Sterol-rich lipid nanoparticles

Abstract

The present invention provides lipid nanoparticles comprising a nucleic acid cargo molecule; a sterol or derivative thereof present in a high content; a neutral lipid; an ionizable lipid; and a hydrophilic polymer-lipid conjugate present in a content of 0.5-3 mol%, each mol% content being a relative value with respect to the total lipid present in the lipid nanoparticle.

Description

[Technical field]

[0001] (Incorporation by reference to priority application) This application claims priority to U.S. Provisional Application No. 63 / 269,815, filed March 23, 2022, which is expressly incorporated by reference herein in its entirety.

[0002] The present disclosure relates to lipid nanoparticle formulations for the delivery of cargo, such as nucleic acids. [Background technology]

[0003] Lipid nanoparticle (LNP) formulations represent a revolution in the field of nucleic acid delivery. An early example of a lipid nanoparticle product approved for clinical use is Onpattro™, a lipid nanoparticle-based short interfering RNA (siRNA) drug aimed at treating polyneuropathy caused by hereditary transthyretin amyloidosis. The success of this LNP delivery system has paved the way for the clinical development of a leading LNP-based COVID-19 mRNA vaccine.

[0004] The Onpattro™ LNP formulation consists of four main lipid components, so-called ionizable amino lipids, distearoylphosphatidylcholine (DSPC), cholesterol, and polyethylene glycol conjugated lipids (PEG lipids), in molar amounts of 50 / 10 / 38.5 / 1.5, respectively. Onpattro™ is still considered the gold standard for comparison in LNP-mediated efficacy studies, and current approaches to LNP design have rarely deviated from the four-component system.

[0005] Of these four components, the ionizable cationic lipids appear to be important for the in vitro and in vivo activity of LNP systems. Thus, most research in this field has focused primarily on improving this lipid component. Ionizable cationic lipids are typically positively charged at low pH, which facilitates association with negatively charged nucleic acids, but are neutral at physiological pH, making them more biocompatible in biological systems. Furthermore, it has been suggested that after lipid nanoparticles are taken up into cells by endocytosis, the ability of these lipids to ionize at low pH allows escape from endosomes. This releases the nucleic acid into the intracellular compartment.

[0006] The ionizable cationic lipid is present at 50 mol% (relative to the total lipids in the LNP) and therefore constitutes the majority of the Onpattro™ formulation. With regard to the remaining three lipid components, it is well known that the PEG-lipid prevents LNP aggregation and cholesterol functions to stabilize the particles. DSPC is a bilayer-forming lipid and is generally accepted to play an important structural role in the LNP membrane.

[0007] It has been reported that DSPC and cholesterol in empty Onpattro™-type LNP systems are present in the outer lipid layer, whereas in siRNA-loaded systems, DSPC and cholesterol are internalized along with siRNA in the hydrophobic core. At low levels of both components (combined DSPC and cholesterol content below 40 mol%), siRNA encapsulation efficiency gradually decreased (Kulkarni et al., 2019, Nanoscale, 11:21733-21739). It was concluded that the presence of both DSPC and cholesterol is essential for stable encapsulation of siRNA in LNPs. Due to concerns regarding cholesterol solubility, studies have only investigated LNPs with cholesterol content up to 40 mol% (1:1 chol / DSPC). Other studies have stated that cholesterol is known to have limited solubility in ionizable cationic lipids above pH 7.4. It has been suggested that DSPC is necessary for stable cholesterol retention, and LNP formulations with chol / DSPC (mol / mol) greater than 40:40 were not considered to avoid ambiguities arising from the presence of excess cholesterol.

[0008] Other studies have investigated the effect of cholesterol on two-component lipoplex systems containing ionizable cationic lipids and cholesterol, but these studies used a 1:1 molar ratio of the two lipid components and did not investigate the effect of increasing the cholesterol content beyond 50 mol% (Cheng and Lee, 2016, Advanced Drug Delivery Reviews, 99:129-137; Sakurai et al., 2001, European Journal of Pharmaceutics and Biopharmaceutics, 52:165-172; Dabkowska et al., 2012, JR Soc. Interface, 9:548-561; Yoshioka et al., 2009, Journal of Liposome Research, 19(2):141-147; ). Other studies have investigated the effect of cholesterol on lipoplexes that form cubic phases, but the lipid dispersions formed (using thin film hydration) contained high levels of phospholipids (Tenchov et al., 2006, Biophysical Journal, 91: 2508-2516).

[0009] In vivo studies examining large unilamellar liposomes (LUVs) containing phospholipids / cholesterol found that cholesterol extended the circulatory lifetime of LUVs, but the effect of cholesterol on extending circulatory lifetime plateaued at 30 mol% (Semple et al., 1996, Biochemistry, 35(8):2521-2525).

[0010] US Patent No. 11,191,849 discloses mRNA-LNPs with 55 mol% ionizable cationic lipid, 41 mol% cholesterol and 3.3 mol% PEG2000-C-DMA. The inflammatory response after LNP administration in mice was reduced compared to the base composition (1.6 / 55 / 33 / 11 mol% PEG-2000-C-DMA / cationic lipid / cholesterol / DSPC) without affecting efficacy. The observed results were partially due to the higher than usual PEG-lipid conjugate content (>3 mol%).

[0011] Despite the aforementioned advances in the field, there is a continuing need for LNP formulations that have desirable properties for delivery of nucleic acids. (overview)

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

[0013] The present disclosure is based in part on the discovery that LNPs with high sterol content and low levels of ionizable lipids have high nucleic acid encapsulation efficiency compared to Onpattro™ LNPs (e.g., significantly more than 38.5 mol% sterols such as cholesterol). Moreover, the LNPs described herein have been found to exhibit in vitro and in vivo nucleic acid delivery capabilities comparable to Onpattro™ LNPs, despite these significant deviations in lipid composition from the "gold standard" benchmark.

[0014] In one example of the present disclosure, LNPs with high levels of sterol or derivatives thereof exhibit improved targeting to the liver over the spleen. In some embodiments, targeting to the liver over the spleen is improved compared to Onpattro™ LNPs.

[0015] According to one aspect of the present disclosure, there is provided a lipid nanoparticle comprising: a nucleic acid cargo molecule; a sterol or derivative thereof present in a content of 49 mol% to 85 mol%; substantially free of phospholipids; an ionizable lipid; and a hydrophilic polymer-lipid conjugate present in a content of 0.5 to 3 mol%, each mol% content being relative to the total lipid present in the lipid nanoparticle.

[0016] In one embodiment, the sterol or derivative thereof is present in a content of at least 50 mol % and / or the ionizable lipid is present in a content of less than 48.5 mol %.

[0017] In another embodiment, the sterol or derivative thereof is present in a content of at least 52 mol % and / or the ionizable lipid is present in a content of less than 46.5 mol %.

[0018] In a further embodiment, the sterol or derivative thereof is present in a content of at least 54 mol % and / or the ionizable lipid is present in a content of less than 44.5 mol %.

[0019] In another embodiment, the sterol or derivative thereof is present in a content of at least 56 mol % and / or the ionizable lipid is present in a content of less than 42.5 mol %.

[0020] In a further embodiment, the sterol or derivative thereof is present in a content of at least 58 mol % and / or the ionizable lipid is present in a content of less than 40.5 mol %.

[0021] In another embodiment, the sterol or derivative thereof is present in a content of at least 60 mol % and / or the ionizable lipid is present in a content of less than 38.5 mol %.

[0022] In a further embodiment, the sterol or derivative thereof is present in a content of at least 62 mol % and / or the ionizable lipid is present in a content of less than 36.5 mol %.

[0023] In a further embodiment, the sterol or derivative thereof is present in a content of at least 63 mol % and / or the ionizable lipid is present in a content of less than 35.5 mol %.

[0024] In another embodiment, the sterol or derivative thereof is present in a content of at least 64 mol % and / or the ionizable lipid is present in a content of less than 34.5 mol %.

