Lipid nanoparticles with nonpolar lipids for delivery of nucleic acids to the liver

Abstract

The present disclosure provides methods of delivering nucleic acid cargo to the liver of an individual, the method comprising administering to the individual a lipid nanoparticle encapsulating the nucleic acid and comprising a non-polar lipid. The lipid nanoparticle can exhibit a vesicular morphology despite the absence or low levels of phospholipids. The present disclosure also provides compositions for delivering nucleic acid cargo to the liver for the treatment of a variety of diseases, disorders, and conditions in an individual.

Description

Technical Field

[0001] This disclosure relates to lipid nanoparticle formulations for delivering goods such as nucleic acids.

[0002] background There is growing interest in using lipid nanoparticle-targeted liver delivery to treat hereditary diseases, cancers, and infectious liver diseases that cause millions of deaths worldwide (Witzigmann et al., 2020, Advanced Drug Delivery Reviews 159:344-363). Many current clinical interventions for hereditary liver diseases and alcoholic liver diseases have limited efficacy, and in many cases, transplantation is the only viable option. However, transplantation can raise concerns about tissue compatibility, introduce surgical risks such as infection, and may require long-term immunosuppression (Zadory et al., 2022, Biomater. Sci., 10:6077-6115). Therefore, lipid nanoparticle (LNP) therapy is an attractive option for avoiding or limiting the use of this invasive procedure.

[0003] An early example of an approved liver-targeted therapy for clinical use is Onpattro. TM This formulation is an LNP-encapsulated short interfering RNA (siRNA) for the treatment of a rare, liver-based genetic disorder known as hereditary thyroxine amyloidosis, which causes amyloid fibrillary deposition in multiple organs. Onpattro TM The LNP formulation consists of four main lipid components: ionizable amino lipids, distearate phosphatidylcholine (DSPC), cholesterol, and polyethylene glycol-conjugated lipids (PEG-lipids), with molar amounts of 50 / 10 / 38.5 / 1.5, respectively. The success of this liver-targeted mRNA-LNP delivery system lays the foundation for the clinical development of LNP-based COVID-19 mRNA vaccines.

[0004] In four Onpattro TM Among lipid components, ionizable cationic lipids are considered the most important for the in vitro and in vivo activity of the LNP system. Therefore, much of the work in this field has focused on improving this lipid component. Ionizable cationic lipids are positively charged at low pH, which facilitates binding to negatively charged nucleic acids, but are neutral at physiological pH, making them more biocompatible in biological systems. Furthermore, it has been proposed that the ability of these lipids to ionize at low pH after being absorbed by cells via endocytosis allows for endosome escape. This, in turn, allows nucleic acids to be released into intracellular compartments.

[0005] Regarding the remaining lipid components, reports indicate that empty Onpattro TM In conventional LNP systems, DSPC and cholesterol are present in the outer lipid layer, but in siRNA-loaded systems, DSPC and cholesterol are internalized together with the siRNA in the hydrophobic core. The presence of DSPC and cholesterol in LNPs has been found to be crucial for the stable encapsulation of siRNA (Kulkarni et al., 2019, Nanoscale, 11:21733-21739). Further, it is suggested that DSPC is needed to stably retain cholesterol in the examined LNP formulations.

[0006] U.S. Patent No. 11,191,849 discloses a composition comprising 55% ionizable cationic lipids, 41% cholesterol, and 3.3% PEG. 2000 -C-DMA liver-targeting mRNA-LNP. With the base composition (1.6 / 55 / 33 / 11% molar ratio of PEG) 2000 Compared to PEG-C-DMA / cationic lipids / cholesterol / DSPC, LNP administration in mice resulted in a reduced inflammatory response without affecting efficacy. The observed results were partly attributed to the high PEG-lipid conjugate content (>3% molar ratio).

[0007] Some recent studies have investigated improving in vivo delivery properties by introducing targeted lipid fractions into LNP formulations. One approach uses selective organ-targeted (SORT) LNPs (Wang et al., 2023, Nat. Protoc, 18:265-291), which are modulated for tissue-specific mRNA delivery by introducing a fifth targeted lipid fraction, such as a permanently charged anionic or cationic lipid. However, this type of LNP still relies on Onpattro to maintain clinical approval. TM The formulation traditionally contains four lipid components. In addition, the introduction of a fifth type, "SORT," namely permanently charged cationic or anionic lipids, can lead to in vivo toxicity.

[0008] Other researchers have examined the use of combinations of triglycerides, such as trioleate, with palmitoylphosphatidylcholine (POPC) in LNPs, although the focus of such research has been on improving the delivery of hydrophobic small molecule drugs (e.g., doxorubicin) rather than nucleic acids (Zhigaltsev et al., 2012, 28:3633-3640). NMR and cryo-EM studies of trioleate / POPC 60 / 40 (mol / mol) LNPs have shown that particles formed by microfluidic mixing consist of POPC in an outer monolayer surrounding a hydrophobic triglyceride core (Zhigaltsev et al., 2012, 28:3633-3640).

[0009] WO 2018 / 119514 describes trioleic acid glyceride-containing LNPs for delivering mRNA to neurons. Studies have shown that cholesterol has a limited effect on efficacy. Furthermore, the mRNA-LNPs detected in the study contained high levels of phospholipids (20 to 40% molar ratio), namely dioleoylphosphatidylethanolamine (DOPE) and distearate phosphatidylcholine (DSPC). US2010 / 0297242 describes particles with LDL-like properties, but similarly uses phospholipids for preparation to impart stability. Additionally, Kulkarni et al. found that incorporation of DOPE, dioleoylphosphatidylcholine (DOPC), sphingomyelin (ESM), and cholesterol into lipid nanoparticles allowed for efficient encapsulation of siRNA; however, the inclusion of trioleic acid glycerides and triolein has been reported to result in ineffective encapsulation (Nanoscale, 2019, 11:21733-21739, Supplementary Figure 2).

[0010] Despite the aforementioned progress in this field, there remains a pressing need for LNP formulations that possess the properties required for nucleic acid delivery to the liver.

[0011] Overview This disclosure solves one or more problems in the art and / or provides useful alternatives thereto.

[0012] This disclosure is based in part on the finding that lipid nanoparticles (LNPs) prepared using nonpolar glycerol lipids or their analogues, but without phospholipids or low levels of nonpolar lipids, possess advantageous nucleic acid delivery properties. Specifically, the LNPs described herein have been found to have advantages over Onpattro. TM Type-based baseline LNP (also referred to in this paper as "Onpattro") TM The "baseline" formulation showed increased nucleic acid delivery to the liver, despite significant deviations in lipid composition from the "gold standard" benchmark.

[0013] In one example of this disclosure, LNPs with a combination of nonpolar lipids and lacking or having low levels of phospholipids exhibited better mRNA expression in the liver compared to the spleen. In some embodiments, compared to Onpattro TM Baseline LNP expression was significantly improved in the liver compared to the spleen.

[0014] Furthermore, LNPs were found to exhibit a surprising morphology as determined by cryo-electron microscopy. As described herein, despite the lack of phospholipids or their low levels, the particles surprisingly exhibited a bubble morphology.

[0015] According to one aspect of this disclosure, lipid nanoparticles are provided comprising: nucleic acid cargo molecules; sterols (including derivatives thereof) in an amount of 0% to 50% molar ratio; ionizable lipids present in an amount of 20% to 70% molar ratio; nonpolar lipids present in an amount of 20% to 80% molar ratio; phospholipids (including derivatives thereof) in an amount of 0 to 5% molar ratio; and a hydrophilic polymer-lipid conjugate in an amount of 0% to 3% molar ratio, wherein each % molar ratio is relative to the total lipids present in the lipid nanoparticles.

[0016] According to another aspect of this disclosure, lipid nanoparticles are provided comprising: encapsulated nucleic acid; ionizable lipid; nonpolar lipid and a uniform, generally spherical hydrophobic core with one or more peripheral vesicle structures as observed by cryo-electron microscopy (cryo-TEM); optionally sterol; optionally hydrophilic polymer-lipid conjugate; and phospholipid in an amount of 0 to 5 mol%.

[0017] In any of the foregoing aspects of this disclosure, the nonpolar lipid is a combination of two or more nonpolar lipids.

[0018] In any of the foregoing aspects of this disclosure or in an example of an embodiment thereof, the nonpolar lipid is a triglyceride.

[0019] In one embodiment, the triglyceride is selected from trioleic acid glyceride, tristearate glyceride, trilaurate glyceride, trilinoleic acid glyceride, trilinolenic acid glyceride, trimyristate glyceride, tripalmitate glyceride, tricaprylic acid glyceride, triarachidonic acid glyceride and oleic acid dipalmitate glyceride.

[0020] In any of the foregoing aspects of this disclosure or any embodiment thereof, the nonpolar lipid is diglyceride.

[0021] In one embodiment, the diglyceride is selected from diglyceride laurate, diglyceride myristate, diglyceride palmitate, diglyceride stearate, diglyceride arachidonicate, diglyceride behenate, diglyceride palmitate, diglyceride oleate, diglyceride linoleate, diglyceride linolenic acid, and diglyceride arachidonicate.

[0022] In any of the foregoing aspects of this disclosure or any embodiment thereof, the nonpolar lipid is a monoglyceride.

[0023] In one embodiment, the monoglyceride is selected from lauroyl-racemic glycerol, myristoyl monoglyceride, palmitate monoglyceride, stearate monoglyceride, arachidonic acid monoglyceride, behenate monoglyceride, palmitoleate monoglyceride, oleate monoglyceride, linoleate monoglyceride, linolenic acid monoglyceride, arachidonic acid monoglyceride, and caprylic acid monoglyceride; and / or, for example: 1-monomyoyl-racemic glycerol, 1-monopalmitoyl-racemic glycerol, 2-monopalmitoyl glycerol, 1-monpalmitoyl-racemic glycerol, 1-monostearoyl-racemic glycerol, 1-monoleoyl-racemic glycerol, 1-monoleinoyl-racemic glycerol, and 1-monolinoyl-racemic glycerol.

[0024] In any of the foregoing aspects of this disclosure or any embodiment thereof, the nonpolar lipid is castor oil.

[0025] In any of the foregoing aspects of this disclosure or any embodiment thereof, the nonpolar lipid is methyl ricinoleate.

[0026] In any of the foregoing aspects of this disclosure or any embodiment thereof, nonpolar lipids are present in amounts ranging from 22% to 38% molar, 25% to 35% molar, 25% to 35% molar, 27% to 32.5% molar, or 28% to 32% molar.

[0027] In any of the foregoing aspects of this disclosure or any embodiment thereof, the ionizable lipid is an amino lipid.

[0028] In any of the foregoing aspects of this disclosure or any embodiment thereof, the ionizable lipids are present in amounts ranging from 23% to 47% molar, 25% to 45% molar, 27% to 43% molar, 28% to 42% molar, 29% to 41.5% molar, 29.5% to 41% molar, or 30% to 40% molar.

[0029] In any of the foregoing aspects of this disclosure or any embodiment thereof, the phospholipid content is less than 4% molar ratio.

[0030] In any of the foregoing aspects of this disclosure or any embodiment thereof, the phospholipid content is less than 3% molar ratio.

[0031] In any of the foregoing aspects of this disclosure or any embodiment thereof, the phospholipid content is less than 2% molar ratio.

[0032] In any of the foregoing aspects of this disclosure or any embodiment thereof, the phospholipid content is less than 1% molar ratio.

[0033] In any of the foregoing aspects of this disclosure or any embodiment thereof, the phospholipid content is less than 0.5% molar ratio.

[0034] In any of the foregoing aspects of this disclosure or any embodiment thereof, the lipid nanoparticles do not have a detectable amount of phospholipids.

[0035] In any of the foregoing aspects of this disclosure or any embodiment thereof, the hydrophilic polymer lipid is present in a content of 0.75% to 2.5% molar ratio, 1% to 2.25% molar ratio, 1.25% to 2% molar ratio, or 1.25% to 1.75% molar ratio.

[0036] In any of the foregoing aspects of this disclosure or any embodiment thereof, sterols are present and are non-cationic lipids.

[0037] In one implementation, the sterol is cholesterol.

[0038] In one embodiment, the sterol is present in amounts of 22% to 38%, 25% to 35%, 27% to 32.5%, or 28% to 32%.

[0039] In an example of any of the foregoing aspects of this disclosure or any embodiment thereof, the LNP has an N / P charge ratio of 1 to 15 for the cationic charge (N) of the ionizable lipid and the anionic charge (P) of the nucleic acid cargo.

[0040] In one embodiment, the N / P charge ratio is 2 to 6 or 3 to 6.

[0041] In one embodiment, the nucleic acid is selected from siRNA, mRNA, vector nucleic acid, antisense oligonucleotide, nucleic acid-protein complex, and nucleic acid-peptide complex.

