol% of individual lipids) and individual lipid molecular weights.Cryo-TEM

[0288] LNPs were concentrated to between 15 - 25 mg / mL estimated total lipid prior to cryo- TEM imaging. A defined volume, for example 2-4 pL, of the resulting LNP solution was applied to a glow-discharged copper grid, and plunge-frozen using an FEI Mark IV Vitrobot™ to generate vitreous ice. These grids were stored in liquid nitrogen until imaged by an FEI Titan Krios™ or an FEI Glacios TEM™. The instrument was operated at 200 kV in low-dose conditions and the resulting images were obtained using a bottom-mount FEI Falcon™ direct electron detector camera at 47-88,000 X magnification with an under-focus of 0.5-2 pm in order to enhance contrast. Tissue homogenate assay

[0289] The LNPs at a luciferase mRNA or pDNA concentration of 0.1 mg / mL were injected intravenously (i.v.) in mice at 1 mg / kg. Organs were harvested at 24 hours after the LNP injections.

[0290] Tissues were removed from the mice and placed in 2 mb tubes and snap frozen in liquid nitrogen. The tissues were subsequently stored at -80°C. An appropriate volume of GLO™ lysis buffer from Promega™ was added to each of the tubes, ensuring that the samples remained frozen before addition of the lysis buffer. Samples were placed in a FastPrep™ homogenizer and the homogenizer was operated at a speed of 6 m / s for 20 seconds and repeated 2 times for a total of three rounds. The homogenized samples were spun down for 10 minutes at 12,000 rpm at room temperature and subsequently 50 pL of homogenate in duplicate was added to a black plate. The plate was transferred to a plate reader and the fluorescence was read at 640 nm excitation / 720 nm emissions. Luminescence was determined by adding 50 pL of Steady Gio™ substrate into the homogenate sample and a luciferase signal was read.Flow cytometry in vivo studies

[0291] The LNPs at the eGFP mRNA or Thy 1.1 concentration of 0.1 mg / mL were injected intravenously (i.v.) in mice at a volume using the formula weight of the mouse (in grams) * 10 pL. Bone marrow was harvested at 24 hours after the LNP injections. 24 hours after the LNP injections. The bone marrow was harvested and processed into a single cell suspension. In particular, the mice were anesthetized with 5% isoflurane until reflex was lost and then exposed to CO2 with 1% air. The marrow was isolated from the femur by centrifugation of the bone for 30 s at 3,810 g and resuspended in FACS buffer (lx sterile PBS (pH 7.4), 2.5 mMethylenediaminetetraacetic acid (EDTA), 0.05% (w / v) sodium azide (NaNs), 2% (v / v) heat- inactivated fetal bovine serum (HI-FBS)).

[0292] After isolation, the bone marrow cells were stained. Eight to ten million cells were added to a well of 96-well round bottom plates and the volume in each well was increased to 150 uL using FACS buffer. Cells were centrifuged at 484 g at 4°C for 5 minutes and the liquid was discarded. Subsequently, cells were incubated with a solution containing 5% rat serum and then a solution containing staining antibodies for 45 minutes. Cells were centrifuged at 484 g at 4°C for5 minutes and the liquid was discarded. The volume was increased to 150 pL and cells were centrifuged at 484 g at 4°C for 5 minutes and the liquid was discarded twice. A volume of 150 pL of propidium iodide (PI) was added at a 1:5,000 dilution (1 mg / mL stock) and the stained, single cells were introduced to a flow cytometer (Cytoflex™, Beckman Coulter™). Single colour setups were used to generate the compensation matrix which was applied to all the samples.

[0293] The flow cytometry data was analyzed using FlowJo™ version 10 (Becton Dickension™6 Company (BD)). The bar graphs for the percentage of eGFP positive (eGFP+) cells were generated using Prism™ version 8 (GraphPad™) software.Example 1: LNPs comprising non-polar glyceride, neutral lipid and lacking PEG (unshielded) have favourable physicochemical characteristics

[0294] This example demonstrates that a lipid nanoparticle comprising a non-polar glyceride (triolein, C18: l), referred to herein as “TAG LNP” and lacking PEG (unshielded) unexpectedly exhibits favourable physicochemical characteristics.