[0025] In a further embodiment, the sterol or derivative thereof is present in a content of at least 65 mol % and / or the ionizable lipid is present in a content of less than 33.5 mol %.

[0026] In yet another embodiment, the sterol or derivative thereof is present in a content of at least 66 mol % and / or the ionizable lipid is present in a content of less than 32.5 mol %.

[0027] In one embodiment, the phospholipid content is less than 1 mol %. In another embodiment, the phospholipid content is less than 0.5 mol %.

[0028] In a further embodiment, the hydrophilic polymeric lipid content is less than 2.5 mol %.

[0029] In one embodiment, the cargo molecule is an siRNA, an mRNA, a vector nucleic acid, an antisense oligonucleotide, or a nucleic acid-protein or peptide complex.

[0030] In further embodiments, the cargo molecule is a siRNA, a vector nucleic acid, or an antisense oligonucleotide.

[0031] In another embodiment, the ionizable lipid is an amino lipid.

[0032] In yet another embodiment, the sterol derivative is a non-cationic lipid.

[0033] In a further embodiment, the lipid nanoparticles have an electron-dense core that is visualized by cryo-TEM.

[0034] In a further embodiment, the lipid nanoparticles are substantially free of neutral lipids.

[0035] In another embodiment, the sterol is cholesterol or the sterol derivative is a cholesterol derivative.

[0036] In a further embodiment, the lipid nanoparticle further comprises tocopherol, for example, the tocopherol may be present at 0.5-15 mol %.

[0037] According to another aspect of the present disclosure, there is provided a method of delivering mRNA or vector DNA for in vivo production of a protein or peptide in the liver, the method comprising administering to a mammal lipid nanoparticles comprising at least 45 mol % sterol or derivative thereof, a neutral lipid and an ionizable lipid, wherein the mRNA or vector DNA is encapsulated within the lipid nanoparticles, administration of the lipid nanoparticles results in liver-specific expression of the protein or peptide encoded by the mRNA or vector DNA, and wherein the lipid nanoparticles increase expression of the protein or peptide encoded by the mRNA or vector DNA in the liver by at least 20-fold compared to the spleen.

[0038] In another aspect, a method of delivering siRNA or antisense oligonucleotides for in vivo silencing of a gene in the liver is provided, the method comprising administering to a mammal lipid nanoparticles comprising at least 45 mol% sterol or a derivative thereof, a neutral lipid and an ionizable lipid, wherein mRNA is encapsulated within the lipid nanoparticles, administration of the lipid nanoparticles results in liver-specific silencing of a protein or peptide encoded by the nucleic acid sequence, and the lipid nanoparticles increase silencing of the nucleic acid in the liver by at least 5-fold compared to the spleen.

[0039] According to one embodiment of the present disclosure, the increased expression of the protein or peptide encoded by the mRNA in the liver relative to the spleen is at least 10% greater than the increased expression of the Onpattro LNP in the liver relative to the spleen.

[0040] According to a further embodiment, the neutral lipid content is 0-10 mol%. In one example of the present disclosure, the neutral lipid content is less than 8 mol%, 7 mol%, 6 mol%, 5 mol% or 4 mol%.

[0041] According to another embodiment, the sterol or its derivative is present in an amount of 45-80 mol %.

[0042] According to another embodiment, the sterol or derivative thereof is present in an amount of 49-65 mol %.

[0043] According to a further embodiment, the hydrophilic polymer lipid conjugate is present in the lipid nanoparticles in a content of 0.5-3 mol %.

[0044] According to one embodiment, the ionizable lipid component in any of the foregoing aspects and / or embodiments comprises a mixture of ionizable cationic lipids and ionizable anionic lipids. [Brief description of the drawings]

[0045] [Figure 1] Figure 1 shows the encapsulation efficiency (%), particle size (nm) and polydispersity index (PDI) of lipid nanoparticle (LNP) formulation DLin-MC3-DMA (MC3) ionizable lipid / cholesterol / PEG-DMG) at respective mol% ratios of X / 98.5 to X / 1.5 encapsulating antisense oligonucleotides targeting c-myc as a function of increasing cholesterol content. Cholesterol content was increased from 33.5 mol% to 63.5 mol%. Nitrogen-to-phosphate (N / P) ratio was 3.

[0046] [Diagram 2] Figure 2A is a graph showing the entrapment (%), particle size (nm) and PDI of LNPs (MC3 ionizable lipid / cholesterol / PEG-DMG) at respective mol% ratios of X / 98.5 to X / 1.5 encapsulating firefly luciferase gene (siLuc) siRNA as a function of increasing cholesterol content. The cholesterol content was increased from 53.5 mol% to 93.5 mol%. The nitrogen-to-phosphate (N / P) ratio was 6.

[0047] Figure 2B is a graph showing the entrapment (%), particle size (nm) and PDI of lipid nanoparticles (MC3 ionizable cationic lipid / cholesterol / PEG-DMG) encapsulating siLuc-siRNA at respective mol% ratios of X / 98.5 to X / 1.5 as a function of increasing MC3 ionizable cationic lipid content. The ionizable cationic lipid content was increased from 5 mol% to 45 mol%. The nitrogen-to-phosphate (N / P) ratio was 6.

[0048] [Diagram 3]1 is a graph showing in vitro luminescence as a function of mRNA dose for Huh7 cells following treatment with MC3 ionizable cationic lipid / cholesterol / PEG-DMG LNPs at a molar ratio of 35 / 63.5 / 1.5 relative to an Onpattro™ LNP formulation (50 / 10 / 38.5 / 1.5; mol / mol MC3 / DSPC / cholesterol / PEG-DMG) encapsulating luciferase mRNA. The nitrogen-to-phosphate (N / P) ratio was 6.

[0049] [Figure 4] Figure 4A is a graph showing the entrapment (%), particle size (nm) and PDI of LNPs (MC3 ionizable cationic lipid / cholesterol / PEG-DMG) at respective mol% ratios from X / 98.5 to X / 1.5 encapsulating firefly luciferase mRNA as a function of increasing cholesterol content. The cholesterol content was increased from 53.5 mol% to 83.5 mol%. The nitrogen-to-phosphate (N / P) ratio was 6.

[0050] Figure 4B is a graph showing the entrapment (%), particle size (nm) and PDI of lipid nanoparticles (MC3 ionizable cationic lipid / cholesterol / PEG-DMG) at respective mol% ratios from X / 98.5 to X / 1.5 encapsulating firefly luciferase mRNA as a function of increasing MC3 ionizable cationic lipid content. The ionizable cationic lipid content was increased from 15 mol% to 45 mol%. The nitrogen-to-phosphate (N / P) ratio was 6.

[0051] [Diagram 5]Figure 5A is a graph showing in vivo luminescence of luciferase mRNA after liver extraction from CD-1 mice after treatment with LNPs (MC3 ionizable cationic lipid / cholesterol / PEG-DMG) encapsulating firefly luciferase mRNA at respective mol% ratios of X / 98.5 to X / 1.5 as a function of increasing cholesterol content for the luciferase mRNA encapsulated Onpatro™ formulation (50 / 10 / 38.5 / 1.5; mol / mol MC3 / DSPC / cholesterol / PEG-DMG). The nitrogen-to-phosphate (N / P) ratio was 6.

[0052] FIG 5B is a graph showing in vivo luminescence of luciferase mRNA after extraction of spleens of CD-1 mice after treatment with luciferase mRNA-containing LNPs (MC3 ionizable cationic lipid / cholesterol / PEG-DMG) in mol% ratios of X / 98.5 to X / 1.5, respectively, as a function of increasing cholesterol content for luciferase mRNA-containing Onpattro™ formulations (MC3 / DSPC / cholesterol / PEG-DMG at 50 / 10 / 38.5 / 1.5; mol / mol). The nitrogen-to-phosphate (N / P) ratio was 6.