[0042] In one embodiment, the nucleic acid is selected from siRNA, vector nucleic acid, and antisense oligonucleotide. In one embodiment, the nucleic acid is mRNA.

[0043] In any of the foregoing aspects of this disclosure or any embodiment thereof, the lipid nanoparticles have a bubble structure as observed by cryo-electron microscopy (cryo-TEM).

[0044] In one embodiment, the lipid nanoparticles have a uniform core of one or more peripheral vesicles that are visualized by cryo-electron microscopy (cryo-TEM).

[0045] In an example of any of the foregoing aspects of this disclosure or any embodiment thereof, the lipid nanoparticles resulted in an increase of at least 10% in the expression of a protein or peptide in the liver of a mammal at 4, 24 and / or 48 hours post-injection, compared to a baseline formulation of a 50 / 10 / 38.5 / 1.5 mol:mol encapsulated nucleic acid norMC3 ionizable lipid / DSPC / cholesterol / PEG-lipid, measured under the same conditions, wherein said expression was measured using luminescence.

[0046] In another aspect of this disclosure, a method is provided for delivering mRNA or vector DNA to produce a protein or peptide in vivo in the liver, the method comprising administering to a mammal any of the foregoing aspects or embodiments described therein, wherein the nucleic acid is mRNA or vector DNA, and wherein administering the lipid nanoparticles results in increased liver-specific expression of the protein or peptide encoded by the mRNA or vector DNA compared to a baseline formulation of 50 / 10 / 38.5 / 1.5 mol:mol of nor-MC3 ionizable lipids / DSPC / cholesterol / PEG-lipids delivering the mRNA or vector DNA.

[0047] In one embodiment, the expression of the mRNA-encoded protein or peptide in the liver is at least 10% higher than that in the spleen, compared to the increase in baseline LNP expression in the liver relative to the spleen as measured under the same conditions.

[0048] In another aspect of this disclosure, a method is provided for delivering siRNA or antisense oligonucleotides to silence genes in vivo in the liver, the method comprising administering to a mammal any of the foregoing aspects or embodiments thereof a lipid nanoparticle, wherein the siRNA or antisense oligonucleotide is encapsulated in the lipid nanoparticle, and wherein the administration of the lipid nanoparticle results in increased liver-specific gene silencing compared to a baseline formulation of 50 / 10 / 38.5 / 1.5 mol:mol of nor-MC3 ionizable lipids / DSPC / cholesterol / PEG-lipids delivering the siRNA or antisense oligonucleotide.

[0049] In another aspect of this disclosure, a method is provided for delivering nucleic acids to hepatocytes to treat a disease, symptom, or disease state, the method comprising contacting the hepatocytes with lipid nanoparticles of any of the foregoing aspects or embodiments thereof in vivo or in vitro.

[0050] In another embodiment of any of the aforementioned methods, the phospholipid content of LNP is less than 4% molar ratio.

[0051] In another embodiment of any of the aforementioned methods, the sterol is present in a molar ratio of 25 to 45% or 25 to 40%.

[0052] In another embodiment of any of the foregoing methods, the hydrophilic polymer-lipid conjugate is present in the lipid nanoparticles at an amount of 0 mol% to 3 mol%. In another embodiment of any of the foregoing methods, the hydrophilic polymer-lipid conjugate is present in the lipid nanoparticles at an amount of 0.5 mol% to 2.0 mol%.

[0053] Brief description of the attached figures Figure 1A shows the effects of TAG LNP and Onpattro on CD-1 mice 4 hours after administration. TM Baseline LNPs exhibited in vitro luminescence at 0.02 mg / kg luciferase in the liver. The cargo was NTx firefly luciferase (fLuc) mRNA. TAG-LNP and Onpattro TM Details of the baseline LNP lipid composition are listed in Table 1 of Example 1.

[0054] Figure 1B shows the effects of TAG LNP and Onpattro on CD-1 mice 4 hours after administration. TM Baseline LNPs exhibited in vitro luminescence at 0.1 mg / kg luciferase in the liver. The cargo was internal NTx fLuc mRNA.

[0055] Figure 1C shows the effects of TAG LNP and Onpattro on CD-1 mice 4 hours after administration. TM Baseline LNPs exhibited in vitro luminescence at 0.5 mg / kg luciferase in the liver. The cargo was internal NTx fLuc mRNA.

[0056] Figure 1D shows the effects of TAG LNP and Onpattro at a dose of 0.5 mg / kg NTx fLuc mRNA. TM Representative whole-body images of CD-1 mice with baseline LNP. Images were taken 4 hours after administration. The intensity scale was the same for both images.

[0057] Figure 2AThe study showed that 4 hours after administration to CD-1 mice, TAG LNP and Onpattro levels in the liver and spleen were [high / low]. TM In vitro luminescence of baseline LNPs. The cargo was NTx fLuc mRNA, and the dose was 0.5 mg / kg mRNA.

[0058] Figure 2B shows Onpattro TM In vivo luminescence of baseline LNP and two distinct TAG LNPs, the two distinct TAG LNPs having ionizable lipid / cholesterol / trioleylglycerol / PEG. 2000 -DMG, but with different ionizable lipids (ionizable amino lipids 1 and 2 in Example 2). Results showed that tissue homogenates from the liver luminescent at a dose of 1 mg / kg 24 hours after administration to CD-1 mice.

[0059] Figure 2C shows the in vivo luminescence of spleen tissue homogenate 24 hours after LNP injection, as shown in Figure 2B.

[0060] Figure 2D shows Onpattro TM In vivo luminescence of baseline LNP and two distinct TAG LNPs, 1 and 3, with different lipid compositions. TAG LNP 1 is a previously described ionizable lipid / cholesterol / trioleylglycerol / PEG. 2000 -DMG formulation, TAG LNP 3, also contains cholesterol hemisuccinate (CHEMS). Results showed that 24 hours after administration to CD-1 mice, tissue homogenates from the liver at a dose of 1 mg / kg exhibited luminescence.

[0061] Figure 2E shows the in vivo luminescence of LNP in spleen tissue homogenate 24 hours after injection, as shown in Figure 2D.

[0062] Figure 3A Table 1 shows the levels of TAG LNP (Table 1) and Onpattro in the whole body and abdomen of CD-1 mice 4, 24, and 48 hours after administration. TM The time course of in vitro luminescence of baseline LNPs. The cargo was MG fLuc mRNA, and the dose was 0.5 mg / kg mRNA.

[0063] Figure 3B Table 1 shows the levels of TAG LNP (Table 1) and Onpattro in the whole body and abdomen of CD-1 mice 4, 24, and 48 hours after administration. TM In vivo chemiluminescence of baseline LNP. The cargo was MG fLuc mRNA, and the dose was 0.5 mg / kg mRNA.

[0064] Figure 4The results showed that after 3 months of storage at 4°C, TAG LNP (Table 1) and Onpattro were administered to CD-1 mice 4 hours later. TM Baseline LNPs exhibited in vitro luminescence in the liver at a concentration of 0.5 mg / kg luciferase. The cargo was internal NTx fLuc mRNA.

[0065] Figure 5 The results showed that after 4 hours of administration to CD-1 mice following cryopreservation at -80°C, TAG LNP (Table 1) and Onpattro TM Baseline LNPs exhibited in vitro luminescence in the liver at a concentration of 0.5 mg / kg luciferase. The cargo was internal NTx fLuc mRNA.

[0066] Figure 6 The TAG LNPs (Table 1) with N / P ratios of 3, 4, 5, and 6 fluoresce in vitro in the liver of CD-1 mice at a rate of 0.5 mg / kg luciferase 4 hours after administration. The cargo is the internal NTx fLuc mRNA.

[0067] Figure 7 Comirnay is shown to contain phospholipids (10% molar ratio DSPC) and lack TAG (denoted as +phospholipid(PL)-TAG). TM The ex vivo luminescence of the type LNP, the TAG-added Comirnaty type LNP (+PL+TAG), and the TAG LNP of the present invention (-PL+TAG) was measured in the liver after administration of 0.1 mg / kg luciferase and in CD-1 mice (iv) for 4 hours. The formulations are listed in Table 3. The cargo is MG fLuc mRNA, and the dosage is 0.1 mg / kg mRNA.

[0068] Figure 8A shows the total bilirubin levels (μmol / L) 4, 24, and 48 hours after administration of TAG LNP and PBS controls, measured from blood samples taken from CD-1 mice at these specific time points. For Onpattro TM Baseline LNP was used, and total bilirubin levels (μmol / L) were measured only 4 hours after administration. In all cases, the cargo was MG fLuc mRNA, and the dose was 0.5 mg / kg mRNA. The formulations are listed in Table 4.

[0069] Figure 8B shows the total alanine aminotransferase (ALT) levels (U / L) 4, 24, and 48 hours after administration of TAG LNP (Table 4) and PBS control, measured from blood samples taken from CD-1 mice at these specific time points. For Onpattro TMBaseline LNP was used, and total alanine aminotransferase (ALT) levels (U / L) were measured only 4 hours after administration. In all cases, the cargo was MG fLuc mRNA, and the dose was 0.5 mg / kg mRNA.

[0070] Figure 8C shows the total aspartate aminotransferase (AST) levels (U / L) 4, 24, and 48 hours after administration of TAG LNP (Table 4) and PBS control, measured from blood samples taken from CD-1 mice at these specific time points. For Onpattro TM Baseline LNP was used, and total aspartate aminotransferase (AST) levels (U / L) were measured only 4 hours after administration. In all cases, the cargo was MG fLuc mRNA, and the dose was 0.5 mg / kg mRNA.

[0071] Figure 8D shows the total alkaline phosphatase (AP) levels (U / L) 4, 24, and 48 hours after administration of TAG LNP (Table 4) and PBS control, measured from blood samples taken from CD-1 mice at these specific time points. For Onpattro TM Baseline LNP levels were measured only 4 hours after administration, with total alkaline phosphatase (AP) levels (U / L) measured. In all cases, the cargo was MG fLuc mRNA, and the dose was 0.5 mg / kg mRNA.

[0072] Figure 8E shows the total albumin levels (g / L) 4, 24, and 48 hours after administration of TAG LNP (Table 4) and PBS control, measured from blood samples taken from CD-1 mice at these specific time points. For Onpattro TM Baseline LNP was used, and total albumin levels (g / L) were measured only 4 hours after administration. In all cases, the cargo was MGfLuc mRNA, and the dose was 0.5 mg / kg mRNA.

[0073] Figure 8F shows the total amylase levels (U / L) 4, 24, and 48 hours after administration of TAG LNP (Table 4) and PBS control, measured from blood samples taken from CD-1 mice at these specific time points. For Onpattro TM Baseline LNP was used, and total amylase levels (U / L) were measured only 4 hours after administration. In all cases, the cargo was MGfLuc mRNA, and the dose was 0.5 mg / kg mRNA.

[0074] Figure 8G shows the total lactate dehydrogenase (LDH) levels (U / L) 4, 24, and 48 hours after administration of TAG LNP (Table 4) and PBS control, measured from blood samples taken from CD-1 mice at these specific time points. For Onpattro TM Baseline LNP levels were measured only 4 hours after administration, with total lactate dehydrogenase (LDH) levels (U / L) measured. In all cases, the cargo was MG fLuc mRNA, and the dose was 0.5 mg / kg mRNA.

[0075] Figure 9A shows the particle size (nm) of the TAG LNP and Onpattro™ baseline LNP formulations across different batches. The lipid composition of the LNPs is listed in Table 4.

[0076] Figure 9B shows TAG LNP and Onpattro TM The baseline LNP formulation shows the polydispersity index (PDI) between different batches of the formulation. LNPs are listed in Table 4.

[0077] Figure 9C shows TAG LNP and Onpattro TM Baseline LNP formulation mRNA encapsulation between different batches of the formulation. LNPs are listed in Table 4. The goods are fLuc mRNA obtained from multiple sources.

[0078] Figure 9D shows TAG LNP and Onpattro TM The fluorescence pair between different batches of the baseline LNP formulation and the apparent pKa of each LNP are shown in Table 4.

[0079] Figure 10A shows a cryo-TEM image of the TAG LNP formulation encapsulating fLuc mRNA. Arrows indicate bubbles. Details of the LNP formulation are listed in Table 4.

[0080] Figure 10B shows the Onpattro encapsulation of fLuc mRNA. TM Cryo-TEM images of the baseline formulation. Details of the LNP formulation are listed in Table 4.

[0081] Figure 11 The in vitro luminescence of phospholipid-free LNPs (containing different nonpolar glycerol lipids) at 0.1 mg / kg luciferase in the liver was shown 4 hours after administration to CD-1 mice. The cargo was internal NTx fLuc mRNA. Details of the LNP formulation are listed in Table 5.