[0295] The two formulations tested were mRNA-LNPs encoding luciferase and the lipids components and molar ratios are set forth in Table 1 below:Table 1: Triacylglycerol mRNA-LNP formulations with or without PEG prepared to examine physicochemical characteristics

[0296] The ionizable lipid 1 included in both formulations was an analogue of lipid 24 as described in U.S. Patent No. 12,121,591 (incorporated herein by reference) in which 8 carbon atoms are distal to the esters in each lipophilic chain.

[0297] The TAG-LNP + PEG formulation was considerably smaller (60 nm vs. 85 nm) relative to the same LNP without PEG and exhibited a decrease in the encapsulation efficiency to 84%. The TAG-LNP with no PEG (unshielded) had a low polydispersity index (PDI), favourable size (85 nm) and greater than 90% encapsulation efficiency.Example 2: Unshielded LNPs having a non-polar glyceride withstand vortexing

[0298] Given the favourable physicochemical data for the TAG-LNP with no PEG in Example 1, the inventors next examined whether the unshielded LNPs with encapsulated luciferase were able to withstand mechanical stress in the form of vortexing. The TAG-LNPs with no PEG were submitted to vortexing for 2 minutes at high (0.5 mg / mL) and low (0.05 mg / mL) concentrations. The results in Figure 2A show that TAG LNPs with no PEG at high and low concentrations were unexpectedly resistant to the mechanical stress from vortexing for 2 min.

[0299] A similar study conducted with gold standard, PEG-containing control LNPs (10 / 50 / 38.5 / 1.5; DSPC / ionizable lipid nor-MC3 / cholesterol / PEG-DMG) encapsulating luciferase showed that the PDI of the formulations increased over a time course of vortexing (Figure 2B). (The ionizable lipid, nMC3, is described in WO 2022 / 246571). After 2 minutes of vortexing postdialysis, the LNPs had a PDI approaching 0.3. At the same time point, the unshielded TAG LNPs had a PDI of less than 0. 1 (Figure 2A).Example 3: The effect of phospholipid phase transition temperature (Tm) on the formulation of LNPs with non-polar glyceride and lacking PEG

[0300] The physicochemical characteristics of unshielded triolein LNPs and formulated with phospholipids having varying phase transition temperatures were next investigated. The unshielded LNPs and contained 1,2-dibehenoylphosphatidylcholine (22:0 PC), dipalmitoylphosphatidylcholine (DPPC), 1 -stearoyl -2 -oleoylphosphatidylcholine (SOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylglycerol (DOPG) and distearoylphosphatidylcholine (DSPC). The LNPs encapsulated luciferase and contained 30 / 20 / 30 / 20 ionizable lipid 1 / phospholipid / triolein / cholesterol.

[0301] Phospholipids with lower Tm, except for DPPC, had low encapsulation efficiency, large size and agglomerated during dialysis or concentration (Figure 3A). (For these particles, it was not possible to measure physicochemical properties and therefore no data is presented in the figure). The LNPs having DSPC with a higher Tmhad favourable physicochemical characteristics.

[0302] A modified LNP preparation method has been developed to facilitate formulation of lipid nanoparticles without PEG as set forth in the Materials and Methods. The results in Figure 3B show that LNPs without PEG prepared using this method and with low Tmlipids were unstable (as represented by the absence of data). The use of different buffers at 25 mM (phosphate buffer (PB), Tris-HCl and sodium acetate) to formulate DSPC LNPs without PEG had an impact on the size of the particles but otherwise produced particles with favourable physicochemical properties.Example 4: The effect of DSPC and cholesterol content on physicochemical properties

[0303] The physicochemical characteristics of unshielded LNPs prepared with non-polar glyceride (triolein in this example) and formulated with varying amounts of DSPC and cholesterol were next investigated. The LNPs prepared for the study are set forth in Table 2 below.Table 2: LNPs investigated for physicochemical properties as a function of DSPC and cholesterol content

[0304] The results of the physicochemical analysis are shown in Figure 4. Unshielded trioleincontaining LNPs prepared with varying amounts of DSPC were found to be robust, with relatively minimal changes in size and encapsulation efficiency. It was found that the triolein LNPs with elevated DSPC (>35 mol%) were less favourable as the particles were more challenging to filter and had decreased encapsulation efficiency, while particles with only 5 mol% DSPC agglomerated. Thus, in some non-limiting examples, it may be desirable to include the neutral lipid (e.g., DSPC) in the particles at less than 40 mol% or most advantageously between 10 and 35 mol%.