[0053] Figure 5C is a graph showing in vivo luminescence of luciferase mRNA after liver extraction from CD-1 mice after treatment with luciferase mRNA-containing LNPs (MC3 ionizable cationic lipid / cholesterol / PEG-DMG) at mol% ratios of X / 98.5 to X / 1.5, respectively, as a function of increasing MC3 lipid content relative to the luciferase mRNA-containing Onpattro™ formulation (50 / 10 / 38.5 / 1.5; mol / mol MC3 / DSPC / cholesterol / PEG-DMG). The nitrogen-to-phosphate (N / P) ratio was 6.

[0054] FIG 5D is a graph showing in vivo luminescence of luciferase mRNA after spleen extraction of CD-1 mice after treatment with luciferase mRNA-containing LNPs (MC3 ionizable cationic lipid / cholesterol / PEG-DMG) at mol% ratios of X / 98.5 to X / 1.5, respectively, as a function of increasing MC3 content relative to the luciferase mRNA-containing Onpattro™ formulation (50 / 10 / 38.5 / 1.5; mol / mol MC3 / DSPC / cholesterol / PEG-DMG). The nitrogen-to-phosphate (N / P) ratio was 6.

[0055] [Figure 6] FIG. 6A is a graph showing in vivo luminescence of luciferase mRNA after liver extraction of CD-1 mice following treatment with mRNA luciferase-containing LNPs in MC3 ionizable cationic lipid / cholesterol / PEG-DMG) at ratios of 35 / 63.5 / 1.5 mol% and 35 / 61.7 / 3.3 mol%, respectively.

[0056] FIG. 6B is a graph showing in vivo luminescence of luciferase mRNA following extraction of spleens from CD-1 mice following treatment with mRNA luciferase-containing LNPs in MC3 ionizable cationic lipid / cholesterol / PEG-DMG) at ratios of 35 / 63.5 / 1.5 mol% and 35 / 61.7 / 3.3 mol%, respectively.

[0057] [Figure 7A] Figure 1 shows the entrapment (%), particle size (nm) and PDI of Onpattro™ (MC3 / Chol / DSPC / PEG-DMG; 50 / 38.5 / 10 / 1.5 mol%), MC3 / Chol / PEG-DMG (42 / 56.5 / 1.5 mol%), MC3 / DSPC / Chol / PEG-DMG (42 / 3 / 53.5 / 1.5 mol%) and MC3 / SPC-Chol(H-001) / Chol / PEG-DMG (42 / 3 / 53.5 / 1.5 mol%). The lipid nanoparticles encapsulated mRNA encoding luciferase and had an N / P of 6.

[0058] [Figure 7B-C] FIG. 7B is a graph showing in vivo luminescence of luciferase mRNA after liver extraction of CD-1 mice treated with mRNA luciferase-containing LNPs Onpattro™ (MC3 / Chol / DSPC / PEG-DMG; 50 / 38.5 / 10 / 1.5 mol%), MC3 / Chol / PEG-DMG (42 / 56.5 / 1.5 mol%), MC3 / DSPC / Chol / PEG-DMG (42 / 3 / 53.5 / 1.5 mol%) and MC3 / SPC-Chol(H-001) / Chol / PEG-DMG (42 / 3 / 53.5 / 1.5 mol%).

[0059] FIG. 7C is a graph showing in vivo luminescence of luciferase mRNA after extraction of spleen from CD-1 mice after treatment with mRNA luciferase-containing LNP Onpattro™ (MC3 / Chol / DSPC / PEG-DMG; 50 / 38.5 / 10 / 1.5 mol%), MC3 / Chol / PEG-DMG (42 / 56.5 / 1.5 mol%), MC3 / DSPC / Chol / PEG-DMG (42 / 3 / 53.5 / 1.5 mol%) and MC3 / SPC-Chol(H-001) / Chol / PEG-DMG (42 / 3 / 53.5 / 1.5 mol%).

[0060] [Figure 8A] Figure 1 shows the entrapment (%), particle size (nm) and PDI of Onpattro™ (MC3 / Chol / DSPC / PEG-DMG; 50 / 38.5 / 10 / 1.5 mol%) and 42 / 3 / 53.5 / 1.5 mol% MC3 / SPC-Chol conjugate / Chol / PEG-DMG formulations with N / P of 5, 6, 7, 8 and 9. The lipid nanoparticles encapsulate mRNA encoding luciferase. The N / P of the Onpatro™ formulation was 6.

[0061] [Figure 8B-C]FIG. 8B is a graph showing in vivo luminescence of luciferase mRNA after liver extraction of CD-1 mice treated with mRNA luciferase-containing LNP Onpattro™ (MC3 / Chol / DSPC / PEG-DMG; 50 / 38.5 / 10 / 1.5 mol%) and 42 / 3 / 53.5 / 1.5 mol% MC3 / SPC-Chol conjugate / Chol / PEG-DMG formulations with N / P of 5, 6, 7, 8, and 9. The lipid nanoparticles encapsulate the mRNA encoding luciferase. The N / P of the Onpatro™ formulation was 6.

[0062] FIG. 8C is a graph showing in vivo luminescence of luciferase mRNA after extraction of spleen from CD-1 mice after treatment with mRNA luciferase-containing LNP Onpattro™ (MC3 / Chol / DSPC / PEG-DMG; 50 / 38.5 / 10 / 1.5 mol%) and 42 / 3 / 53.5 / 1.5 mol% MC3 / SPC-Chol conjugate / Chol / PEG-DMG formulations with N / P of 5, 6, 7, 8, and 9. The lipid nanoparticles encapsulate the mRNA encoding luciferase. The N / P of the Onpatro™ formulation was 6.

[0063] [Figure 9] FIG. 9A is a graph showing the entrapment (%), particle size (nm) and PDI of Onpattro™ (MC3 / Chol / DSPC / PEG-DMG; 50 / 38.5 / 10 / 1.5 mol%) and three formulations of MC3 / Chol / SPC-Chol / PEG-DMG with PEG-DMG content of 1.0 mol% (42 / 54 / 3 / 1.0 mol:mol); 1.5 mol% (42 / 53.5 / 3 / 1.5 mol:mol) and 2.0 mol% (42 / 53.0 / 3 / 2.0). The lipid nanoparticles encapsulate mRNA encoding luciferase. The N / P for each formulation was 6.

[0064] FIG. 9B is a graph showing in vivo luminescence of luciferase mRNA after liver extraction of CD-1 mice after treatment with mRNA luciferase-containing LNP Onpattro™ (MC3 / Chol / DSPC / PEG-DMG; 50 / 38.5 / 10 / 1.5 mol%) and three formulations of MC3 / Chol / SPC-Chol / PEG-DMG with PEG-DMG content of 1.0 mol% (42 / 54 / 3 / 1.0 mol:mol); 1.5 mol% (42 / 53.5 / 3 / 1.5 mol:mol); and 2.0 mol% (42 / 53.0 / 3 / 2.0). The lipid nanoparticles encapsulate the mRNA encoding luciferase.

[0065] [Figure 10A] 1 is a graph showing the physicochemical properties of mRNA formulations showing entrapment (%), particle size (nm) and PDI for Onpattro™ (MC3 / Chol / DSPC / PEG-DMG; 50 / 38.5 / 10 / 1.5 mol%) and 42 / 2.7 / 54.3 / 1.0 mol% ionizable cationic lipid / SPC-Chol / Chol / PEG-DMG formulations. The ionizable cationic lipids were norMC3, C108, C109, C123, C124, C136, C137 and C142 as described in Example 8.

[0066] [Figure 10B-C] FIG. 10B is a graph showing in vivo luminescence of luciferase mRNA following liver extraction of CD-1 mice after treatment with mRNA luciferase-containing LNP Onpattro™ (MC3 / Chol / DSPC / PEG-DMG; 50 / 38.5 / 10 / 1.5 mol%) and a high cholesterol formulation of 42 / 2.7 / 54.3 / 1.0 mol% ionizable cationic lipid / SPC-Chol / Chol / PEG-DMG as described in Example 8.