[0082] Detailed Explanation nonpolar lipids As used herein, the term "nonpolar lipid" refers to lipids that are neutral and uncharged at physiological pH, lack a head group moiety (e.g., phosphate ester), and contain one or more lipophilic chains of at least 10 carbon atoms in length that impart an overall hydrophobic character to the lipid. Nonpolar lipids include, but are not limited to, esterified fatty acids and glycerides as described below.

[0083] In one example of this disclosure, the nonpolar lipid has the structure of Formula I:

[0084] Formula I Or its tautomers or stereoisomers, wherein R1, R2, and R3 are independently H or C1-C each time they appear. 26 Alkyl or C1-C6 alkenyl having 1 to 6 double bonds, wherein at least one, at least two, or all of R1-X, R2-Y, and R3-Z are C1-C6 alkenyl groups bonded to heteroatom substituents represented by X, Y, and Z. 26 Alkyl or alkenyl, wherein at least one of R1, R2, and R3 comprises at least 10 carbon atoms and is terminated with a methyl group; and Where X, Y, and Z are either direct bonds or independently selected from -O-; -(C=O)O-; -O(C=O)-; -C(=O)-; -O(C=O)O-; -S(O) x -;-SS-;-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) x NR'-;-NR'S(O) x The heteroatom substituents R'- and -S(O)xNR'-, wherein R' is independently selected from H, C1-C each time it appears. 15 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 groups of R1, R2 and R3 are optionally substituted.

[0085] The term "optionally substituted" in relation to the alkyl or alkenyl groups of R1, R2, or R3 means that at least one hydrogen atom of the alkyl or alkenyl group can be replaced by a non-hydrogen atom or atomic group (i.e., a "substituent"), and / or the alkyl group can be interrupted by a non-carbon atom or one or more substituents (i.e., a -(CH)2- group), provided that the nonpolar lipid retains sufficient hydrophobicity to form the hydrophobic core of the LNP. Substituents that replace hydrogen atoms can include -OR', -SR', or -NR'. Substituents that can replace the methylene -(CH)2- group include, but are not limited to, -O-, -S-, and -NR'-, where 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, X, Y, and Z are independently selected from -(C=O)O- and -O(C=O)-. In one embodiment, the nonpolar lipid is a nonpolar glycerol lipid or its analogue, including triglycerides, diglycerides, monoglycerides, or mixtures thereof.

[0086] In one embodiment, the nonpolar glycerol lipid is a triglyceride (TAG), which refers to a glycerol ester having three fatty acid chains covalently bonded to the glycerol backbone via their respective ester bonds. In one embodiment, all three fatty acids bonded to the glycerol backbone are identical. In other embodiments, the three fatty acids of the triglyceride are different, for example, having different lengths and / or degrees of saturation.

[0087] In some embodiments, TAGs can be characterized by the 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 and U represents an unsaturated fatty acid. In other embodiments, asymmetric TAGs are used in LNPs. 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 asymmetric.

[0088] In embodiments of this disclosure, the triglyceride in the LNP is a trioleic acid glyceride, a symmetrical triglyceride derived from glycerol and three unsaturated fatty acid oleic acid units. The IUPAC name for trioleic acid glyceride is 2,3-bis[[(Z)-octadec-9-enoyl]oxy]propyl(Z)-octadec-9-enolate, and synonyms include glyceroltrioleate, glycerol trioleyl, trielaidin, trioleoylglycerol, and trioleylglycerol.

[0089] Non-limiting examples of triglycerides include trioleic acid glyceride, tristearate glyceride, trilaurate glyceride, trilinoleic acid glyceride, trilinolenic acid glyceride, trimyristate glyceride, tripalmitate glyceride, tricaprylic acid glyceride, triarachidonic acid glyceride, trisanhexanoate glyceride, tripalmitate glyceride, triarachidonic acid glyceride, distearate oleate glyceride, distearate linoleate glyceride, palmitic oleate stearate glyceride, palmitic dioleate glyceride, stearic dioleate glyceride, palmitic dilinoleate glyceride, and stearic oleate linoleate glyceride. In some embodiments, a mixture of two or more triglycerides is included in the LNP.

[0090] In some embodiments, the triglycerides are naturally derived. Examples of naturally sourced fatty acids used in some embodiments include soybean oil, castor oil, sunflower oil, canola oil, and palm oil, as well as omega-3 and omega-6 fatty acids. Furthermore, examples of omega-3 fatty acids used in some embodiments include, but are not limited to, alpha-linolenic acid and docosahexaenoic acid. Examples of omega-6 fatty acids used in some embodiments include, but are not limited to, linoleic acid and gamma-linolenic acid.

[0091] Other commercially available triglycerides include Captex® and Sterotex®. In some embodiments, TAG is selected from Captex. TM 8000, Captex TM GTO and Captex TM 1000.

[0092] In some embodiments, the number of carbon atoms on the aliphatic tail 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 MCTs or mixtures thereof. In some embodiments, the medium-chain triglyceride comprises one or more fatty acids selected from oleic acid, caprylic acid, capric acid, caprylic acid, and / or lauric acid. In some embodiments, the MCT is of high purity. In some embodiments, the MCT has a purity equal to or greater than about the following by weight percentage: 90%, 95%, 97%, 98%, 99%, 100%, or a range including and / or covering the above values. In some embodiments, the MCT (or LCT) is present in the lipid-based particulate composition at a dry weight percentage equal to or greater than about the following: 10%, 20%, 30%, 35%, 40%, 45%, 50%, or a range including and / or covering the above values.

[0093] In some embodiments, the triglycerides comprise fatty acids with a length of more than 12 carbons. In some embodiments, the lipid component comprises long-chain triglycerides (LCTs) with a length or range (including and / or covering the above values) greater than or equal to 13, 14, 15, 16, 17, 18, 19, or 20 carbons.

[0094] The term "diacylglycerol lipid" or "dacylglycerol" (DAG) as used herein refers to a glycerol ester having two fatty acid chains covalently bonded to the glycerol backbone via their respective ester bonds. In some embodiments, DAG includes rac-1,3 or sn-1,2 lipids.

[0095] Exemplary diglycerides include, but are not limited to, diglycerides of laurate, diglycerides of myristate, diglycerides of palmitate, diglycerides of stearate, diglycerides of arachidonic acid, diglycerides of behenicol, diglycerides of palmitoleate, diglycerides of oleate, diglycerides of linoleic acid, diglycerides of linolenic acid, diglycerides of arachidonic acid, diglycerides of caprylate, 1-stearoyl-3-oleoyl-glycerol, 1-stearoyl-2-oleoyl-sn-glycerol, or combinations thereof.

[0096] As used herein, the term "monoacylglycerol lipid" or "monoglyceride" (MAG) refers to a glycerol ester having a fatty acid chain covalently bonded to the glycerol backbone via an ester bond. In one embodiment, the monoacylglycerol lipid is 1-monoacylglycerol or 2-monoacylglycerol, depending on the position of the ester bond on the glycerol backbone.

[0097] Exemplary monoacylglycerol lipids used for incorporation into LNPs include, but are not limited to, monoacyl laurate, monoacyl myristate, monoacyl palmitate, monoacyl stearate, monoacyl arachidonicate, monoacyl behenate, monoacyl palmitate, monoacyl oleate, monoacyl linoleate, monoacyl linoleate, monoacyl arachidonicate, and monoacyl caprylate, and / or, for example, 1-monomyristoyl-racemic glycerol, 1-monopalmitoyl-racemic glycerol, 2-monopalmitoyl glycerol, 1-monopalmitoyl-racemic glycerol, 1-monostearoyl-racemic glycerol, 1-monooleoyl-racemic glycerol, 1-monolinoleoyl-racemic glycerol, 1-monolinoleoyl-racemic glycerol, or combinations thereof.

[0098] Sterols In some non-limiting instances, lipid nanoparticles include sterols.

[0099] The term "sterol" refers to naturally occurring or synthetic steroids. This term includes cholesterol, phytosterols, animal and plant sterols, and their derivatives.

[0100] The term "sterol derivative" refers to modified sterols or their precursors, including triterpenes.

[0101] The term “cholesterol” refers to a naturally occurring or synthetic compound that has a sterane skeleton and a hydroxyl group bonded to one of its rings, usually the A ring.

[0102] Cholesterol derivatives can be naturally occurring or synthetic, including but not limited to cholesterol molecules having a sterane structure and one or more other functional groups, including derivatization of a terminal hydroxyl group.

[0103] In another embodiment, the LNP may comprise a triterpenoid. Non-limiting examples include squalene, yarrow A, polypodatetraene, malabaricane, lanostane, cucurbitacin, hopane, oleanane, and ursolic acid.

[0104] In some embodiments, the cholesterol derivative is a phytosterol. The phytosterol may be β-sitosterol, 3-sitosterol, campesterol, stigmasterol, alginosterol, or stigmasterol, or a salt or ester thereof.

[0105] Cholesterol derivatives include β-sitosterol, 3-sitosterol, campesterol, stigmasterol, fucosterol, or stigmasterol, dihydrocholesterol, α-cholesterol, epi-cholesterol, sterol, cholesterol, cholesterol, cholesterol, cholesterol, cholesterol, cholesterol, cholesterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, 3β[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxapropylcyclosterol, 23-oxapropylcyclosterol, 24-oxapropylcyclosterol, cycloaltiol, 22-ketosterol, 20- Hydroxysterols, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol, 3,6,9-trioxaoctane-1-ol-cholest-3β-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanosterol, lupeosterol, sitocalciferol, calcipotriol, coccosterol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, brassosterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, yeast sterol, diosgenin, fucosterol, coccosterol or their salts or esters.

[0106] Sterols, cholesterol, or their derivatives can be conjugated with another component, such as an amino acid or an alkyl group.

[0107] In one embodiment, based on the total lipids present in the lipid nanoparticles, sterols or their derivatives are present in a molar ratio of 0% to 50%, 15% to 50%, 20% to 45%, or 25% to 40%.

[0108] In one embodiment, total lipids, cholesterol or its derivatives present in the lipid nanoparticles are present in a molar ratio of 0% to 50%, 15% to 50%, 20% to 45%, or 25% to 40%.

[0109] In some embodiments, the LNP comprises cholesterol esters, which contain fatty acids conjugated via ester groups. For example, the cholesterol ester content may be 0 to 10% molar ratio, 0 to 8% molar ratio, 0 to 5% molar ratio, or 0 to 2% molar ratio.

[0110] In another embodiment, the LNP has a low level of cationic cholesterol lipids, such as 3β[N-(N'N'-dimethylaminoethyl)carbamoyl cholesterol hydrochloride (DC-cholesterol). For example, the cationic cholesterol content may be less than 10% molar ratio, less than 8% molar ratio, less than 5% molar ratio, or less than 2% molar ratio.

[0111] LNPs may also contain tocopherols as an additional component. Tocopherols include α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, or their salts or esters. Tocopherols may be present in molar ratios of 0.5% to 20%, 1% to 15%, or 2% to 10%.

[0112] Ionizable lipids The LNPs disclosed herein possess ionizable lipids. These ionizable lipids can be charged at low pH and have essentially no net charge at physiological pH. This allows for electrostatic interactions between the lipids and negatively charged nucleic acid cargo during initial formulation. Since the ionizable lipids are near neutral at physiological pH, toxicity and renal clearance are reduced. Without being theoretically limited, the acidic environment of the endosome after endocytic uptake leads to an increase in the net positive charge of the ionizable amino lipids, which promotes fusion with anionic lipids at the endosome membrane and subsequent membrane destabilization and release of nucleic acid-based therapeutic agents into the cytoplasm to exert their effects.

[0113] In some embodiments, it is desirable to include less than 50% molar of ionizable lipids in the LNP. In some embodiments, the ionizable lipid content is 5% to 50% molar, 8% to 47% molar, 10% to 50% molar, 15% to 45% molar, or 15% to 35% molar of the total lipids present in the lipid nanoparticles.

[0114] In some embodiments, the ionizable lipid content may be less than 48% molar ratio, less than 45% molar ratio, less than 40% molar ratio, less than 35% molar ratio, less than 30% molar ratio, less than 25% molar ratio, less than 20% molar ratio, less than 15% molar ratio, less than 10% molar ratio, or less than 5% molar ratio. In some embodiments, the lower limit of the ionizable lipid content may be greater than 5% molar ratio, greater than 8% molar ratio, greater than 10% molar ratio, greater than 12% molar ratio, greater than 14% molar ratio, greater than 15% molar ratio, greater than 16% molar ratio, greater than 18% molar ratio, or greater than 20% molar ratio. Any of the upper limits may be combined with any of the lower limits to obtain a suitable ionizable lipid content in the LNP.