[0305] In some non-limiting examples, it may be advantageous to include greater than 15 mol% sterol (e.g., cholesterol) in the LNPs since particles with 15 mol% sterol had lower encapsulation efficiency than those with 25 and 30 mol% sterol. (Figure 4).Example 5: LNPs with various nitrogen-to-phosphate (N / P) have favourable physicochemical properties

[0306] This example demonstrates that unshielded LNPs with non-polar glyceride and formulated with varying nitrogen-to-phosphate exhibit favourable physicochemical properties.

[0307] The unshielded LNPs having ionizable lipid 1 / DSPC / triolein / cholesterol molar ratios at 30 / 20 / 30 / 20 (samples 1-5) and 30 / 25 / 25 / 20 (samples 6-10) were prepared with nitrogen-to- phosphate ratios of 2, 3, 4, 6 and 9. The formulations are set forth in Table 3 below:Table 3: LNPs investigated for physicochemical properties as a function of N / P

[0308] The physicochemical properties of the LNPs of Table 3 having varying N / P were all within acceptable ranges as shown in Figure 5.

[0309] The foregoing results indicate that the unshielded LNPs with non-polar glyceride are robust in that they exhibit favourable physiochemical properties over a range of changes to the molar ratios of lipids in the formulations (Figure 4) and N / P values (Figure 5).Example 6: LNPs formulated with various ionizable cationic lipids have favourable physicochemical properties

[0310] This example demonstrates that unshielded LNPs with non-polar glyceride and formulated with varying ionizable lipids exhibit favourable physicochemical properties.Unshielded LNPs having ionizable lipid / DSPC / triolein / cholesterol molar ratios at 30 / 20 / 30 / 20 (mol / mol) of ionizable lipid 2, 3 or 4 / DSPC / triolein / cholesterol molar ratios were prepared. The ionizable lipids 2, 3 and 4 tested are set out in the table below:Table 4: Ionizable lipids tested in non-polar glyceride LNPs

[0311] The physicochemical properties of the unshielded LNPs tested with various ionizable lipids were all within acceptable ranges (Figure 6). An additional four unshielded LNPs having the same 30 / 20 / 30 / 20 (mol / mol) composition (see above) and differing ionizable lipids were tested and also had physicochemical properties that were within acceptable ranges (data not shown).

[0312] This data, combined with the data of Example 5, demonstrate that the unshielded LNPs with non-polar glyceride are robust formulations that exhibit favourable physicochemical properties over a range of compositional variations.Example 7: Unshielded LNPs with non-polar glyceride exhibit favourable extrahepatic protein expression

[0313] This example demonstrates that unshielded LNPs with non-polar glyceride unexpectedly exhibit protein expression in the bone marrow equivalent to long -circulating formulations with PEG as well as low liver expression.

[0314] The following unshielded LNPs with non-polar glyceride were prepared as set forth in Table 5 below. The cargo was luciferase mRNA and the dose injected to CD-I mice was 1 mg / kg.Table 5: Unshielded LNPs with non-polar glyceride examined for protein expression in bulk tissue

[0315] The results are shown in Figures 7A, 7B, and 7C. The unshielded LNPs with non-polar glyceride do not lead to relevant protein expression in the liver at 4 or 24 hours (Figure 7A). The bone marrow / liver ratio was 14 for LNP 4 at 24 h. Unexpectedly, the unshielded LNPs with nonpolar glyceride showed protein expression in the bone marrow equivalent to long-circulating LNP (IcLNP™) having ionizable lipid / DSPC / cholesterol at both 4- and 24-hours post-injection (Figure 7C).