[0067] FIG. 10C is a graph showing in vivo luminescence of luciferase mRNA following extraction of spleens from CD-1 mice after treatment with mRNA luciferase-containing LNP Onpattro™ (MC3 / Chol / DSPC / PEG-DMG; 50 / 38.5 / 10 / 1.5 mol%) and a high cholesterol formulation of 42 / 2.7 / 54.3 / 1.0 mol% ionizable cationic lipid / SPC-Chol / Chol / PEG-DMG as described in Example 8.

[0068] [Figure 11A] 1 shows the physicochemical properties of plasmid DNA formulations showing the entrapment (%), particle size (nm) and PDI of Onpattro™ (MC3 / Chol / DSPC / PEG-DMG; 50 / 38.5 / 10 / 1.5 mol%) and 42 / 2.7 / 54.3 / 1.0 mol% ionizable cationic lipid / SPC-Chol / Chol / PEG-DMG formulations. The ionizable cationic lipids were norMC3, C123, C124 and C136 as described in Example 8. The N / P ratio was 6.

[0069] [Figure 11B] 1 is a graph showing in vitro luminescence as a function of plasmid DNA dose for Huh7 cells following treatment with ionizable cationic lipid (nMC3, C124, and 136) / H-001 / cholesterol / PEG-DMG LNPs at a molar ratio of 42 / 2.7 / 54.3 / 1.0 relative to an Onpattro™ LNP formulation (50 / 10 / 38.5 / 1.5; mol / mol MC3 / DSPC / cholesterol / PEG-DMG) encapsulating luciferase plasmid DNA. The N / P ratio was 6.

[0070] [Figure 12]1 shows the physicochemical properties of siRNA formulations showing the entrapment (%), particle size (nm) and PDI of the 42 / 2.7 / 54.3 / 1.0 mol% ionizable cationic lipid / SPC-Chol / Chol / PEG-DMG formulation. The ionizable cationic lipids were nMC3, C123, C124 and C136 as described in Example 8. The N / P ratio was 9.

[0071] [Figure 13A] 1 shows the physicochemical properties of a 42 / 2.7 / 54.3 / 1.0 mol% ionizable cationic lipid / SPC-Chol / Chol / PEG-DMG formulation and an mRNA formulation showing Onpattro™ entrapment (%), particle size (nm) and PDI. The N / P ratio was 6.

[0072] [Figure 13B-C] FIG 13B is a graph showing the percentage of mCherry uptake in various non-parenchymal cell types in the liver after treatment with a molar ratio of 42 / 2.7 / 54.3 / 1.0 nMC3 / H-001 / cholesterol / PEG-DMG relative to an Onpattro™ LNP formulation (50 / 10 / 38.5 / 1.5; mol / mol nMC3 / DSPC / cholesterol / PEG-DMG). The N / P ratio was 6.

[0073] FIG 13C is a graph showing the percentage of mCherry uptake in hepatocytes in the liver after treatment with nMC3 / H-001 / cholesterol / PEG-DMG at a molar ratio of 42 / 2.7 / 54.3 / 1.0 relative to the Onpattro™ LNP formulation (nMC3 / DSPC / cholesterol / PEG-DMG at 50 / 10 / 38.5 / 1.5; mol / mol) encapsulating mCherry mRNA. The N / P ratio was 6. (Detailed Description) Sterol or sterol derivative

[0074] As described herein, the LNPs of the present disclosure have sterol or sterol derivative levels that significantly exceed those used in conventional four-component Onpattro™-like formulations for nucleic acid delivery.

[0075] The term "sterol" refers to natural or synthetic steroids. This term includes plant sterols, animal sterols and their derivatives.

[0076] The term "sterol derivative" refers to modified sterols or precursors thereof, including triterpenes.

[0077] The term "cholesterol" refers to a natural or synthetic compound that has a gonan skeleton with a hydroxyl moiety attached to one of the rings, usually the A ring.

[0078] LNPs may also include "cholesterol derivatives." Cholesterol derivatives may be naturally occurring or artificial and include, but are not limited to, cholesterol molecules having a gonane structure and one or more additional functional groups.

[0079] In another embodiment, the LNPs can comprise triterpenes. Non-limiting examples include squalene, achilleol A, polypodatetraene, malabarican, lanostane, cucribitacin, hopane, oleanane, and urosolic acid.

[0080] The LNP may further comprise tocopherol as an additional component. In certain embodiments, the cholesterol derivative is a plant sterol. The plant sterol may be β-sitosterol, 3-sitosterol, campesterol, stigmasterol, fucosterol, stigmastanol, or a salt or ester thereof.

[0081] In certain embodiments, the cholesterol derivative is β-sitosterol, β-sitosterol acetate, 3-sitosterol, campesterol, stigmasterol, fucosterol or stigmastanol, dihydrocholesterol, ent-cholesterol, epi-cholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 3β[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholester ...

[0043] The present invention is selected from the group consisting of 5-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol, 3,6,9-trioxaoctane-1-ol-cholesteryl-3e-ol, dehydroergosterol, 9,11-dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, cytocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroegocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fucosterol, fecosterol, daucosterol, or a salt or ester thereof.

[0082] The sterol or cholesterol derivative may be conjugated to another moiety, such as an amino acid or an alkyl group.

[0083] In one embodiment, cholesterol or a derivative thereof is present at 48 mol% to 85 mol%, 50 mol% to 85 mol%, 52 mol% to 80 mol%, 54 mol% to 80 mol%, 56 mol% to 80 mol%, 58 mol% to 80 mol%, 60 mol% to 80 mol% or 62 mol% to 80 mol%, or is cholesterol or a derivative thereof, based on the total lipid present in the lipid nanoparticle.

[0084] In another embodiment, cholesterol or its derivatives are present in an amount of more than 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, 60 mol%, 61 mol% or 62 mol%. The upper limit of the cholesterol or cholesterol derivative content may be 88 mol%, 87 mol%, 86 mol%, 85 mol%, 84 mol%, 83 mol%, 82 mol%, 81 mol%, 80 mol%, 79 mol%, 78 mol%, 77 mol%, 76 mol% or 75 mol%. The cholesterol or cholesterol derivative content may include any combination of the above lower and upper limits.

[0085] In one embodiment, cholesterol is present at 48 mol% to 85 mol%, 50 mol% to 85 mol%, 52 mol% to 80 mol%, 54 mol% to 80 mol%, 56 mol% to 80 mol%, 58 mol% to 80 mol%, 60 mol% to 80 mol% or 62 mol% to 80 mol%, or is cholesterol, based on the total lipid present in the lipid nanoparticle.

[0086] In another embodiment, cholesterol is present at more than 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, 60 mol%, 61 mol% or 62 mol%. The upper limit of cholesterol content may be 88 mol%, 87 mol%, 86 mol%, 85 mol%, 84 mol%, 83 mol%, 82 mol%, 81 mol%, 80 mol%, 79 mol%, 78 mol%, 77 mol%, 76 mol% or 75 mol%. The cholesterol content may include any combination of the above lower and upper limits.

[0087] Tocopherols include α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol or salts or esters thereof. Tocopherols may be present at 0.5 mol % to 20 mol %, 1 mol % to 15 mol %, or 2 mol % to 10 mol %.

[0088] In some embodiments, the LNPs contain low levels of cholesteryl esters that include fatty acids conjugated via ester groups, for example, the cholesteryl ester content may be less than 10 mol%, less than 8 mol%, or less than 5 mol%, or less than 2 mol%.