[0115] In some embodiments, the ionizable lipid is an "ionizable cationic lipid," which refers to a lipid that is electrostatically neutral at physiological pH and accepts a proton to become positively charged. In some embodiments, the cationic lipid has a pKa of 6.0 to 8.5 or 6.0 to 7.5.

[0116] In some embodiments, the cationic lipid has an amino group. In some cases, the cationic lipid contains a protonable tertiary amine (e.g., pH-titrile) head group. Non-limiting examples of such lipids include, but are not limited to, thiolipin lipids, such as MF019 and DODMA described herein. Other lipids that can be used to implement this disclosure include MC3- and KC2-type lipids well known to those skilled in the art. In other embodiments, the ionizable lipid is selected from one or more lipids described in WO 2022 / 246555; WO 2022 / 246568; WO 2022 / 24657; WO 2023 / 147657; WO 2022 / 155728; WO 2023 / 215989; WO2024 / 065041; WO 2024 / 065042; WO 2024 / 130421; WO 2024 / 065043; and US 2024 / 0294462, each of which is incorporated herein by reference.

[0117] In one embodiment, the ionizable cationic lipid comprises an ionizable amino head group and at least two lipophilic groups, wherein at least one lipophilic group comprises a heteroatom, such as an ester or one or more sulfur atoms. In some embodiments, at least one lipophilic group comprises distally branched 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 commonly owned and co-pending WO2023 / 215989; WO2024 / 065041; WO2024 / 065042; WO2024 / 130421; and WO 2024 / 065043. The functional group comprising one or more heteroatoms can be biodegraded in vivo.

[0118] In some implementations, it is desirable to include less than 50% molar of cationic lipids in the LNP. That is, the content of ionizable lipids can be less than 50% molar, less than 45% molar, less than 40% molar, less than 35% molar, less than 30% molar, less than 25% molar, less than 20% molar, less than 15% molar, less than 10% molar, or less than 5% molar.

[0119] In some embodiments, the cationic lipid content is 5% to 50% of the total lipids present in the lipid nanoparticles, or 8% to 47% of the total lipids, or 10% to 50% of the total lipids, or 15% to 45% of the total lipids, or 15% to 35% of the total lipids.

[0120] Ionizable lipid components may include ionizable anionic lipids as part of the ionizable lipid content. An example of such lipid is cholesterol hemisuccinate (CHEMS). Other examples of ionizable anionic lipids are described in co-pending and co-owned WO 2024 / 192528, the entire contents of which are incorporated herein by reference.

[0121] Hydrophilic polymer-lipid conjugate In one embodiment, the lipid nanoparticles comprise a hydrophilic polymer lipid conjugate capable of incorporating into the LNP. In some instances, the conjugate comprises a vesicle-forming lipid, hydrophilic polymer chain having a polar head group and covalently linked to the head group. The lipid comprises any portion having at least one hydrophobic portion. Examples of hydrophilic polymers include polyethylene glycol (PEG), polyvinylpyrrolidone, polyethylene methyl ether, polyhydroxypropyl methacrylate, polyhydroxypropyl methacrylamide, polyhydroxyethyl acrylate, polymethacrylamide, polydimethylacrylamide, polymethyl methacrylate, polyethyl methacrylate, polyethyl methacrylate, polyhydroxypropyl methacrylate, polycarboxylic acid, polyethyl methacrylate, polyhydroxypropyl methacrylate, polysarcosine, and polyasparagine. In one embodiment, the hydrophilic polymer lipid conjugate is a PEG-lipid conjugate. The hydrophilic polymer lipid conjugate may also be a naturally occurring or synthetic oligosaccharide-containing molecule, such as monosialotetrahexosylganglioside (G... M1 The ability of a given hydrophilic-polymer lipid conjugate to enhance the cycle life of the LNP described herein can be readily determined by those skilled in the art using known methods.

[0122] The hydrophilic polymer lipid conjugate can be present in nanoparticles at a molar ratio of 0.5% to 5%, 0.5% to 3%, 0.5% to 2.5%, 0.5% to 2.0%, or 0.5% to 1.8% of the total lipids.

[0123] The hydrophilic polymer lipid conjugate may exist in nanoparticles at a total lipid content of 0% to 5%, 0% to 3%, 0% to 2.5%, 0% to 2.0%, or 0% to 1.8%.

[0124] In another embodiment, the hydrophilic polymer-lipid conjugate is a PEG-lipid conjugate present in nanoparticles at a molar ratio of 0.5% to 5%, or 0.5% to 3%, or 0.5% to 2.5%, or 0.5% to 2.0%, or 0.5% to 1.8% of the total lipids.

[0125] In another embodiment, the hydrophilic polymer-lipid conjugate is a PEG-lipid conjugate present in nanoparticles at a total lipid molar ratio of 0% to 5%, or 0% to 3%, or 0% to 2.5%, or 0% to 2.0%, or 0% to 1.8%.

[0126] As described below, hydrophilic polymer-lipid conjugates can be conjugated with targeting ligands at their distal ends.

[0127] Low phospholipid content The lipid nanoparticles are “substantially phospholipid-free,” meaning they contain less than 3% molar amount of phospholipids, such as neutral phospholipids. In one example, the lipid nanoparticles have a phospholipid content of 3% molar amount or less. Examples of neutral phospholipids include phosphatidylcholine or phosphatidylethanolamine, such as distearylphosphatidylcholine (DSPC), distearylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and dipalmitoylphosphatidylcholine (DPPC). In some embodiments, the phospholipids are phospholipid-sterol conjugates, such as SPC-cholesterol, OPC-cholesterol, or PPC-cholesterol conjugates. Other phospholipid-sterol conjugates are described in US 2011 / 0177156, which is incorporated herein by reference.

[0128] In one embodiment, the lipid nanoparticles have phospholipids in a molar ratio of less than 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or 0%. Most advantageously, the LNPs are phospholipid-free, but small amounts may not affect the properties of the LNPs described herein.

[0129] In another embodiment, "substantially free of neutral lipids" means that the lipid nanoparticles have a molar ratio of any polar neutral lipids from 0 to 3%, excluding cholesterol or cholesterol derivatives and nonpolar glycerides or their analogues. The term "neutral lipids" refers to any of a variety of polar lipids present at physiological pH in an uncharged or neutral zwitterionic form, including vesicle-forming lipids. In another example, the lipid nanoparticles have a molar ratio of less than 8, 6, 4, or 2% neutral lipids, such as phospholipids and / or phospholipid conjugates.

[0130] Examples of polar neutral lipids include sphingomyelins, diacylphosphatidylcholine such as distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and dipalmitoylphosphatidylcholine (DPPC), diacylphosphatidylethanolamine such as dioleoylphosphatidylethanolamine (DOPE), ceramides, cephalins, triglycerides, and diacylglycerols. In some embodiments, the neutral lipid is a phospholipid-sterol conjugate, such as SPC-cholesterol or PPC-cholesterol conjugates or those described in US 2011 / 0177156.

[0131] In one embodiment, the lipid nanoparticles have any polar neutral lipids in a molar ratio of less than 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or 0%. Most advantageously, the LNP does not contain polar neutral lipids, but small amounts may be included in the formulation without affecting the properties of the LNP described herein.

[0132] Other components LNPs may contain additional lipid components or modifications to sterols (e.g., cholesterol), sterol derivatives (e.g., cholesterol derivatives), and / or hydrophilic polymer-lipid conjugates.

[0133] For example, the surface of the LNP can be grafted to contain a targeting ligand. The targeting ligand can be conjugated to cholesterol, cholesterol derivatives, and / or hydrophilic polymer-lipid conjugates. The targeting ligand can be conjugated to the distal end of the hydrophilic polymer-lipid conjugate. In some embodiments, the targeting ligand can be used to target receptors on cells in vivo. In some embodiments, the targeting ligand can be conjugated to at least an amount of phospholipids that may be contained in the LNP. In such embodiments, the phospholipid-targeting ligand conjugate is typically present at a molar ratio of less than 3%.

[0134] Ligands include peptides, polypeptides, or proteins, and may include antibodies or fragments thereof. In one embodiment, the ligand may be a single-chain antibody fragment. Examples of ligands are described in WO 2024 / 119279, which is incorporated herein by reference.

[0135] Nanoparticle preparation and morphology Lipid nanoparticles can be prepared using a variety of suitable methods, such as rapid mixing / ethanol dilution. Examples of preparation methods are described in Jeffs, LB, et al., Pharm Res, 2005, 22 (3):362-72; and Leung, AK, et al., The Journal of Physical Chemistry. C, Nanomaterials and Interfaces, 2012, 116(34): 18440-18450, which are each incorporated herein by reference in their entirety.

[0136] For example, methods for preparing lipid nanoparticles may include dissolving lipid components (e.g., ionizable lipids, phosphatidylcholine, and sterols or derivatives thereof) in an organic solvent (e.g., ethanol) at appropriate ratios. Aqueous buffers containing nucleic acids are prepared separately at a suitable pH to ensure that the head groups (e.g., amino groups) of the ionizable lipids are protonated to promote electrostatic interactions with both the negatively charged cargo and the positively charged ionizable lipids. This charge interaction improves nucleic acid encapsulation.

[0137] In some embodiments, the aqueous phase containing nucleic acids is subsequently combined with an organic solvent-lipid mixture containing lipids. The combination of the aqueous phase and the organic solvent-lipid mixture can be performed in a mixing device (e.g., an inline mixer), such as a T-joint mixer with a pump (e.g., a T-tube mixer), a herringbone micromixer, a ring mixer, a multi-inlet vortex mixer, or other suitable mixing devices known to those skilled in the art. In some embodiments, a mixing device refers to a device comprising two or more inlets that meet in a central mixing region and an outlet through which the mixture exits the device. LNP formation can occur on and / or after the mixture of the aqueous phase and the organic solvent-lipid mixture (Kulkarni et al., 2019, Nanoscale, 11(18):9023-9031, which is incorporated herein by reference).

[0138] The aqueous phase typically contains a buffer. Non-limiting examples of suitable buffers include MES, sodium acetate, or phosphate-buffered saline (PBS). Examples of suitable solvents for preparing organic solvent-lipid mixtures are organic solvents, including ethanol, isopropanol, methanol, and acetone.

[0139] The aqueous phase and organic solvent lipid mixture can be introduced into the mixer as two separate corresponding streams via a pump. The volumetric flow rate of each stream can be the same or different, and the corresponding flow rate of each stream can be adjusted to achieve optimal mixing and / or LNP formation.

[0140] In some embodiments, LNPs are prepared by solvent injection. In one embodiment, this method involves dissolving the lipids in an organic solvent and then gradually diluting the resulting solution with an aqueous solution (e.g., a buffer). This controlled gradual dilution is achieved by mixing the aqueous and lipid streams together in a container.

[0141] The lipid nanoparticles may have an average size of 40 to 150 nm, 40 to 140 nm, or 45 to 130 nm, or any range thereof. In another embodiment, the lipid nanoparticles have a PDI of less than 0.20, less than 0.15, less than 0.12, or less than 0.10.

[0142] The nitrogen-to-phosphorus ratio of the lipid nanoparticles can be from 1 to 12. In another embodiment, the nitrogen-to-phosphorus ratio of the lipid nanoparticles can be from 1 to 6.

[0143] LNPs typically include a “core” region that can be characterized as electronically dense, and optionally have one or more peripheral bubbles visualized by cryo-TEM microscopy.

[0144] "Bubble" refers to the protrusions of lipid nanoparticles, which are usually spherical or hemispherical in shape.

[0145] Morphology was visually evaluated using cryo-TEM microscopy as described in the materials and methods of this embodiment.

[0146] Instances of this form are in Figure 7 As shown in A. Bubbles can appear as spherical or hemispherical protrusions composed of lipid nanoparticles (see Figure A). Figure 7 A). In some embodiments, the nucleus is hydrophobic, and one or more vesicle protrusions extend from the nucleus. The vesicles may have aqueous compartments and contain nucleic acids. In one embodiment, a nonpolar glycerol ester or its analogue forms the hydrophobic nucleus of the LNP. In one embodiment, the nucleus formed from the nonpolar glycerol ester lipid or its analogue is homogeneous as observed by cryo-TEM microscopy, but has a periphery with one or more vesicles.

[0147] In some embodiments, LNPs are not lipid complexes. Lipid complexes are prepared by mixing pre-formed cationic liposomes with nucleic acids in an aqueous solution and can exhibit undesirable properties, such as cargo localization on particle surfaces. Lipid complexes lack the core of the LNP particles described above. Furthermore, LNPs have defined size, shape, and morphology, while lipid complexes lack such defined physical properties. (See Kubota et al., 2017, Int. J. Nanomedicine, 12:5121-5133 and Kulkarni et al., 2018, Nucleic Acid Therapeutics, 28(3):146-157, each incorporated herein by reference).