[0316] A second study was conducted to assess enhanced extrahepatic delivery to the spleen and bone marrow. The formulations investigated are set forth in Table 6 below. The LNP dose was 1 mg / kg injected to CD-I mice and the endpoint was 24 hours.Table 6: PEG-containing and unshielded LNPs with non-polar glyceride examined for extrahepatic expression

[0317] As shown in Figures 8A-C and the BM / liver ratios of Table 6, unexpectedly, high bone marrow activity was observed for unshielded non-polar glyceride formulations with almost no activity seen in the liver.Example 8: Unshielded LNPs with non-polar glyceride exhibit enhanced transfection of macrophages

[0318] This example demonstrates that unshielded LNPs with non-polar glyceride unexpectedly exhibited enhanced transfection of bone marrow macrophages relative to PEG-containing and unshielded benchmark formulations.

[0319] The LNPs examined are set forth in Table 7 below.Table 7: Unshielded LNPs with non-polar glyceride examined for transfection of macrophages

[0320] The physicochemical properties of the above LNPs were all within acceptable ranges. The results are shown in Figure 9A.

[0321] Figure 9B shows the bone marrow eGFP+cells neutrophil and macrophage populations. As can be seen, the percentage of cells positive for eGFP was about 40% for the unshielded ionizable lipid 1 / DSPC / triolein / cholesterol sample. Similar results were observed for spleen macrophages (Figure 9C).Example 9: Extrahepatic protein expression of unshielded, non-polar glyceride LNPs is maintained with variations in lipid molar ranges

[0322] This example investigates the impact of variations in molar percentages of lipid components in unshielded LNPs with non-polar glyceride on extrahepatic delivery. In particular, LNPs with elevated DSPC and lower levels of TAG (relative to the previous examples) were also found to have enhanced delivery to the bone marrow. Unexpectedly, the protein expression was often elevated relative to a similar formulation incorporating PEG.

[0323] The formulations investigated are set forth in Table 8 below.Table 8: Unshielded LNPs with non-polar glyceride examined for liver and bone marrow delivery

[0324] The physicochemical properties of the above LNPs were all within acceptable ranges. The results are shown in Figure 10A.

[0325] The in vivo extrahepatic data is summarized in Figures 10B and 10C. Luminescence was measured at 24 hours post-injection at a dose of 1 mg / kg for the formulations of Table 8.

[0326] As can be seen in Figure 10B, each formulation tested exhibited similar luminescence in the liver. However, as shown in Figure 10C, in the bone marrow, unshielded LNPs 3 and 4 (without PEG) exhibited higher luminescence intensity than a similar PEG-containing formulation (LNP 5). Thus, similar to the unshielded LNPs tested in earlier examples, the LNPs with comparatively lower TAG (e.g., 5 and 10 mol%) also exhibited favourable bone marrow expression.

[0327] A subsequent study was conducted to assess extrahepatic delivery of the low TAG- containing, unshielded LNPs in other organs and tissues besides the bone marrow. The LNPs tested are set forth in Table 9 below.Table 9: Unshielded LNPs with non-polar glyceride examined for liver, bone marrow, heart, lung and abdominal delivery

[0328] The physicochemical properties of the above LNPs were all within acceptable ranges. The results are shown in Figure 11 A.

[0329] As can be seen in Figures 11B-11G, luminescence intensity for unshielded, non-polar glyceride LNPs (LNPs 3 and 4) was higher than the PEG-containing LNP controls (LNPs 1 and 2) in most tissues and organs.Example 10: Examination of in vivo bone marrow expression of LNPs formulated with a new panel of ionizable lipids

[0330] The previous examples investigated the extrahepatic delivery of LNPs with non-polar glyceride lacking PEG that were formulated with ionizable lipids 1-5. The studies were expanded to explore the extrahepatic expression of nucleic acid for the triolein (TAG). This included unshielded LNPs formulated with a new panel of ionizable lipids, namely lipids 6-8, along with lipids 2 and 4 examined in previous examples. The nucleic acid expression was comparable relative to a control long-circulating formulation incorporating PEG.

[0331] The ionizable lipid 6 examined in this example (lipid T-07 described in co-owned PCT / CA2025 / 050901, which is incorporated herein by reference), was formulated in TAG LNPs lacking PEG at the molar ratios below (Table 10, LNP 3). In addition, a similar formulation was prepared with ionizable lipid 2 (Table 6), as well as a long-circulating control (IcLNP™)formulation containing PEG as indicated in the table below. The N / P for each formulation was 6 for the gold standard Onpattro™-like control LNP, and 9 for the remaining LNPs.Table 10: Unshielded TAG-LNPs formulated with ionizable lipid 6 examined for extrahepatic delivery

[0332] The physicochemical properties of LNPs 1, 2 and 3 of Table 10 above were all within acceptable ranges (Figure 12A).