[0089] In another embodiment, the LNPs have low levels of cationic cholesterol lipids, such as 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol). For example, the cationic cholesterol content may be less than 10 mol%, less than 8 mol%, less than 5 mol%, or less than 2 mol%. Ionizable lipids

[0090] The LNPs of the present disclosure have ionizable lipids and include one or more combinations of such lipids. The ionizable lipids may be charged at low pH and have substantially no net charge at physiological pH. This allows electrostatic interactions between the lipids and the negatively charged nucleic acid cargo upon initial formulation. The ionizable lipids are close to neutral at physiological pH, which reduces toxicity and renal clearance. Without being limited by theory, after intracellular uptake by endocytosis, the acidic environment of the endosome increases the net positive charge of the ionizable amino lipids, which promotes fusion with anionic lipids of the endosomal membrane, which then destabilizes the membrane and releases the nucleic acid-based therapeutic into the cytoplasm to exert its effects.

[0091] In some embodiments, it is desirable to include less than 50 mol% ionizable lipids in the LNPs, hi certain embodiments, the ionizable lipid content is between 5 mol% and 50 mol%, or between 8 mol% and 47 mol%, or between 10 mol% and 50 mol%, or between 15 mol% and 45 mol%, or between 15 mol% and 35 mol% of the total lipid present in the lipid nanoparticle.

[0092] In certain embodiments, the ionizable lipid content may be less than 48 mol%, less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol%, or less than 5 mol%. In certain embodiments, the lower limit of the ionizable lipid content may be greater than 5 mol%, greater than 8 mol%, greater than 10 mol%, greater than 12 mol%, greater than 14 mol%, greater than 15 mol%, greater than 16 mol%, greater than 18 mol%, or greater than 20 mol%. Any one of the upper limits may be combined with any one of the lower limits to obtain a suitable ionizable lipid content in the LNP.

[0093] As used herein, the term "cationic lipid" refers to a lipid that, in an electrostatically neutral form at a particular pH, such as physiological pH, can accept or donate a proton to become electrostatically positively charged, and whose electrostatically neutral form has a calculated logarithm of the partition coefficient between water and 1-octanol (i.e., cLogP) greater than 8. In some embodiments, the pKa of the cationic lipid is between 5.0 and 7.0.

[0094] In some embodiments, the cationic lipid has an amino group. In some cases, the cationic lipid comprises a protonatable tertiary amine (e.g., pH-titratable) head group, a C16-C18 alkyl chain, an ether bond between the head group and the alkyl chain, and 0-3 double bonds. Such lipids include, but are not limited to, sulfur lipids such as MF019 and DODMA described herein. Other lipids that can be used in the practice of the present disclosure include MC3-type lipids and KC2-type lipids that are well known to those skilled in the art. In further embodiments, the ionizable lipid is selected from one or more lipids described in WO2022 / 246555; WO2022 / 246568; WO2022 / 24657; PCT / CA2023 / 050129 filed January 31, 2023; WO2022 / 155728; 63 / 340,687 filed May 11, 2022; 63 / 410,281 filed September 27, 2022; 63 / 410,261 filed September 27, 2022; 63 / 434,506 filed December 22, 2022; 63 / 410,273 filed September 27, 2022; and 63 / 445,854 filed February 15, 2023, each of which is incorporated herein by reference.

[0095] In one embodiment, the ionizable cationic lipid comprises an ionizable amino head group and at least two lipophilic groups, at least one of which comprises a heteroatom such as an ester or one or more sulfur atoms. In some embodiments, at least one lipophilic group comprises a distal branch 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 of which comprises one or more sulfur atoms and / or ester groups) are described in co-owned and co-pending applications 63 / 340,687, filed May 11, 2022; 63 / 410,281, filed September 27, 2022; 63 / 410,261, filed September 27, 2022; 63 / 434,506, filed December 22, 2022; and 63 / 410,273, filed September 27, 2022. Functional groups containing one or more heteroatoms may be biodegradable in vivo.

[0096] In some embodiments, it is desirable to include less than 50 mol% cationic lipids in the LNPs, i.e., the ionizable lipid content can be less than 50 mol%, less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol%, or less than 5 mol%. In certain embodiments, the cationic lipid content is between 5 mol% and 50 mol%, or between 8 mol% and 47 mol%, or between 10 mol% and 50 mol%, or between 15 mol% and 45 mol%, or between 15 mol% and 35 mol% of the total lipid present in the lipid nanoparticle.

[0097] The ionizable lipid component may include an ionizable anionic lipid as part of the ionizable lipid content. One example of such lipid is cholesteryl hemisuccinate (CHEMS). Further examples of ionizable anionic lipids are described in the co-pending and co-owned US provisional patent entitled "Ionizable Anionic Lipids" filed on March 23, 2023, which is incorporated herein by reference in its entirety. Hydrophilic polymer lipid conjugates

[0098] In one embodiment, the lipid nanoparticle comprises a hydrophilic polymer lipid conjugate that can be incorporated into the LNP. The conjugate comprises a vesicle-forming lipid with a polar head group and a hydrophilic polymer chain covalently attached to the head group. Examples of hydrophilic polymers include polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinylmethylether, polyhydroxypropylmethacrylate, polyhydroxypropylmethacrylamide, polyhydroxyethylacrylate, polymethacrylamide, polydimethylacrylamide, polymethyloxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polysarcosine, and polyaspartamide. In one embodiment, the hydrophilic polymer lipid conjugate is a PEG lipid conjugate. The hydrophilic polymer lipid conjugate can be, for example, a monosialoganglioside (G M1 The oligosaccharide-containing molecule may be a naturally occurring or synthetic oligosaccharide-containing molecule such as a saccharide-containing polypeptide. The ability of a particular hydrophilic polymer-lipid conjugate to extend the circulatory life of the LNPs described herein can be readily determined by one of skill in the art using known methodologies.

[0099] The hydrophilic polymer lipid conjugate may be present in the nanoparticles at 0.5 mol% to 5 mol%, or 0.5 mol% to 3 mol%, or 0.5 mol% to 2.5 mol%, or 0.5 mol% to 2.0 mol%, or 0.5 mol% to 1.8 mol% of the total lipid.

[0100] In another embodiment, the hydrophilic polymer lipid conjugate is a PEG-lipid conjugate present in the nanoparticles at 0.5 mol% to 5 mol%, or 0.5 mol% to 3 mol%, or 0.5 mol% to 2.5 mol%, or 0.5 mol% to 2.0 mol%, or 0.5 mol% to 1.8 mol% of the total lipid.

[0101] As described below, the hydrophilic polymer-lipid conjugate may be conjugated at its distal end to a targeting ligand.

[0102] Low phospholipid content The lipid nanoparticles are "substantially free of phospholipids", i.e., the lipid nanoparticles have less than 3 mol% of phospholipids, such as neutral phospholipids. In one embodiment, the lipid nanoparticles have a phospholipid content of 3 mol% or less. Examples of neutral phospholipids include phosphatidylcholine or phosphatidylethanolamine, such as distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC). In some embodiments, the phospholipid is a phospholipid-sterol conjugate, such as SPC-cholesterol, OPC-cholesterol or PPC-cholesterol conjugate. Additionally, phospholipid-sterol conjugates are described in US2011 / 0177156, which is incorporated herein by reference.

[0103] An example of a suitable phospholipid that is an SPC-Chol conjugate is NTX-H-0001, described below. [ka]

[0104] In one embodiment, the lipid nanoparticles have 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 mol% phospholipids. Most advantageously, the LNPs are free of phospholipids, although small amounts do not affect the LNP properties described herein.

[0105] In some disclosed examples, the inclusion of small amounts of phospholipids in high sterol-containing LNPs has been found to improve liver targeting compared to Onpatro™.

[0106] In another embodiment, the lipid nanoparticles are "substantially free of neutral lipids" means that the lipid nanoparticles have less than 3 mol% of any neutral lipids, except cholesterol or cholesterol derivatives. The term "neutral lipids" refers to any of a number of lipid species, including vesicle-forming lipids, that exist in either uncharged or neutral zwitterionic form at physiological pH. In another embodiment, the lipid nanoparticles have less than 8, 6, 4 or 2 mol% of neutral lipids, such as phospholipids and / or phospholipid conjugates.