[0148] Therefore, according to certain embodiments, the LNP disclosed herein has a defined average particle size of 40 to 150 nm, 40 to 140 nm, or 45 to 150 nm. In some embodiments, the LNP herein has a PDI of less than 0.25, less than 0.20, less than 0.18, less than 0.16, less than 0.15, or less than 0.14.

[0149] As used herein, the term "encapsulation" in relation to the incorporation of nucleic acid cargo into LNPs refers to any association of the nucleic acid with any lipid component or compartment of the lipid nanoparticle. However, this excludes the localization of the nucleic acid on the particle surface, as in lipid complexes. In some instances of this disclosure, the nucleic acid is present within the vesicles of the LNP.

[0150] Nucleic acid goods In one embodiment, the cargo is a nucleic acid. Nucleic acids include, but are not limited to, RNA, including small interfering RNA (siRNA), small nuclear RNA (snRNA), microRNA (miRNA), messenger RNA (mRNA), or DNA, such as vector DNA and linear DNA. The length of the nucleic acid can vary and can include nucleic acids with a length of 1 to 50,000 nucleotides. Nucleic acids can be in any form, including single-stranded DNA or RNA, double-stranded DNA or RNA, or hybrids thereof. Single-stranded nucleic acids include antisense oligonucleotides. Nucleic acids can be conjugated to another molecule, including a targeting moiety. Examples of such nucleic acid conjugates are antibody-nucleic acid conjugates or oligosaccharide-nucleic acid conjugates, such as GalNAc-nucleic acid conjugates.

[0151] In one embodiment, the cargo is mRNA, which includes polynucleotides encoding 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.

[0152] The mRNAs used herein include both modified and unmodified mRNAs. In one embodiment, the mRNA comprises one or more coding and non-coding regions. The mRNA can be purified from natural sources, generated and optionally purified using a recombinant expression system, or can be chemically synthesized.

[0153] In embodiments where the mRNA is a chemically synthesized molecule, the mRNA can include nucleoside analogs, such as analogs with chemically modified bases or sugars, and / or backbone-modified analogs. In some embodiments, the mRNA is or contains natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiopyrimidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 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-methylcyt ...methylcytidine, 2-aminouridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminouridine, C5-bromouridine, C5-fluorouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminouridine, C5-bromouridine, C5-bromouridine, C5-fluorouridine, C5-bromour -aminoadenosine, 7-deadenosine, 7-deadenosine, 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., thiophosphate and 5'-N-phosphoramide bond).

[0154] The mRNA disclosed herein can be synthesized according to any of a variety of known methods. For example, in some embodiments, the mRNA can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a library of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitors.

[0155] In some implementations, the in vitro synthesized mRNA can be purified before encapsulation to remove unwanted impurities, including various enzymes and other reagents used during mRNA synthesis.

[0156] This disclosure can be used to encapsulate mRNA of various lengths. In some embodiments, this disclosure can be used to encapsulate in vitro synthesized mRNA of lengths ranging from about 1 to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about 8 to 15 kb.

[0157] Typically, mRNA synthesis involves adding a "cap" at the 5' end and a "tail" at the 3' end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of the tail protects the mRNA from degradation by exonucleases.

[0158] In some embodiments, the mRNA includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as iron-reactive elements. In some embodiments, the length of the 5' untranslated region can be between about 50 and 500 nucleotides.

[0159] In some embodiments, the 3' untranslated region includes one or more polyadenylation signals, protein binding sites that affect the stability of mRNA localization in the cell, or one or more binding sites for miRNA. In some embodiments, the length of the 3' untranslated region can be between 50 and 500 nucleotides or longer.

[0160] In another embodiment, the mRNA is circular. Advantageously, such mRNA lacks both 5' and 3' ends, and is therefore more stable in vivo due to its resistance to degradation by exonucleases. Circular mRNAs can be prepared by any known method, including Deviatkin et al., 2023, “Cap-Independent Circular mRNA Translation Efficiency,” Vaccines, 11(2), 238, which is incorporated herein by reference. Translation of circular mRNAs proceeds via a cap-independent initiation mechanism.

[0161] While some implementations may require mRNA provided by in vitro transcription reactions, other mRNA sources, such as mRNA produced by bacteria, fungi, plants, and / or animals, are also considered.

[0162] The mRNA sequence may contain a reporter gene sequence, although the inclusion of a reporter gene sequence in a drug formulation for administration is optional. This sequence can be incorporated into the mRNA for in vitro studies or for in vivo studies in animal models to assess biodistribution.

[0163] In another embodiment, the cargo is siRNA. The siRNA is incorporated into endogenous cellular mechanisms to cause mRNA degradation, thereby preventing transcription. Since RNA is readily degraded, its incorporation into the delivery vector can reduce or prevent this degradation, thereby facilitating delivery to the target site.

[0164] The siRNAs covered by this disclosure can be used to specifically inhibit the expression of a variety of target polynucleotides. siRNA molecules targeting specific polynucleotides can be readily prepared according to methods known in the art. siRNA target sites can be selected, and the corresponding siRNAs can be chemically synthesized, produced by in vitro transcription, or expressed from vectors or PCR products. A variety of different siRNA molecules can be used to target specific genes or transcripts. siRNAs can be double-stranded RNAs or hybrid molecules containing RNA and DNA, such as one RNA strand and one DNA strand. siRNAs can have various lengths, such as 15 to 30 nucleotides or 20 to 25 nucleotides. In some embodiments, the siRNA is double-stranded and has a 3' overhang or a 5' overhang. In some embodiments, the overhang is UU or dTdt 3'. In specific embodiments, the siRNA comprises a stem-loop structure.

[0165] In other embodiments, the cargo molecule is microRNA or small nuclear RNA. MicroRNAs (miRNAs) are short, non-coding RNA molecules that are transcribed from genomic DNA but not translated into proteins. These RNA molecules are believed to play a role in the regulation of gene expression by binding to regions of target mRNAs. The binding of miRNAs to target mRNAs can downregulate gene expression, for example, by inducing translational repression, deadenylation, or degradation of the target mRNA. Small nuclear RNAs (snRNAs) are typically longer non-coding RNA molecules involved in gene splicing. snRNA molecules may have therapeutic importance in diseases that result from splicing defects.

[0166] Examples of nucleic acid cargo include, but are not limited to, antisense oligonucleotides, ribozymes, microRNA, mRNA, ribozymes, tRNA, tracrRNA, sgRNA, snRNA, siRNA, shRNA, ncRNA, miRNA, mRNA, precondensed DNA, pDNA, or aptamers.

[0167] In another embodiment, the cargo is a DNA vector. The encapsulated DNA vector can be administered to an individual to repair, enhance, block, or reduce the expression of cellular proteins or peptides. In another embodiment, the encapsulated DNA vector can be administered to an individual for disease diagnosis. The DNA vector can be localized to target cells (e.g., rapidly dividing cells), and the expression of the encoded DNA can be used to provide a measurable signal. Therefore, the nucleotide polymer can be a nucleotide sequence comprising genomic DNA, cDNA, or RNA.

[0168] As will be understood by those skilled in the art, a vector may encode a promoter region, an operator region, or a structural region. A DNA vector may contain double-stranded DNA or may consist of a DNA-RNA hybrid. Non-limiting examples of double-stranded DNA include structural genes, genes including operator control and termination regions, and self-replicating systems such as vector DNA.

[0169] Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes, and oligonucleotides that form triplet strands. For extended activity, single-stranded nucleic acids will preferably have some or all of the nucleotide bonds replaced by stable non-phosphodiester bonds, including, for example, thiophosphate, dithiophosphate, phosphoselenate, or O-alkyl phosphate triester bonds.

[0170] DNA vectors may comprise nucleic acids modified in one or more sugar moieties and / or one or more pyrimidine or purine bases. Such sugar modifications may include substituting one or more hydroxyl groups with halogens, alkyl groups, amines, azides, or functionalization into ethers or esters. In another embodiment, the entire sugar may be replaced by a spatially and electronically similar structure, including aza-sugars and carbocyclic sugar analogs. Modifications to the purine or pyrimidine base moieties include, for example, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substituents known to those skilled in the art.

[0171] In some embodiments, DNA vectors may be modified with modifying molecules such as peptides, proteins, steroids, or glycosides. Modifying DNA vectors with such molecules can facilitate delivery to target sites of interest. In some embodiments, this modification causes the DNA vector to translocate across the nuclear region of the target cell. As an example, modifiers may bind to specific portions of the DNA vector (typically not encoding the target gene), but may also have peptides or other modifiers that exhibit nuclear homing effects such as nuclear localization signals. A non-limiting example of a modifier is a steroid-peptide nucleic acid conjugate, as described in Rebuffat et al., 2002, Faseb J. 16(11):1426-8, which is incorporated herein by reference. DNA vectors may contain sequences encoding different proteins or peptides. Promoters, enhancers, stress or chemically regulated promoters, antibiotic-sensitive or nutrient-sensitive regions, and sequences encoding therapeutic proteins may be included as needed. Non-coding sequences may also be present in DNA vectors.

[0172] The nucleic acids used in this disclosure can be isolated from natural sources, obtained from sources such as ATCC or GenBank libraries, or prepared by synthetic methods. Synthetic nucleic acids can be prepared by various solution or solid-phase methods. Generally, solid-phase synthesis is preferred. Detailed descriptions of methods for solid-phase synthesis of nucleic acids via chemical reactions of phosphite-triesters, phosphate-triesters, and H-phosphonates are widely available.

[0173] In one embodiment, the DNA vector is double-stranded DNA and contains more than 700 base pairs, more than 800 base pairs, more than 900 base pairs, or more than 1,000 base pairs.

[0174] Improved liver-specific expression In some embodiments, the LNP exhibits “liver-specific expression” of a protein or peptide encoded by the cargo nucleic acid, meaning that the cargo nucleic acid increases the expression of the protein or peptide in the liver by at least 1.5-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or at least 60-fold more than that in the spleen. In some embodiments, the cargo nucleic acid is mRNA or vector DNA. In one example, the nucleic acid is mRNA.

[0175] Improvement in liver-specific expression can be compared to "Onpattro" TM The baseline formulation or baseline LNP is evaluated. This term refers to a formulation with ionizable lipids / DSPC / chol / PEG at 50 / 10 / 38.5 / 1.5 mol / mol. 2000 -DMG lipids in LNP, wherein the ionizable lipid is nor-MC3 (see compositions in Table 1 of this document).

[0176] In other implementations, relative to Onpattro TM Baseline formulations containing low levels of hydrophilic polymer-lipid conjugates can improve liver-specific expression of LNPs with elevated levels of sterols or sterol derivatives. For example, the amount of the liver-targeting hydrophilic polymer-lipid conjugate achieving this improvement can be less than 2.5% molar, less than 2.25% molar, less than 2.0% molar, less than 1.75% molar, less than 1.5% molar, less than 1.25% molar, less than 1.00% molar, less than 0.75% molar, less than 0.50% molar, less than 0.25% molar, or less than 0.20% molar.

[0177] Or, or otherwise, under otherwise identical conditions, measuring and encapsulating the same cargo, the increased expression of proteins or peptides encoded by nucleic acid cargo (e.g., mRNA or vector DNA) in the liver relative to the spleen compared to Onpattro TM Baseline LNP expression was at least 5% or 10% higher in the liver than in the spleen.

[0178] In one implementation, with Onpattro TM Compared to fresh samples of the baseline formulation, administration of lipid nanoparticles to fresh samples resulted in a 5-fold increase in liver-specific expression of proteins or peptides encoded by encapsulated mRNA or vector DNA (less than one month after preparation).

[0179] In one implementation, with Onpattro TM Compared to fresh samples of the baseline formulation, administration of lipid nanoparticles to fresh samples resulted in a 5-fold increase in liver-specific gene silencing.

[0180] In one implementation, with Onpattro TM Compared to frozen samples of the baseline formulation, administration of lipid nanoparticles to frozen samples resulted in a 4-fold increase in liver-specific expression of proteins or peptides encoded by encapsulated mRNA or vector DNA.

[0181] In one implementation, with Onpattro TM Compared to frozen samples of the baseline formulation, frozen samples administered with lipid nanoparticles resulted in a 4-fold increase in liver-specific gene silencing.

[0182] Editing of hepatocyte genetic material LNPs can contain nucleic acids that encode proteins or peptides that form part of a “gene editor” or “editor”, including but not limited to products or compositions that edit genetic material, such as inserting, deleting, modifying (e.g., epigenetic editing), or replacing nucleic acids in the genetic material of an organism at a site-specific location.

[0183] The editor can be used for in vitro or in vivo genetic modification of hepatocytes, including post-translational modifications.