[0333] The TAG-LNPs exhibited enhanced expression of luciferase in the spleen and bone marrow (Figures 12C and 12D) and reduced expression in the liver relative to the control LNP (Figure 12B).

[0334] In a subsequent study, unshielded TAG-LNPs with ionizable lipids 2, 4, 6, 7 and 8 were further examined for extrahepatic delivery. The TAG-LNPs were diluted during formulation at 1:2 (v:v) with Tris buffer as per the modified method in the Materials and Methods. The LNPs investigated are set forth Table 11 below:Table 11: Unshielded TAG-LNPs with ionizable lipids 2, 4, 6, 7 and 8 examined for extrahepatic delivery

[0335] The physicochemical properties of LNPs 1, 2, 3, 4, 5, 6, 7 and 8 of Table 11 above were all within acceptable ranges (Figure 13A). As shown in Figures 13B-D, the TAG-LNPs often exhibited enhanced expression of luciferase in the spleen and bone marrow (Figures 13C and 13D) and reduced expression of the reporter in the liver relative to the control LNP (Figure 13B). Expression of luciferase was particularly pronounced in the bone marrow for the unshielded TAG- LNPs relative to the LNP control (LNP 1).Example 11: Examination of transfection of cells of the bone marrow, blood and spleen by flow cytometry by unshielded TAG-LNPs formulated with new ionizable lipids

[0336] Flow cytometry was conducted with unshielded TAG-LNPs formulated with ionizable lipids 2, 5 and 6. The control and TAG-LNPs of Table 12 were prepared encapsulating Thy 1.1 cargo to assess cell expression of the marker in vivo in bone marrow and blood. Thy 1.1 expression was assessed at 24 hours post-injection and the dose was 1 mg / kg. The N / P for LNP 1 was 6 and for LNPs 2-6 it was 9. Phosphate buffered saline (PBS) was used as a control.Table 12: Unshielded TAG-LNPs examined in flow cytometry studies

[0337] The physicochemical data in Figure 14 show that the LNP sizes, polydispersity index (PDI) and encapsulation percentages of LNPs 1-6 of Table 12 above were all within acceptable ranges.

[0338] The data in Figures 15A-C shows transfection of hematopoietic stem and progenitor cells (HSPC) in the bone marrow (BM). Despite lacking PEG, the unshielded TAG-LNPs (LNPs 3-6) exhibited similar percentage Thy 1.1 expression as the PEG-containing, long -circulating IcLNP™ control (LNP 2). Notably, the unshielded TAG-LNPs had significantly higher expression in HSPC than the control LNP.

[0339] Figure 16A-F show the results of transfection of various cells types of the bone marrow, namely monocytes, macrophages, neutrophils, CDl lb+cells, T cells and B cells. In most cell types, the TAG-LNPs (LNPs 3-6) exhibited similar percentages of Thy 1. 1 expression as the PEG- containing, long-circulating IcLNP™ control (LNP 2). Figures 17A-F show results of transfectionof monocytes, macrophages, neutrophils, CD1 lb+cells, T cells and B cells in the blood. In some cell types, the TAG-LNPs (LNPs 3-6) exhibited comparable percentages of Thyl.l expression as the PEG-containing, long-circulating IcLNP™ control (LNP 2). In most cases, however, the unshielded TAG-LNPs had significantly higher expression in each cells type measured in the bone marrow and blood than the gold-standard, Onpattro™-type control LNP.Example 12: cryo-TEM images of unshielded LNPs with non-polar glyceride

[0340] An unshielded lipid nanoparticle with non-polar glyceride (triolein) was assessed by cryotransmission electron microscopy (cryo-TEM) as set forth in the Materials and Methods to visualize their morphology. The lipid nanoparticle was ionizable lipid 5 / DSPC / TAG / cholesterol at molar ratios of 30 / 45 / 5 / 20. The LNP was prepared by the modified method described in the Materials and Methods and the N / P was 9.