[0107] Examples of neutral lipids include sphingomyelin, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), diacylphosphatidylcholine such as dipalmitoylphosphatidylcholine (DPPC), diacylphosphatidylethanolamine such as dioleoylphosphatidylethanolamine (DOPE), ceramide, cephalin, triglyceride, and diacylglycerol. In some embodiments, the neutral lipid is a SPC-cholesterol or PPC-cholesterol conjugate, or a phospholipid-sterol conjugate such as those described in US2011 / 0177156.

[0108] Examples of suitable neutral lipids that are SPC-Chol conjugates include NTX-H-0001, described above.

[0109] In one embodiment, the lipid nanoparticles have 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 mol% of any neutral lipid. Most advantageously, there are no neutral lipids in the LNPs, although small amounts may be included in the formulation without affecting the properties of the LNPs described herein.

[0110] Additional Ingredients The LNPs can include additional lipid components or modifications to a sterol (eg, cholesterol), a sterol derivative (eg, cholesterol derivative), and / or a hydrophilic polymer-lipid conjugate.

[0111] For example, the surface of the LNP may be grafted to include a targeting ligand. The targeting ligand may be conjugated to cholesterol, cholesterol derivatives, and / or hydrophilic polymer-lipid conjugates. The targeting ligand may be conjugated to the distal end of a hydrophilic polymer-lipid conjugate. The targeting ligand may be used to target receptors on cells in vivo. In some embodiments, the targeting ligand may be conjugated to any amount of phospholipids that may be included in the LNP. In such embodiments, the phospholipid targeting ligand conjugate is typically present at less than 3 mol%.

[0112] Ligands include peptides, polypeptides, or proteins, including antibodies or fragments thereof, hi one embodiment, the ligand may be a single chain antibody fragment.

[0113] Preparation of nanoparticles Lipid nanoparticles can be prepared using various suitable methods, such as rapid mixing / ethanol dilution process.Examples of preparation methods are disclosed 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, each of which is incorporated herein by reference in its entirety.

[0114] For example, a method for preparing lipid nanoparticles may include dissolving lipid components (e.g., ionizable lipid, sterol, and hydrophilic polymer lipid) in an organic solvent (e.g., ethanol) in an appropriate ratio. An aqueous buffer solution is separately prepared at an appropriate pH so that the head groups (e.g., amino groups) of the ionizable lipids are protonated and promote electrostatic interactions with the negatively charged cargo and the positively charged ionizable lipids. Such charge interactions improve encapsulation.

[0115] In some embodiments, the aqueous phase is then mixed with the organic solvent-lipid mixture. Mixing of the aqueous phase and the organic solvent-lipid mixture can be performed in a mixing device (e.g., an in-line mixer), such as a T-junction mixer (e.g., a T-tube mixer) equipped with a specialized pump, a herringbone micromixer, a toroidal 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 that includes two or more inlets that meet in a central mixing region and an outlet through which the mixture exits the device. Formation of LNPs can occur during mixing of the aqueous phase and the organic solvent-lipid mixture and / or after such mixing. (Kulkarni et al., 2019, Nanoscale, 11(18):9023-9031, which is incorporated herein by reference).

[0116] The aqueous phase usually comprises a buffer solution.Non-limiting examples of suitable buffer solutions include MES or phosphate buffered saline (PBS).Examples of suitable solvents for preparing organic solvent-lipid mixture include organic solvents such as ethanol, isopropanol, methanol and acetone.

[0117] The aqueous phase and the organic solvent-lipid mixture can be introduced into the mixer as two separate streams via pumps. The volumetric flow rates of each stream can be the same or different, and the respective flow rates of each stream can be adjusted to achieve optimal mixing and / or LNP formation.

[0118] In some embodiments, LNPs are prepared by solvent injection. In one embodiment, such a method involves dissolving lipids in an organic solvent, followed by stepwise dilution of the resulting solution with an aqueous solution (e.g., a buffer solution). This controlled stepwise dilution is achieved by mixing the aqueous stream and the lipid stream in a vessel.

[0119] The average size of the lipid nanoparticles can be 40-120 nm, 45-110 nm, or any range therebetween. In another embodiment, the PDI of the lipid nanoparticles is less than 0.2, less than 0.15, less than 0.12, or less than 0.10. The nitrogen to phosphate ratio of the lipid nanoparticles is 3 or 6.

[0120] LNPs generally include a "core" region, which may include an electron-dense region and optionally an aqueous portion as visualized by cryo-TEM microscopy. Without limitation, the electron-dense region in the core may be partially surrounded by an aqueous portion in an enclosed space, may be completely surrounded or enveloped by an aqueous portion in the core, or may have a solid core without an aqueous portion as observed by cryo-TEM. The core may include nucleic acid and ionizable lipids. In one embodiment, cholesterol, cholesterol derivatives and / or tocopherol are present in the outer lipid layer and / or core of the LNP.

[0121] In some embodiments, the LNP is not a lipoplex. Lipoplexes are prepared by mixing preformed cationic liposomes and nucleic acids in an aqueous solution and may exhibit undesirable properties such as localization of cargo to the particle surface. Lipoplexes lack the core of the LNP particle described above. Furthermore, LNPs have a defined size, shape, and morphology, whereas lipoplexes 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 of which is incorporated herein by reference).

[0122] Thus, according to some embodiments, the LNPs disclosed herein have a defined average particle size ranging from 40 to 120 nm, or 45 to 100 nm, or 50 to 90 nm, hi some embodiments, the LNPs herein have a PDI of less than 0.20, less than 0.18, less than 0.16, less than 0.15, or less than 0.14.

[0123] As used herein, the term "encapsulation" with respect to incorporating nucleic acid cargo into LNP refers to any association of nucleic acid with any lipid component or compartment of lipid nanoparticle.However, this excludes the localization of nucleic acid on the particle surface as in the case of lipoplex.In some examples of the present disclosure, nucleic acid is present in the core of LNP.Without being limited by theory, nucleic acid may be present in micelles in the core of LNP.

[0124] Nucleic Acid Cargo 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 or linear DNA. The length of the nucleic acid can vary and can include nucleic acids from 1 to 50,000 nucleotides in length. The nucleic acid can be in any form, including single-stranded DNA or RNA, double-stranded DNA or RNA, or hybrids thereof. Single-stranded nucleic acids include antisense oligonucleotides. The nucleic acid can be conjugated to another molecule that includes 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.

[0125] In one embodiment, cargo is mRNA, which comprises the polynucleotide that codes for at least one peptide, polypeptide or protein. mRNA includes, but is not limited to, small activating RNA (saRNA) and trans-amplifying RNA (taRNA), as described in co-pending US provisional application No. 63 / 195,269 "mRNA Delivery Using Lipid Nanoparticles", which is incorporated herein by reference.

[0126] As used herein, mRNA includes both modified and unmodified mRNA.In one embodiment, mRNA comprises one or more coding regions and non-coding regions.MRNA can be purified from natural sources, or produced using recombinant expression systems and optionally purified, or chemically synthesized.

[0127] In those embodiments where the mRNA is a chemically synthesized molecule, the mRNA may include nucleoside analogs, such as analogs with chemically modified bases or sugars, and / or backbone modifications. In some embodiments, the mRNA may include natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deoxyur ... are or contain: zaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0128] The mRNA of the present invention can be synthesized according to any of a variety of known methods.For example, the mRNA in certain embodiments can be synthesized by in vitro transcription (IVT).Briefly, IVT is usually carried out using a linear or circular DNA template that contains a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.

[0129] In some embodiments, in vitro synthesized mRNA may be purified prior to encapsulation to remove undesirable impurities, including various enzymes and other reagents used during mRNA synthesis.

[0130] The present disclosure may be used to encapsulate mRNAs of various lengths, in some embodiments, the present disclosure may be used to encapsulate in vitro synthesized mRNAs ranging in length from about 1-20 kb, about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb.