[0184] Gene editors include, but are not limited to, Cas-based (e.g., CRISPR or non-CRISPR), transcription activator-like effector nucleases (TALENs), macroTALs, zinc finger nucleases (ZFNs), RNA-acting adenosine deaminases (ADARs), primary immune editors, base editors, epigenetics, transposases, macronucleases, the ARCUS gene editing system, or any variants or combinations thereof. However, these edits are exemplary, and this disclosure includes any product or composition that can modify the genetic material of hepatocytes (including RNA transcripts and non-coding regions) to treat, prevent, or improve a condition or disease. Unrestricted, editors may include those designed using methods known to those skilled in the art as directed nuclease editors (DNEs).

[0185] CAS-based editors include CRISPR and non-CRISPR gene editing systems. Furthermore, editors include DNA-cutting editors and epigenetic editing systems that modify nucleic acid markers, as described below.

[0186] CRISPR nucleic acid editors most advantageously comprise nucleic acids (e.g., mRNA) encoding one or more proteins from the class II Cas nuclease family and guide RNA. The nuclease encoded by the nucleic acid is an enzyme with DNA endonuclease activity and is capable of directionally cleaving a desired nucleic acid target via a suitable guide RNA. The nuclease and guide RNA form a complex called a ribonucleoprotein (RNP). In some embodiments, the nuclease is a class II CRISPR enzyme, which is further subdivided into type II, type V, and type VI. According to one embodiment, the mRNA encodes a Cas protein, which is part of a type II CRISPR / Cas system, such as the Cas9 protein or the Cpf1 protein.

[0187] In another embodiment, the mRNA encodes a Cas protein, such as Cas12a, which is part of a type V CRISPR / Cas system. In another embodiment, the mRNA encodes a Cas protein called Cas13a, which is an RNA endonuclease that cleaves single-stranded RNA.

[0188] Guide RNA directs Cas nucleases to target sequences on target nucleic acid molecules, where the guide RNA hybridizes to the target sequence and the Cas nuclease cleaves or regulates the sequence. In some embodiments, the guide RNA binds to two types of nucleases, thereby providing cleavage specificity.

[0189] The guide RNA of the CRISPR / Cas9 nuclease system includes CRISPR RNA (crRNA) or tracr RNA (tracr). In some embodiments, the crRNA can include a target sequence that is complementary to and hybridizes with a target sequence on a target nucleic acid molecule. The crRNA can also include a flagpole that is complementary to and hybridizes with a portion of the tracrRNA. In some embodiments, the crRNA can correspond to the structure of naturally occurring crRNA transcribed from a bacterial CRISPR locus, wherein the target sequence acts as a spacer sequence in the CRISPR / Cas9 system. The flagpole corresponds to a portion of a repeating sequence adjacent to the spacer sequence above the CRISPR locus.

[0190] The guide RNA of an RNP can target any sequence of interest via the target sequence of the crRNA. In some embodiments, the target sequence of the guide RNA and the target sequence on the target nucleic acid molecule can be 100% complementary. In other embodiments, the target sequence of the guide RNA and the target sequence on the target nucleic acid molecule can contain at least one mismatch.

[0191] The length of the target sequence can depend on the RNP system and the components used. For example, different Cas proteins from different bacterial species have a variety of optimal target sequence lengths. Therefore, a target sequence can include 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 nucleotides. In some embodiments, the target sequence can contain 18 to 24 nucleotides. In some embodiments, the target sequence can contain 19 to 21 nucleotides. In some embodiments, the target sequence can contain 20 nucleotides.

[0192] In some implementations, nucleic acid editors include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb111, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologues or modified forms thereof.

[0193] As described above, embodiments of this disclosure also include non-CRISPR, Cas-based nucleic acid editors. Cas-based editors may include Cas enzymes fused with deaminases (Luo et al., 2020, Microbial Cell Factories, 19(93), incorporated herein by reference). Examples are cytosine base editors or adenine base editors produced by fusing the endonuclease Cas to the cytosine deaminase pmCDA1 or the heterodimeric adenine deaminase TadA-TadA. Other non-limiting examples are Cas fused with reverse transcriptases (Mohr et al., 2018, Mol Cell., 72(4):700-714, incorporated herein by reference).

[0194] Fanzor is a eukaryotic RNA-guided endonuclease that can serve as a nucleic acid editor. (See Saito et al., 2023, Nature 620:660-668, which is incorporated herein by reference). In some implementations, the Fanzor protein uses RNA as a guide to precisely target DNA and can be modified to edit hepatocytes using the LNPs described herein. In some examples, compact Fanzor systems can have the ability to facilitate more improved delivery than CRISPR-Cas systems.

[0195] In embodiments where the editor is a TALEN, the LNP comprises a fragment or variant of a nucleic acid encoding a peptide having a transcription activator-like (TAL) effector DNA-binding domain. In one embodiment, the editor comprises a nucleic acid encoding a peptide having nuclease activity such as endonuclease activity. In one embodiment, the peptide having nuclease activity is a type II restriction 1-like endonuclease, such as Fokl endonuclease.

[0196] In embodiments where 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 thereof, or a variant thereof; and / or nuclease activity, such as endonuclease activity. In one embodiment, the zinc finger binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, or more zinc fingers. In one embodiment, the peptide having nuclease activity is a type II restriction 1-like endonuclease, such as Fokl endonuclease.

[0197] RNA-acting adenosine deaminases (ADARs) are another editor covered by embodiments of this disclosure that can be used for post-transcriptional modification of RNA. Examples include ADAR1 and ADAR2. ADAR1 can catalyze the post-transcriptional deamination of C6 of adenosine in dsRNA, converting them to inosine (see Song et al., 2022, PMC, 13(1):e1665, incorporated herein by reference).

[0198] Large nucleases are enzymes in the endonuclease family that can induce homologous recombination, generate mutations, and alter reading frames. Large nucleases include homing endonucleases of intron or protein intron endonucleases. In one embodiment, the large nuclease is derived from the LAGLIDADG family, GIY-YIG endonucleases, HNH endonucleases, His-Cys box endonucleases, or PD-(D / E)XK endonucleases. Large nucleases can be combined with components of other gene editing systems. In one embodiment, a DNA-binding domain from a transcription activator-like (TAL) effector is combined with the large nuclease to produce “megaTAL”. In another embodiment, the large nuclease can be fused with a DNA end-processing enzyme to facilitate error-prone non-homologous end joining.

[0199] ARCUS nuclease is a gene-editing system based on I-CreI, used in Chlamydomonas reinhardtii (…). Chlamydomonas reinhardtii ARCUS nucleases are homing endonucleases that evolved in the genome. In some implementations, the nucleases are capable of self-inactivation after gene editing, thereby reducing off-target effects. In some implementations, ARCUS nucleases are capable of producing unique cleavage sites that are four-base pairs with a 3' overhang and can perform gene insertion, gene excision, gene repair, or a combination thereof.

[0200] Epigenetic editing is also included in the examples of this disclosure. This editing of genetic material does not cut nucleic acids, but rather alters epigenetic markers to “modify” DNA. Changing the epigenetic characteristics of a cell can be used to modify its epigenetic signature and alter its transcriptional profile. In some embodiments, the epigenetic editing system can target and edit one or more methylation sites on a nucleic acid sequence. In some embodiments, a genomic homing protein having engineered or naturally occurring nuclease function for gene editing can be mutated and adapted to function solely as a delivery system. 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. Targeting proteins that recognize DNA sequences can be attached to effector proteins that alter epigenetic markers, such as methylation. Examples of targeting proteins include transcription activator-like effectors (TALEs), zinc finger proteins, and Cas systems including, but not limited to, CRISPR-Cas. Non-limiting examples of effector proteins include TET1, which induces cytosine demethylation at CpG sites; LSD1, which induces demethylation of H3K4me1 / 2, which in turn causes the indirect effect of deacetylation of H3K27; and CIB1 / CRY2, a leuco dye / blue light activated complex that allows chromatin to be modified upon irradiation.

[0201] Other examples of effector proteins include DNA methyltransferases, fragments (e.g., biologically active fragments) or variants thereof (e.g., DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3L or CpG methyltransferase (M.SSSL)); or multicomb repressive complexes or components thereof, such as PRC1 or PRC2, or PR-DUB, or fragments thereof (e.g., biologically active fragments) or variants thereof.

[0202] In one embodiment, the epigenetic editor comprises molecules that modify chromatin structure and / or modify histones. In one embodiment, the epigenetic regulator is a chromatin-modifying molecule, such as the SWI / SNF remodeling complex or a component thereof. In one embodiment, the epigenetic regulator is a histone-modifying and / or histone-acetylated molecule, such as a histone-modifying enzyme or a fragment thereof (e.g., a biologically active fragment) or a variant thereof, such as HMT, HDM, HAT, or HDAC.

[0203] The following examples are intended to illustrate the preparation and properties of specific lipid nanoparticle formulations, but are in no way intended to limit the scope of the invention.

[0204] Unless otherwise stated, the article “a” or “an” as used in this article means both singular and plural.

[0205] Example Materials and methods LNP formulations LNPs were prepared by dissolving mRNA in 25 mM sodium acetate, pH 4.0, while a lipid fraction was dissolved in anhydrous ethanol at a specified molar ratio. The lipids in ethanol and the reporter (firefly luciferase) mRNA in the buffer were combined at a 1:3 volume ratio using a three-way connector with dual syringe pumps. The solution was propelled through the three-way connector at a combined flow rate of 20 mL / min (5 mL / min for the lipid-containing syringe and 15 mL / min for the mRNA-containing syringe). The mixture was then dialyzed overnight using a Spectro / Por dialysis membrane (molecular weight cutoff 12000–14000 Da or 3000–5000 Da) against at least approximately 100 volumes of 1× phosphate-buffered saline, pH 7.4. Alternatively, Amicon Ultra could be used as needed. TM 10000 MWCO (molecular weight cutoff), LNP concentrated by regenerated cellulose concentrator.

[0206] To store LNPs at low temperatures, first perform buffer exchange to a low-salt (e.g., 15 mM TRIS) pH 7 buffer containing 20% ​​sucrose (w / v). Then store the LNPs in the sucrose buffer at -80°C. After thawing, remove the LNPs from the -80°C freezer and allow them to warm to room temperature on a workbench.

[0207] By directing mRNA-LNP ( F i Add RiboGreen TM Measure fluorescence and compare the value with that obtained by passing 2% Triton X-100 ( F t Encapsulation efficiency is calculated by comparing the total mRNA content obtained from LNP lysis with the unencapsulated mRNA content: %encapsulation = ( F t - F i ) / F t ×100.

[0208] Using Zetasizer Ultra Red TM (Malvern PANalytical TM ) Characterizes particle size and polydispersity index (PDI).

[0209] Measurement of luminescence in in vivo and in vitro organs / tissues For all in vivo studies, LNP was administered intravenously (iv) in CD-1 mice (6 to 8 weeks old, male and female) at a concentration of 0.02 to 0.5 mg / kg mRNA, using a volume of 2 μL (molecular weight in grams). The mRNA used in the studies was firefly luciferase (fLuc), which is of endogenous origin (NanoVation Therapeutics). TM , NTx fLuc) or from RNA Technologies and Therapeutics ( https: / / www.rnatechnology.com / ; MGfLuc). Four hours after injection, the fluorescein substrate (15 mg / mL) was injected intraperitoneally at a dose of 150 mg / kg. An in vivo imaging system (PhotonIMAGER Optima, BioSpaceLab) was used. TM ; https: / / biospacelab.com In vivo imaging of mice was performed within 10 minutes of substrate administration. Organs were then harvested, placed in Petri dishes, and luminescence was measured using an in vivo imaging system. For the liver, in vitro bioluminescence was determined by summing the bioluminescence of five individual liver lobes. The reported bioluminescence rates were normalized to per mg tissue weight.

[0210] For kinetic studies, the dose was 0.5 mg / kg MG fLuc, and the substrate fluorescein was administered intraperitoneally at 4, 24, and 48 hours after injection, followed by in vivo imaging and tail vein bleeding at each time point.

[0211] Tissue homogenate assay LNP at a concentration of 0.1 mg / mL was administered intravenously (iv) to mice. Organs were collected 24 hours after LNP injection.

[0212] Tissue was removed from mice and placed in 2 mL tubes and rapidly frozen in liquid nitrogen. The tissue was then frozen at -80°C. Appropriate volumes of Promega... TM GLO TM Add lysis buffer to each tube, ensuring the sample is frozen before adding the lysis buffer. Place the sample in FastPrep. TM The homogenizer was run at 6 m / s for 20 seconds, repeated twice for a total of three rounds. The homogenized sample was then rotated at 12,000 rpm for 10 minutes at room temperature, after which the homogenized product was added to a black plate. The plate was transferred to a plate reader, and fluorescence was read at 640 nm excitation / 720 nm emission. The fluorescence was then measured using 50 μL of Steady Globe. TM The substrate was added to the homogenized sample and the luciferase signal was read to determine the luminescence.