[0341] The morphology of the unshielded TAG-LNPs is shown in figure 18. The LNPs have a core with an aqueous region and a distinctive electron dense region with a surrounding lipid layer resembling a bilayer. The morphology of the unshielded TAG-LNPs may contribute to their in vivo stability.

Claims

Claims:

1. A lipid nanoparticle comprising:(i) nucleic acid cargo;(ii) ionizable lipid present at a content of between 15 mol% and 40 mol%;(iii) neutral lipid having at least two tails and a head group, the neutral lipid present at a content of 10 to 55 mol%;(iii) a non-polar lipid content of between 5 mol% and 40 mol%, wherein the non-polar lipid is a glyceride; and(iv) sterol at a content of between 4 mol% and 45 mol%, wherein the lipid nanoparticle is unshielded and wherein each mol% content is relative to total lipid present in the lipid nanoparticle.

2. A lipid nanoparticle comprising:(i) nucleic acid cargo;(ii) ionizable lipid;(iii) neutral lipid having at least two tails and a head group, the neutral lipid having a phase transition temperature of at least 25 degrees as measured by differential scanning calorimetry;(iii) a non-polar lipid; and(iv) sterol, wherein the lipid nanoparticle is unshielded and wherein each mol% content is relative to total lipid present in the lipid nanoparticle.

3. The lipid nanoparticle of claim 1 or 2, wherein the non-polar lipid content includes a combination of two or more non-polar lipids.

4. The lipid nanoparticle of claims 1, 2 or 3, wherein the non-polar lipid is a triglyceride.

5. The lipid nanoparticle of claim 4, wherein the triglyceride is selected from triolein, tristearin, trilaurin, trilinoein, trilinolenin, trimyristin, tripalmitin, tricaprylin, triarachidin and oleoyldipalmitin.

6. The lipid nanoparticle of claim 1, 2 or 3, wherein the non-polar lipid is a diglyceride.

7. The lipid nanoparticle of claim 6, wherein the diglyceride is selected from glycerol dilaurate, glycerol dimyristate, glycerol dipalmitate, glycerol distearate, glycerol diarachidate, glycerol dibehenate, glycerol dipalmitoleate, glycerl dioleate, glycerol dilinoleate, glycerol dilinolenate and glycerol diarachidonate.

8. The lipid nanoparticle of claim 1, 2 or 3, wherein the non-polar lipid is a monoglyceride.

9. The lipid nanoparticle of claim 8, wherein the monoglyceride is selected from lauroyl-rac- glycerol, glycerol monomyristate, glycerol monopalmitate, glycerol monostearate, glycerol monoarachidate, glycerol monobehenate, glycerol monopahnitoleate, glycerol monooleate, glycerol monolinoleate, glycerol monolinolenate, glycerol monoarachidonate, and glycerol monocaprylate, and / or for example 1 -monomyristoyl -rac glycerol, 1 -mono-palm itoyl-rac- glycerol, 2-monopalm itoylglycerol, 1 -mono-palm itolenyl -rac -glycerol, 1 -monostearoyl -rac- glycerol, 1 -monoleoyl -rac-glycerol, 1- monolinoleoyl-rac-glycerol and 1-monolinolenoyl-rac- glycerol.

10. The lipid nanoparticle of any one of claims 1 to 4, wherein the non-polar lipid is castor oil.

11. The lipid nanoparticle of claim 1 or 2, wherein the non-polar lipid is methyl ricinoleate.

12. The lipid nanoparticle of claim 1, 2 or 3, wherein the non-polar lipid is present at a content between 10 mol% and 40 mol%.

13. The lipid nanoparticle of claim 1, 2 or 3, wherein the non-polar lipid is present at a content between 10 mol% and 35 mol%.

14. The lipid nanoparticle of claim 1, 2 or 3, wherein the non-polar lipid is present at a content between 10 mol% and 32.5 mol%.

15. The lipid nanoparticle of claim 1, 2 or 3, wherein the non-polar lipid is present at a content between 30 mol% and 32 mol%.

16. The lipid nanoparticle of any one of claims 1 to 15, wherein the ionizable lipid is an amino lipid.

17. The lipid nanoparticle of any one of claims 1 to 16, wherein the ionizable lipid is present at a content between 23 mol% and 47 mol%.

18. The lipid nanoparticle of any one of claims 1 to 17, wherein the ionizable lipid is present at a content between 25 mol% and 45 mol%.