[0131] Typically, mRNA is synthesized by adding a "cap" to the 5' end and a "tail" to the 3' end. The presence of a cap is advantageous in that it provides resistance to nucleases found in eukaryotic cells. The presence of a "tail" serves to protect the mRNA from degradation by exonucleases.

[0132] In some embodiments, the mRNA comprises 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 (e.g., iron response elements). In some embodiments, the 5' untranslated region may be about 1-500 nucleotides in length, or 50-500 nucleotides in length or longer.

[0133] In some embodiments, the 3' untranslated region includes a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location within the cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region may be 1-500 nucleotides in length, or 50-500 nucleotides in length or more.

[0134] In certain embodiments, mRNA provided from an in vitro transcription reaction may be desirable, although other sources of mRNA are contemplated, such as mRNA produced from bacteria, fungi, plants, and / or animals.

[0135] The mRNA sequence may include a reporter gene sequence, although including a reporter gene sequence in the pharmaceutical formulation for administration is optional. Such sequences may be incorporated into the mRNA for in vitro studies to assess expression and biodistribution or in vivo studies in animal models.

[0136] In another embodiment, cargo is siRNA.siRNA is incorporated into endogenous cellular machinery, causing mRNA degradation and preventing transcription.Since RNA is easily degraded, its incorporation into delivery vehicle can reduce or prevent such degradation, thereby facilitating delivery to target site.

[0137] The siRNAs included in the embodiments of the present disclosure may be used to specifically inhibit the expression of a wide variety of target polynucleotides. siRNA molecules targeting a particular polynucleotide for any therapeutic, prophylactic, or diagnostic application may be readily prepared following procedures known in the art. siRNA target sites may be selected and the corresponding siRNAs may be chemically synthesized, generated by in vitro transcription, or expressed from vectors or PCR products. A wide variety of siRNA molecules may be used to target a particular gene or transcript. siRNAs may be double-stranded RNA or may be hybrid molecules that contain both RNA and DNA (e.g., one RNA strand and one DNA strand). siRNAs may be of various lengths, such as 1-30 nucleotides long, 15-30 nucleotides long, or 20-25 nucleotides long. In certain embodiments, the siRNA is double-stranded and has a 3' overhang or a 5' overhang. In certain embodiments, the overhang is UU or dTdT3'. In certain embodiments, the siRNA comprises a stem-loop structure.

[0138] In further embodiments, the cargo molecule is a microRNA or small nuclear RNA. MicroRNA (miRNA) is a short non-coding RNA molecule that is transcribed from genomic DNA but is not translated into protein. These RNA molecules are believed to play a role in regulating gene expression by binding to a region of the target mRNA. When miRNA binds to the target mRNA, gene expression can be downregulated, such as by inducing translational repression, deadenylation, or degradation of the target mRNA. Small nuclear RNA (snRNA) is a longer non-coding RNA molecule that is usually involved in gene splicing. snRNA molecules can play an important role in the treatment or diagnosis of diseases caused by splicing defects.

[0139] In another embodiment, the cargo is a DNA vector. The encapsulated DNA vector can be administered to a subject for the purpose of repairing, enhancing, inhibiting, or reducing the expression of a cellular protein or peptide. In another embodiment, the encapsulated DNA vector can be administered to a subject for the diagnosis of a disease. The DNA vector can be localized to a target cell (e.g., a rapidly dividing cell) and utilize the expression of the encoded DNA to provide a measurable signal. Thus, the nucleotide polymer can be a nucleotide sequence, including genomic DNA, cDNA, or RNA.

[0140] As will be understood by those skilled in the art, vectors can code for promoter regions, operator regions, or structural regions.DNA vectors can comprise double-stranded DNA or can be composed of DNA-RNA hybrids.Non-limiting examples of double-stranded DNA include structural genes, genes that comprise operator control and termination regions, and self-replicating systems such as vector DNA.

[0141] Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes, and triplex-forming oligonucleotides. To prolong activity, single-stranded nucleic acids most advantageously have some or all of the nucleotide linkages replaced with stable non-phosphodiester linkages, including, for example, phosphorothioate, phosphorodithioate, phosphoroselenate, or O-alkyl phosphotriester linkages.

[0142] The DNA vector may contain nucleic acids in which one or more sugar moieties and / or one or more pyrimidine or purine bases are modified. Such sugar modifications may include replacing one or more hydroxyl groups with halogen, alkyl groups, amines, azide groups, or functionalizing as ethers or esters. In another embodiment, the entire sugar may be replaced with a sterically and electronically similar structure, including azasugars and carbocyclic sugar analogs. Modifications of purine or pyrimidine base moieties include, for example, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substitutes known to those skilled in the art.

[0143] In certain embodiments, the DNA vector may be modified with a modifying molecule, such as a peptide, protein, steroid, or sugar moiety. Modifying the DNA vector with such molecules may facilitate delivery to the desired target site. In some embodiments, such modifications cause the DNA vector to translocate beyond the nucleus of the target cell. As an example, the modifier may be attached to a specific portion of the DNA vector (usually a portion that does not code for the gene of interest), but may also have a peptide or other modifier with a nuclear homing effect, such as a nuclear localization signal. A non-limiting example of a modifier is the steroid-peptide nucleic acid conjugate described in Rebuffat et al., 2002, Faseb J. 16(11):1426-8, which is incorporated herein by reference. The DNA vector may contain sequences encoding various proteins or peptides. Promoters, enhancers, stress or chemically regulated promoters, antibiotic or nutrient sensitive regions, and encoding sequences for therapeutic proteins may be included as needed. Non-encoding sequences may also be present in the DNA vector.

[0144] 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 a variety of solution or solid-phase methods. Solid-phase synthesis is generally preferred. Known procedures for the solid-phase synthesis of nucleic acids with phosphite triester, phosphate triester, and H-phosphonate chemistries are widely available.

[0145] In one embodiment, the DNA vector is double stranded DNA and comprises 700 or more base pairs, 800 or more base pairs, 900 or more base pairs, or 1000 or more base pairs. Improved liver-specific expression

[0146] In some embodiments, the LNPs show "liver-specific expression" of the protein or peptide encoded by the cargo nucleic acid, meaning that the cargo nucleic acid increases the expression of the protein or peptide by 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 in the liver compared to the spleen. In some embodiments, the cargo nucleic acid is an mRNA or vector DNA. In one example, the nucleic acid is an mRNA.

[0147] In some embodiments, the inclusion of small amounts of neutral lipids may improve liver-specific expression of LNPs with elevated levels of sterol or sterol derivatives compared to Onpattro™. For example, to improve liver targeting, the neutral lipid content may be 0-10 mol%, 0.5-10 mol%, 1 mol%-8 mol%, 1 mol%-5 mol%, or 1 mol%-3.5 mol%.

[0148] In further embodiments, the inclusion of low levels of hydrophilic polymer lipid conjugates may improve liver-specific expression of LNPs with elevated levels of sterol or sterol derivatives compared to Onpattro™. For example, the hydrophilic polymer lipid conjugate content to achieve such improved liver targeting may be less than 2.5 mol%, less than 2.25 mol%, less than 2.0 mol%, less than 1.75 mol%, less than 1.5 mol%, or less than 1.25 mol%.

[0149] Alternatively or additionally, the increase in expression of a protein or peptide encoded by a nucleic acid cargo (e.g., mRNA or vector DNA) in the liver relative to the spleen is at least 5% or 10% greater than the increase in expression in the liver relative to the spleen in Onpattro™ LNP encapsulating the same cargo, measured under otherwise identical conditions.

[0150] These examples are intended to illustrate the preparation of specific lipid nanoparticle preparations and their characteristics and are not intended to limit the scope of the invention.

[0151] As used herein, the articles "a" or "an" are intended to include both the singular and the plural, unless otherwise specified. EXAMPLES

[0152] Example 1: LNPs with higher cholesterol levels have higher encapsulation efficiency In this example, we investigate the effect of increasing cholesterol content and decreasing ionizable lipid content on the physical properties of lipid nanoparticles lacking phospholipids.