[0213] Apparent pKa Apparent pKa was measured using a modified 6-(p-toluidine)-2-naphthalenesulfonic acid (TNS) assay from previous studies in other groups (Shobaki et al., 2018, International Journal of Nanomedicine, 13:8395-8410; Jayaraman et al., 2012, Angew Chem Int. Ed., 51:8529-8533, which are incorporated herein by reference for the purpose of determining apparent pKa). In the modified method, a series of buffer solutions consisting of 130 mM NaCl, 10 mM ammonium acetate, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), and 10 mM HEPES were prepared in pH ranges from 2.5 to 10.9, with different pH increments. LNP was also prepared from 0.15 to 0.2 mM. Subsequently, 0.12 mM TNS solution was mixed in triplicate with 175 μL LNP at each buffer pH in a black polystyrene 96-well plate to produce final concentrations of 6.25 and 12 μM lipids and TNS in each well, respectively. The mixture was then processed using a BMG LabTech CLARIOstar PLUS. TM Fluorescence was measured using a microplate reader at λex = 321 nm and λem = 445 nm. Subsequently, it was analyzed using Prism... TM Fluorescence was plotted against pH using an S-shaped curve fit, where pKa was determined as the pH value at which the maximum fluorescence intensity was 50%.

[0214] Cryo-TEM Prior to cryo-electron microscopy imaging, LNP was concentrated to an estimated total lipid concentration of 5 to 10 mg / mL. A predetermined volume, e.g., 2 to 4 μL, of the resulting LNP solution was added to a glow discharge copper grid plate and subjected to FEI Mark IV Vitrobot. TM Impregnation freezing was used to generate glassy ice. These grids were stored in liquid nitrogen until imaging via Glacios TEM. The instrument was operated at 200 kV under low-dose conditions and using a bottom-mounted FEI Falcon. TM The resulting image is obtained by a direct electronic detector camera with a magnification of 47 to 88,000× and an underfocus of 0.5 to 2 μm to enhance contrast.

[0215] Determination of N / P ratio of lipid nanoparticles The N / P ratio describes the ratio of anionic charge (P) to cationic charge (N) within oligonucleotide-containing lipid nanoparticles. To calculate the total lipid weight required to obtain a specific N / P ratio, the relationship between the average molecular weight (P) per anionic charge of the single nucleic acid base / phosphate / ribose monomer (~300 g / mol) and the molecular weight per cationic charge of the ionizable lipid (N) is first calculated. From this molar ratio, the required weight of ionizable lipid is calculated as: required N / P * molar ratio calculated * ionizable lipid molecular weight (g / mol) * required oligonucleotide weight (g). Thus, the total lipid weight (including the uncharged lipid component) is calculated based on the desired lipid composition (i.e., the % molar ratio of each lipid) and the molecular weight of each lipid.

[0216] Example 1: LNPs with nonpolar glycerides and lacking phospholipids (TAG LNPs) showed improved cargo liver delivery compared to the gold standard baseline lipid nanoparticles. This embodiment demonstrates that, compared to the gold standard Onpattro TM The baseline formulation, comprising triglycerides (trioleylglycerol, C18:1) and phospholipid-deficient lipid nanoparticles (referred to herein as "TAG LNP"), surprisingly exhibited significantly improved nucleic acid delivery to the liver. Onpattro was selected. TM The baseline formulation was used as the baseline because it is a known formulation specifically designed for hepatic orientation and contains 10% phospholipids (DSPC). The two formulations tested are listed in Table 1 below: Table 1: Preparation of triglyceride (C18:1) mRNA-LNP formulations with or without phospholipids for in vivo expression detection of liver mRNA

[0217] As described in jointly owned and jointly pending WO 2022 / 246571, Onpattro TM The ionizable lipid contained in the baseline LNP is nor-MC3 (nMC3), and the ionizable lipid 1 in the TAG-LNP of the present invention is compound 24 of the commonly owned U.S. Patent No. 12,121,591 (incorporated herein by reference).

[0218] LNP also contains 0.1% molar of the fluorescent lipid probe, DiR (1,1'-bis(octadecyl-3,3,3',3'-tetramethylindole)tricarbonylcyanine iodide). DiR is added to the TAG-LNP composition at the expense of 0.05% molar of cholesterol and 0.05% molar of triglycerides. DiR is added to Onpattro at the expense of 0.1% molar of DSPC. TM In the baseline composition.

[0219] TAG-LNP and Onpattro TM Baseline LNP was administered intravenously to CD-1 mice at doses of 0.02, 0.1, and 0.5 mg / kg luciferase mRNA, followed by administration of the substrate (luciferin) 4 hours later, as described in Materials and Methods.

[0220] Figures 1A through D show the results for 0.02 to 0.5 mg / kg luciferase mRNA (NTx fluc). Surprisingly, onpattro was observed relative to 10% molar ratio DSPC. TM The baseline formulation, at doses of 0.02 mg / kg, 0.1 mg / kg, and 0.5 mg / kg fLuc mRNA, showed 7.5-fold, 11.3-fold, and 4.4-fold bioluminescence in the liver, respectively. Figure 1D shows representative in vivo, whole-body bioluminescence images (0.5 mg / kg NTx fLuc).

[0221] Example 2: LNPs with nonpolar glycerides and lacking phospholipids (TAG LNP) relative to Onpattro TM The baseline formulation showed improved liver selectivity relative to the spleen. The results of Example 1 revealed a luminescent signal in the spleen. Therefore, the results of an in vitro liver and spleen luminescence assessment were performed using a 0.5 mg / kg fLuc mRNA dose to determine TAG-LNP and Onpattro TM The degree of liver selectivity of the baseline formulation relative to the spleen.

[0222] Figure 2A The results showed that TAG LNP (0.5 mg / kg fLuc mRNA; Table 1) exhibited 50-fold luminescence in the liver relative to the spleen, while Onpattro, with a 10% molar ratio of phospholipids, showed significantly higher luminescence. TM The baseline formulation (0.5 mg / kg fLuc mRNA) showed only 26 times the luminescence of the spleen.

[0223] Therefore, the results indicate that TAG LNP exhibits superior hepatic orientation relative to the spleen when compared to the Onpattro™ baseline formulation.

[0224] In separate studies, the hepatic orientation of the aforementioned TAG-LNPs relative to the spleen was also examined for those with different ionizable amino lipids (lipid 2 in Table 2) or those also containing cholesterol hemisuccinate (CHEMS). Other formulations tested (TAG-LNP 2 and TAG-LNP 3) are listed in Table 2 below. In vivo luciferase expression in the liver and spleen was assessed by quantifying luminescence in tissue homogenates, as described in the Materials and Methods section above.

[0225] Table 2: Preparation of triglyceride (C18:1) mRNA-LNP formulations with different ionizable lipids and CHEM for in vivo detection of liver mRNA expression.

[0226] In tissue homogenate studies, TAG-LNP 2, containing ionizable lipid 2, also exhibited hepatic orientation relative to the spleen (Fig. 2B and 2C). Furthermore, TAG-LNP 3, containing the additional lipid component CHEMS, showed higher hepatic expression relative to the spleen (Fig. 2D and 2E).

[0227] Example 3: Expression kinetics of LNPs containing nonpolar glycerides and lacking phospholipids Next, the expression pharmacokinetics of the TAG LNP formulation (Table 1) were examined. The TAG LNP formulation containing MG fLuc was administered intravenously to CD-1 mice according to the materials and methods. The dose was 0.5 mg / kg MG fLuc, and the substrate fluorescein was administered intraperitoneally at 4, 24, and 48 hours after injection, followed by in vivo imaging and tail vein bleeding at each time point.

[0228] In vivo luminescence results showed Figure 3A and Figure 3B The whole-body and abdominal luminescence were similar, indicating that most fLuc expression originated from the liver (and spleen), and the results showed that the maximum luminescence signal was maintained at least 24 hours after LNP administration.

[0229] Example 4: Effects of long-term storage at 4°C Next, we examine TAG LNP relative to Onpattro. TM Stability of baseline LNP formulations. After storing LNPs at +4°C for 3 months, TAG LNP (Table 1) or Onpattro with NTx fLuc as cargo was administered intravenously to CD1 mice. TM The baseline LNP was then administered, followed by the substrate (luciferin) 4 hours after administration, as described in Materials and Methods.

[0230] Figure 4 The results showed that, compared with Onpattro in the liverTM Compared to baseline LNPs, TAG LNPs showed 58x luciferase expression after long-term storage at 4°C. This surprising result indicates that, compared to Onpattro TM Compared to baseline LNPs, the long-term stability of mRNA in TAGLNPs was significantly improved.

[0231] Example 5: Effect of freeze-thaw cycles on LNP activity Next, the effects of freezing / thawing LNP samples were examined. After thawing, CD-1 mice were intravenously injected with TAGLNP (Table 1) or Onpattro containing NTx fLuc as cargo. TM The baseline LNP was then administered, followed by the substrate (luciferin) 4 hours after administration, as described in Materials and Methods.

[0232] Figure 5 The results showed that, after freezing / thawing, Onpattro in the liver... TM Compared to baseline LNPs, TAGLNPs maintained superior luciferase expression at 4.1x. This result indicates that, compared to Onpattro... TM Compared to baseline LNPS, cryogenic storage of TAG LNP does not adversely affect its in vivo efficacy in the liver.

[0233] Example 6: Effect of different N / P ratios in TAG-LNP Next, the effects of different N / P ratios were examined. CD-1 mice were intravenously injected with NTx fLuc as cargo TAG LNP with N / P ratios ranging from 3 to 6 (Table 1), and then the substrate (fluorescein) was administered 4 hours later as described in Materials and Methods.

[0234] Figure 6 The results showed that TAG LNP maintained similar levels of mRNA expression in the liver across all N / P ratios tested.

[0235] Example 7: Effects of phospholipids on liver delivery of TAG LNP To determine the effect of phospholipids (PL) on hepatic delivery of triglycerides (TAG) LNPs, the following three LNP formulations were prepared.

[0236] Table 3: Testing lipid formulations to examine the effect of phospholipids on hepatic delivery of TAG LNP

[0237] Figure 7 The display shows a DSPC (Comirnaty) with a molar ratio of 9.4%. TMAdding a TAG to baseline LNPs (-PL) only provided a modest enhancement in hepatic delivery as measured by luminescence (compare +PL-TAG with +PL+TAG formulations, left and middle columns). However, adding a TAG to phospholipid-free LNPs (-PL) significantly increased hepatic luminescence signal (-PL+TAG, right column).

[0238] Results after 4 hours showed that a significant improvement in liver luminescence was achieved in the absence of phospholipids by TAG-containing LNPs.

[0239] Example 8: Hepatotoxicity Study Table 4 shows the preparation of TAG LNP and Onpattro. TM Baseline LNP was used to assess liver toxicity. Mice were administered LNP at 0.5 mg / kgf Luc mRNA. Total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin, amylase, and lactate dehydrogenase (LDH) were measured.

[0240] Table 4: Preparation of triglyceride (C18:1) mRNA-LNP formulations with or without phospholipids for the detection of liver toxicity.

[0241] The results shown in Figures 8A to G indicate that no acute liver toxicity was observed with TAG-LNP.

[0242] Example 9: TAG LNP batch consistency To assess the batch-to-batch variability of the physicochemical properties of TAG LNP, Onpattro was prepared as shown in Table 4 above. TM Baseline and TAG LNPs were studied. The formulation volume (2 to 20 mL scale), initial total lipid concentration (5 to 20 mM), fLuc mRNA source (internal NTx mRNA or from RNA Technologies and Therapeutics (MG mRNA), device used to prepare LNPs (T-mixer with microfluidic chip), and preparer were varied.

[0243] The results showed that particle size (nm), polydispersity index (PDI), and mRNA encapsulation percentage did not vary significantly between batches (Figures 9A to C).

[0244] Onpattro TM The apparent pKa of the baseline LNP is 6.38, while that of the TAG LNP is 6.58 (Fig. 9D).

[0245] Example 10: The morphology of TAG LNP differs from that of OnpattroTM morphology of baseline formulation The morphology of LNPs in Table 4 was evaluated using Cryo-TEM. (TAG LNP and Onpattro) TM Cryo-TEM images of the baseline LNP are shown in Figures 10A and 10B, respectively.