19. The lipid nanoparticle of any one of claims 1 to 18, wherein the ionizable lipid is present at a content between 27 mol% and 43 mol%.

20. The lipid nanoparticle of any one of claims 1 to 19, wherein the ionizable lipid is present at a content between 28 mol% and 42 mol%.

21. The lipid nanoparticle of any one of claims 1 to 20, wherein the ionizable lipid is present at a content between 29 mol% and 41.5 mol%.

22. The lipid nanoparticle of any one of claims 1 to 21, wherein the ionizable lipid is present at a content between 29.5 mol% and 41 mol%.

23. The lipid nanoparticle of any one of claims 1 to 11, wherein the ionizable lipid is present at a content between 30 mol% and 40 mol%.

24. The lipid nanoparticle of any one of claims 1 to 23, wherein the neutral lipid content is greater than 12 mol%.

25. The lipid nanoparticle of any one of claims 1 to 24, wherein the neutral lipid content is greater than 15 mol%.

26. The lipid nanoparticle of any one of claims 1 to 25, the neutral lipid content is between 10 and 40 mol%.

27. The lipid nanoparticle of any one of claims 1 to 26, wherein the neutral lipid is a phospholipid.

28. The lipid nanoparticle of claim 27, wherein the phospholipid is a phosphocholine selected from a phosphatidylcholine and a sphingolipid.

29. The lipid nanoparticle of any one of claims 1 to 28, wherein the lipid nanoparticle has less than 0.75 mol% of a surface stabilizer based on a total lipid content of the lipid nanoparticle.

30. The lipid nanoparticle of claim 29, wherein the surface stabilizer is present at a content between 0 mol% and 0.50 mol%.

31. The lipid nanoparticle of claim 29, wherein the surface stabilizer is present at a content between 0 mol% and 0.40 mol%.

32. The lipid nanoparticle of claim 29, wherein the surface stabilizer is present at a content between 0 mol% and 0.30 mol%.

33. The lipid nanoparticle of claim 29, wherein the surface stabilizer is present at a content between 0 mol% and 0.20 mol%.

34. The lipid nanoparticle of any one of claims 1 to 33, wherein the sterol is present at a content of less than 30 mol%.

35. The lipid nanoparticle of any one of claims 1 to 34, wherein the sterol is cholesterol.

36. The lipid nanoparticle of any one of claims 1 to 35, wherein the sterol is present at a content of greater than 15 mol%.

37. The lipid nanoparticle of any one of claims 1 to 36, wherein the sterol is present at a content between 18 mol% and 40 mol%.

38. The lipid nanoparticle of any one of claims 1 to 37, wherein the sterol is present at a content between 20 mol% and 40 mol%.

39. The lipid nanoparticle of any one of claims 1 to 38, wherein the sterol is present at a content between 25 mol% and 38 mol%.

40. The lipid nanoparticle of any one of claims 1 to 39, wherein an N / P charge ratio of the cationic charge (N) of the ionizable lipid to the anionic charge (P) of the nucleic acid cargo is between 1 and 15.

41. The N / P charge ratio of claim 40, wherein the N / P charge ratio is between 2 and 9.

42. The N / P charge ratio of claims 40 or 41, wherein the ratio is between 3 and 9.

43. The lipid nanoparticle of any one of claims 1 to 42, wherein the nucleic acid is selected from an siRNA, mRNA, a vector nucleic acid, an antisense oligonucleotide, a nucleic acid-protein complex, and a nucleic acid-peptide complex.

44. The lipid nanoparticle claim 43, wherein the nucleic acid is selected from siRNA, vector nucleic acid, and an antisense oligonucleotide.

45. The lipid nanoparticle of claim 43, wherein the nucleic acid is mRNA.

46. The lipid nanoparticle of any one of claims 1 to 45, wherein the lipid nanoparticle has a solid core as visualized by cryogenic electron microscopy (cryo-TEM).

47. The lipid nanoparticle of claim 46, wherein the lipid nanoparticle has a lipid layer surrounding the core as visualized by cryogenic electron microscopy (cryo-TEM).

48. The lipid nanoparticle of any one of claims 1 to 47 the lipid nanoparticle resulting in a ratio of bone marrow / liver expression that is between 1.5 and 30.

49. The lipid nanoparticle of any one of claims 1 to 48, the lipid nanoparticle resulting in a ratio of bone marrow / liver expression that is between 1.5 and 25.

50. The lipid nanoparticle of claim 49, wherein the ratio of bone marrow / liver expression is between 4 and 20.

51. The lipid nanoparticle of claim 49, wherein the ratio of bone marrow / liver expression is between 5 and 20.

52. The lipid nanoparticle of any one of claims 1 to 48, the lipid nanoparticle having a ratio of bone marrow / liver expression that is greater than 1.5 or greater than 2.0.

53. The lipid nanoparticle of claim 1, wherein the neutral lipid is a phosphatidylglycerol or sphingolipid and wherein the phase transition temperature of the neutral lipid is at least 25 °C as measured by differential scanning calorimetry (DSC).

54. The lipid nanoparticle of claim 1, wherein dimyristoylphosphatidylcholine (DMPC) is present at 0 to 10 mol%, 0 to 8 mol%, 0 to 7 mol%, 0 to 6 mol%, 0 to 5 mol%, 0 to 4 mol% or 0 to 3 mol% relative to the total lipid content of the lipid nanoparticle.

55. A method for delivery of mRNA or vector DNA for in vivo expression of protein or peptide in the bone marrow or spleen, the method comprising administering to a mammal a lipid nanoparticle of any one of claims 1 to 54.

56. The method of claim 55, wherein a ratio of bone marrow / liver expression is between 2 and 30.

57. The method of claim 55, wherein a ratio of bone marrow / liver expression is between 4 and 20.

58. The method of claim 55, wherein a ratio of bone marrow / liver expression is between 5 and 20.

59. The method of claim 55, wherein a ratio of bone marrow / liver expression is greater than 1.5 or is greater than 2.

60. A method for delivery of siRNA or antisense oligonucleotide for in vivo silencing of a gene in the bone marrow or spleen, the method comprising administering to a mammal a lipid nanoparticle of any one of claims 1 to 54, wherein the siRNA or antisense oligonucleotide is encapsulated within the lipid nanoparticle and wherein the administering of the lipid nanoparticle results in an increased bone marrow- or spleen-specific silencing of the gene as compared as compared to a benchmark that is an otherwise identical LNP lacking a surface stabilizer and encapsulating the siRNA or antisense oligonucleotide.

61. A method for delivering a nucleic acid to a macrophage to treat a disease, disorder or condition, the method comprising contacting the lipid nanoparticle of any one of claims 1 to 52 with the macrophage in vivo or in vitro.

62. A lipid nanoparticle of any one of claims 1 to 54 for use to deliver mRNA or vector DNA for in vivo expression of protein or peptide in the bone marrow or spleen to a subject in need of mRNA or vector DNA to treat, ameliorate or prevent a condition or disease by expression of said protein or peptide.

63. The lipid nanoparticle of claim 62, wherein a ratio of bone marrow / liver expression in the subject is between 2 and 30.

64. The lipid nanoparticle of claim 63, wherein a ratio of bone marrow / liver expression is between 4 and 20.

65. The lipid nanoparticle of claim 64, wherein a ratio of bone marrow / liver expression is between 5 and 20.

66. The lipid nanoparticle of claim 65, wherein a ratio of bone marrow / liver expression is greater than 1.5 or is greater than 2.

67. A lipid nanoparticle of any one of claims 1 to 54 for use to deliver siRNA or antisense oligonucleotide in a subject to treat, ameliorate or prevent a condition or disease in vivo by silencing of a gene in the bone marrow or spleen, wherein the siRNA or antisense oligonucleotide is encapsulated within the lipid nanoparticle and wherein the administering of the lipid nanoparticle results in an increased bone marrow- or spleen-specific silencing of the gene as compared as compared to a benchmark that is an otherwise identical LNP lacking a surface stabilizer and encapsulating the siRNA or antisense oligonucleotide.

68. Use of a lipid nanoparticle of any one of claims 1 to 54 to deliver a nucleic acid to a macrophage to treat, ameliorate or prevent a disease, disorder or condition in a subject, the use comprising contacting the lipid nanoparticle with the macrophage in vivo, ex vivo or in vitro.

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