[0246] The images show that the LNP contains small vesicular structures as indicated by the arrows (Fig. 10A). In contrast, Onpattro TM The baseline LNP lacks bubbles and is a solid nucleus, which is consistent with previous studies on Onpattro. TM The observations were consistent with those of the baseline formulation. These results were surprising because LNPs without conventional bilayer-forming lipids (e.g., DSPC phospholipids) would not be expected to form a stable membrane around the internal aqueous LNP compartment.

[0247] Example 11: Formulation of triacylglycerol (TAG), diacylglycerol (DAG), monoacylglycerol (MAG), methyl ricinoleate (MR), and castor oil (CO) in phospholipid-free lipid nanoparticles This embodiment demonstrates that various nonpolar glycerides containing monoacyl chains (monoacylglycerol or MAG), two acyl chains (diacylglycerol or DAG), three acyl chains (triacylglycerol or TAGs), cholesterol esters, and esterified fatty acids can be formulated into phospholipid-free lipid nanoparticles. The tested formulations are listed in Table 5 below: Table 5: Studies of LNPs containing nonpolar glycerides to evaluate the compatibility and potency of phospholipid-free LNPs

[0248] Figure 11 The results show that phospholipid-free LNPs can be formulated using a variety of structurally different nonpolar lipids, including glycerides and esterified fatty acids. In CD-1 mice, after intravenous administration of NTx fLuc as cargo, the DAG-, CE-, MR-, and CO-LNPs tested in this example showed similar expression potency in the liver as the original TAG-LNPs.

Claims

1. Lipid nanoparticles, comprising: (i) Nucleic acid cargo molecules; (ii) Sterols in a molar ratio of 0% to 50%; (iii) Ionizable lipids present in amounts ranging from 20% to 70% molar ratio; (iv) Nonpolar lipids present in amounts ranging from 20% to 80% molar ratio; (v) Phospholipids in a molar ratio of 0 to 5%; and (vi) Hydrophilic polymer-lipid conjugates in a molar ratio of 0% to 3%. Each percentage molar ratio is relative to the total lipids present in the lipid nanoparticles.

2. Lipid nanoparticles, comprising: (i) Encapsulated nucleic acid; (ii) Ionizable lipids; (iii) Nonpolar lipids and a uniform, approximately spherical hydrophobic core of one or more peripheral vesicle structures as observed by cryo-electron microscopy (cryo-TEM); (iv) Optional sterol; (iv) optionally, hydrophilic polymer-lipid conjugates; and (v) Phospholipids in a molar ratio of 0 to 5%.

3. The lipid nanoparticles of claim 1 or 2, wherein the nonpolar lipid is a combination of two or more nonpolar lipids.

4. The lipid nanoparticles of claim 1, 2 or 3, wherein the nonpolar lipid is a triglyceride.

5. The lipid nanoparticles of claim 4, wherein the triglyceride is selected from trioleic acid glyceride, tristearate glyceride, trilaurate glyceride, trilinoleic acid glyceride, trilinolenic acid glyceride, trimyristate glyceride, tripalmitate glyceride, tricaprylic acid glyceride, triarachidonic acid glyceride and dipalmitate oleate.

6. The lipid nanoparticles of claim 1, 2 or 3, wherein the nonpolar lipid is a diglyceride.

7. The lipid nanoparticles of claim 6, wherein the diglyceride is selected from diglyceride laurate, diglyceride myristate, diglyceride palmitate, diglyceride stearate, diglyceride arachidonicate, diglyceride behenate, diglyceride palmitate, diglyceride oleate, diglyceride linoleate, diglyceride linolenic acid, and diglyceride arachidonicate.

8. The lipid nanoparticles of claim 1, 2 or 3, wherein the nonpolar lipid is a glycerol monoester.

9. The lipid nanoparticles of claim 8, wherein the monoglyceride is selected from lauroyl-racemic glycerol, myristoyl monoglyceride, palmitate monoglyceride, stearate monoglyceride, arachidonic acid monoglyceride, behenate monoglyceride, palmitoleate monoglyceride, oleate monoglyceride, linoleate monoglyceride, linolenic acid monoglyceride, arachidonic acid monoglyceride, and caprylic acid monoglyceride; and / or, for example: 1-monomyoyl-racemic glycerol, 1-monopalmitoyl-racemic glycerol, 2-monopalmitoyl glycerol, 1-monpalmitoyl-racemic glycerol, 1-monostearoyl-racemic glycerol, 1-monoleoyl-racemic glycerol, 1-monoleinoyl-racemic glycerol, and 1-monolinoyl-racemic glycerol.

10. The lipid nanoparticles according to any one of claims 1 to 4, wherein the nonpolar lipid is castor oil.

11. The lipid nanoparticles of claim 1 or 2, wherein the nonpolar lipid is methyl ricinoleate.

12. The lipid nanoparticles of claim 1, 2 or 3, wherein the nonpolar lipid is present in an amount of 22% to 38% molar ratio.

13. The lipid nanoparticles of claim 1, 2 or 3, wherein the nonpolar lipid is present in an amount of 25% to 35% molar ratio.

14. The lipid nanoparticles of claim 1, 2 or 3, wherein the nonpolar lipid is present in an amount of 27% to 32.5% molar ratio.

15. The lipid nanoparticles of claim 1, 2 or 3, wherein the nonpolar glycerol ester or its analogue is present in an amount of 28% to 32% molar ratio.

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

17. The lipid nanoparticles according to any one of claims 1 to 16, wherein the ionizable lipid is present in an amount of 23% to 47% molar ratio.

18. The lipid nanoparticles according to any one of claims 1 to 17, wherein the ionizable lipid is present in an amount of 25% to 45% molar ratio.

19. The lipid nanoparticles according to any one of claims 1 to 18, wherein the ionizable lipid is present in an amount of 27% to 43% molar ratio.

20. The lipid nanoparticles according to any one of claims 1 to 19, wherein the ionizable lipid is present in an amount of 28% to 42% molar ratio.

21. The lipid nanoparticles according to any one of claims 1 to 20, wherein the ionizable lipid is present in an amount of 29% to 41.5% molar ratio.

22. The lipid nanoparticles according to any one of claims 1 to 21, wherein the ionizable lipid is present in an amount of 29.5% to 41% molar ratio.

23. The lipid nanoparticles according to any one of claims 1 to 11, wherein the ionizable lipid is present in an amount of 30% to 40% molar ratio.

24. The lipid nanoparticles according to any one of claims 1 to 23, wherein the phospholipid content is less than 4% molar ratio.

25. The lipid nanoparticles according to any one of claims 1 to 24, wherein the phospholipid content is less than 3% molar ratio.

26. The lipid nanoparticles according to any one of claims 1 to 25, wherein the phospholipid content is less than 2% molar ratio.

27. The lipid nanoparticles according to any one of claims 1 to 26, wherein the phospholipid content is less than 1% molar ratio.

28. The lipid nanoparticles according to any one of claims 1 to 27, wherein the phospholipid content is less than 0.5% molar ratio.

29. The lipid nanoparticles according to any one of claims 1 to 28, wherein the lipid nanoparticles do not have a detectable amount of phospholipids.

30. The lipid nanoparticles according to any one of claims 1 to 29, wherein the hydrophilic-polymer lipid is present in an amount of 0.75% to 2.5% molar ratio.

31. The lipid nanoparticles according to any one of claims 1 to 30, wherein the hydrophilic-polymer lipid is present in an amount of 1% to 2.25% molar ratio.

32. The lipid nanoparticles according to any one of claims 1 to 31, wherein the hydrophilic-polymer lipid is present in an amount of 1.25% to 2% molar ratio.

33. The lipid nanoparticles according to any one of claims 1 to 32, wherein the hydrophilic-polymer lipid is present in an amount of 1.25% to 1.75% molar ratio.

34. The lipid nanoparticles according to any one of claims 1 to 33, wherein the sterol is present and is a non-cationic lipid.

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

36. The lipid nanoparticles according to any one of claims 1 to 35, wherein the sterol is present in an amount of 22% to 38% molar ratio.

37. The lipid nanoparticles according to any one of claims 1 to 36, wherein the sterol is present in an amount of 25% to 35% molar ratio.

38. The lipid nanoparticles according to any one of claims 1 to 37, wherein the sterol is present in an amount of 27% to 32.5% molar ratio.

39. The lipid nanoparticles according to any one of claims 1 to 38, wherein the sterol is present in an amount of 28% to 32% molar ratio.

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

41. The N / P charge ratio as claimed in claim 40, wherein the N / P charge ratio is 2 to 6.

42. The N / P charge ratio as claimed in claim 40 or 41, wherein the ratio is 3 to 6.

43. The lipid nanoparticles according to any one of claims 1 to 40, wherein the nucleic acid is selected from siRNA, mRNA, carrier nucleic acid, antisense oligonucleotide, nucleic acid-protein complex and nucleic acid-peptide complex.

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

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

46. ​​The lipid nanoparticles of claim 1, wherein the lipid nanoparticles have a bubble structure visualized by cryo-electron microscopy (cryo-TEM).

47. The lipid nanoparticles of claim 1, wherein the lipid nanoparticles have a uniform core of one or more peripheral vesicles visualized by cryo-electron microscopy (cryo-TEM).

48. The lipid nanoparticles of any one of claims 1 to 47, compared with a baseline formulation of a 50 / 10 / 38.5 / 1.5 mol:mol formulation of encapsulated nucleic acid-encapsulated nor-MC3 ionizable lipid / DSPC / cholesterol / PEG-lipid, measured under the same conditions, wherein the lipid nanoparticles result in an increase of at least 10% in the expression of a protein or peptide in the liver of a mammal at 4 hours, 24 hours and / or 48 hours post-injection, wherein said expression is measured using luminescence.

49. A method of delivering mRNA or vector DNA to produce a protein or peptide in vivo in the liver, the method comprising administering to a mammal the lipid nanoparticles of any one of claims 1 to 48, wherein the nucleic acid is mRNA or vector DNA, and wherein, compared to a baseline formulation of 50 / 10 / 38.5 / 1.5 mol:mol of nor-MC3 ionizable lipids / DSPC / cholesterol / PEG-lipids delivering the mRNA or vector DNA, administration of the lipid nanoparticles results in increased liver-specific expression of the protein or peptide encoded by the mRNA or vector DNA.

50. A method of delivering siRNA or antisense oligonucleotides to silence genes in vivo in the liver, the method comprising administering to a mammal lipid nanoparticles according to any one of claims 1 to 48, wherein the siRNA or antisense oligonucleotide is encapsulated in the lipid nanoparticles, and wherein administration of the lipid nanoparticles results in increased liver-specific gene silencing compared to a baseline formulation of 50 / 10 / 38.5 / 1.5 mol:mol of nor-MC3 ionizable lipids / DSPC / cholesterol / PEG-lipids delivering the siRNA or antisense oligonucleotide.

51. A method of delivering nucleic acids to hepatocytes to treat a disease, symptom, or disease state, the method comprising contacting the hepatocytes with the lipid nanoparticles of any one of claims 1 to 48, either in vivo or in vitro.

52. The method of claim 49, wherein the expression of the mRNA-encoded protein or peptide in the liver is at least 10% higher than that in the spleen compared to the increase in expression of baseline LNP in the liver relative to the spleen as measured under the same conditions.

53. The method of any one of claims 49 to 52, comprising administering the lipid nanoparticles at a dose of 0.075 mg / kg to 5 mg / kg.

54. The method of any one of claims 49 to 53, comprising administering the lipid nanoparticles at a dose of 0.075 mg / kg to 4 mg / kg.

55. The method of any one of claims 49 to 54, comprising administering the lipid nanoparticles at a dose of 0.075 mg / kg to 3 mg / kg.

56. The method of any one of claims 49 to 55, comprising administering the lipid nanoparticles at a dose of 0.075 mg / kg to 2 mg / kg.

57. The method of any one of claims 49 to 56, comprising administering the lipid nanoparticles at a dose of 0.075 mg / kg to 1.5 mg / kg.

58. The method of any one of claims 49 to 57, comprising administering the lipid nanoparticles at a dose of 0.075 mg / kg to 1 mg / kg.

59. The method of any one of claims 49 to 58, comprising administering the lipid nanoparticles at a dose of 0.075 mg / kg to 0.75 mg / kg.

60. The method according to any one of claims 49 to 59, wherein the phospholipid content is less than 4% molar ratio.

61. The method according to any one of claims 49 to 60, wherein the sterol is present in a molar ratio of 25 to 45%.

62. The method of claim 61, wherein the sterol is present in a molar ratio of 25 to 40%.

63. The method as claimed in any one of claims 49 to 62, wherein the hydrophilic polymer-lipid conjugate is present in the lipid nanoparticles in an amount of 0% to 3% molar ratio.

64. The method of claim 63, wherein the hydrophilic polymer-lipid conjugate is present in the lipid nanoparticles at a molar ratio of 0.5% to 2.0%.

Images