[0153] Phospholipid-free or substantially phospholipid-free LNPs containing various concentrations of cholesterol were prepared by injecting a lipid mixture (ionizable lipid (DLin-MC3-DMA, hereafter "MC3"), cholesterol, PEG-DMG) at a water:ethanol (v / v) ratio of 3:1 and an amine to phosphate (N / P) ratio of 6, through a T-junction, together with an aqueous phase containing the nucleic acid cargo, from a range of 15-45 mol% ionizable lipid dissolved in ethanol, at respective mol% ratios of X / 98.5 to X / 1.5, to a final concentration of 10 mM. The flow rates were set at 5 mL / min for the lipid phase and 15 mL / min for the aqueous phase containing mRNA dissolved in 25 mM sodium acetate (pH 4), resulting in an output flow rate of 20 mL / min. The resulting formulation was then dialyzed against 1000-fold pile volume of phosphate-buffered saline (pH 7.4) for 24 h to remove ethanol from the formulation. The nucleic acid cargo was an antisense oligonucleotide targeting c-myc or an siRNA targeting the firefly luciferase gene (siLuc).

[0154] Figure 1 shows the effect of increasing the mol% of cholesterol in phospholipid-free LNPs (containing ionizable lipid, cholesterol, and PEG-DMG) encapsulating antisense oligonucleotides at various mol% ratios from X / 98.5 to X / 1.5 (ionizable lipid / cholesterol / PEG-DMG).

[0155] Strikingly, the phospholipid-free LNPs with the highest cargo (antisense oligonucleotide) encapsulation rates contained the highest levels of cholesterol investigated in our dataset, namely, 53.5 mol%, 58.5 mol%, and 63.5 mol%. Notably, the encapsulation efficiency of each of these formulations approached 100%. These LNPs also exhibited good PDI values ​​(<0.1) and appropriate particle sizes (~60 nm).

[0156] Figure 2A shows the effect on the physical properties of phospholipid-free LNPs of increasing the cholesterol content beyond the values ​​examined in Figure 1, i.e., from 53.5 mol% to 93.5 mol%. Surprisingly, when the cholesterol content of the phospholipid-free LNPs was in the range of 53.5 mol% - 83.5 mol%, the encapsulation efficiency of the cargo (siLuc) approached 100%. LNP size and PDI were also within acceptable limits within this cholesterol mol% range. When cholesterol was above 83.5 mol%, the LNPs exhibited high PDI values ​​(>0.2) and the particle size of the LNPs exceeded 100 nm.

[0157] FIG. 2B plots the physical property data of FIG. 2A, but as a function of ionizable lipid (MC3) content (instead of cholesterol content). As noted, the benchmark Onpattro™ formulation contains high levels of ionizable lipid (50 mol%), with the remaining lipid components being DSPC (10 mol%), cholesterol (38.2 mol%), and PEG-DMG (1.5 mol%). Surprisingly, however, the phospholipid-free LNPs investigated herein showed encapsulation efficiencies approaching 100% even with MC3 contents as low as 15 mol%, with PDI values ​​below 0.2 and particle sizes ranging from 50 nm to 75 nm. The data also show that LNPs with MC3 mol% between 20% and 45% consistently showed encapsulation levels of approximately 100%.

[0158] Example 2: Phospholipid-free luciferase mRNA-LNPs increase cholesterol levels, decrease MC3 levels, and have comparable in vitro efficacy to Onpattro™ formulations This example shows that MC3 / cholesterol / PEG-DMG (35 / 63.5 / 1.5 mol:mol) LNPs encapsulating luciferase mRNA have in vitro potency comparable to the Onpattro™ formulation in assays measuring luminescence and mRNA dosage in tissue culture.

[0159] The mRNA-encapsulated MC3 / cholesterol / PEG-DMG (35 / 63.5 / 1.5 mol:mol) and Onpattro™ (MC3 / distearoylphosphatidylcholine (DSPC) / cholesterol / PEG-DMG 50 / 10 / 38.5 / 1.5) LNPs were prepared by injecting the respective mol% ratios of lipid mixtures (ionizable lipids, cholesterol, PEG-DMG) dissolved in ethanol with an aqueous phase containing mRNA encoding luciferase through a T-junction at a water:ethanol (v / v) ratio of 3:1 and an amine to phosphate (N / P) ratio of 6. The flow rates were set at 5 mL / min for the lipid phase and 15 mL / min for the aqueous phase containing mRNA dissolved in 25 mM sodium acetate (pH 4), resulting in an output flow rate of 20 mL / min. The resulting formulation was then dialyzed against 1000 volumes of phosphate buffered saline (pH 7.4) for 24 hours to remove ethanol from the formulation.

[0160] Huh7 cells were seeded at 8,000 cells per well in a clear flat-bottom 96-well plate after reaching approximately 80% confluency. After 24 hours of culture, the initial medium was replaced with medium containing luciferase mRNA-encapsulated LNPs at doses ranging from 0.03 μg / mL to 10 μg / mL. Each cell treatment was performed in triplicate. After 24 hours, the medium was aspirated and 100 μL of GloLysisBuffer™ (Promega™) was added to each well. Each plate was then incubated at 37°C for 10 minutes, after which 50 μL from each well was transferred to a white flat-bottom 96-well plate and luminescence was measured. Next, 50 μL of Steady-GloLuciferase™ substrate was added to each well, after which luminescence values ​​were immediately measured on a BioTek Synergy Neo2 Hybrid Multi-Mode Reader™.

[0161] The results shown in Figure 3 indicate that the luminescence intensity of luciferase expressed from mRNA was comparable across the dose range of Luc-mRNA LNPs between the MC3 / Cholesterol / PEG-DMG (35 / 63.5 / 1.5 mol:mol) and Onpattro™ formulations, which is surprising in part because the MC3 / Cholesterol / PEG-DMG formulation contains 15 mol% less MC3 ionizable lipid and no phospholipids than the Onpattro™ formulation.

[0162] Example 3: Phospholipid-free luciferase mRNA-LNPs increase cholesterol levels, decrease MC3 levels, and have comparable in vivo efficacy to Onpattro™ formulations This example shows that various LNPs with higher cholesterol content and lower ionizable lipid content compared to Onpattro™ and encapsulating luciferase mRNA have in vivo efficacy comparable to the Onpattro™ formulation.

[0163] Phospholipid-free MC3 / cholesterol / PEG-DMG (X / 98.5-X / 1.5 mol ionizable lipid / cholesterol / PEG-DMG) and Onpattro™ formulations encapsulating Luc-mRNA (MC3 / cholesterol / DSPC / PEG-DMG 50 / 38.5 / 10 / 1.5) were prepared as described in Example 2.

[0164] Figures 4A and 4B show the biophysical data for the formulations tested. Similar to the formulations tested in Example 2, the encapsulation efficiency of formulations containing 53.5-83.5 mol% cholesterol approached 100% for each LNP (Figure 4A), despite the corresponding reduction in levels of MC3 ionizable lipid (45 mol%-15 mol%, see Figure 4B). PDI and size were within acceptable ranges for each formulation tested.

[0165] For in vivo studies, the final dosage of mRNA in the formulation was 1mg / kg mRNA. Luc-mRNA formulations were administered to CD-1 mice, and the liver and spleen were removed 4 hours after injection. mRNA luminescence was measured by the Promega™ Steady-Glo™ Luciferase Assay System.

[0166] Figures 5A-D show the luminescence in liver and spleen for each formulation investigated. Figures 5A and 5B show the luminescence intensity / mg tissue as a function of increasing cholesterol content in liver and spleen, respectively. As cholesterol was increased from 53.5 mol% to 83.5 mol%, the luminescence intensity / mg liver for each formulation tested became similar. Furthermore, the luminescence intensity of formulations with increased cholesterol content was similar to that of the Onpattro™ formulation. Figures 5C and 5D show the same data plotted as a function of increasing ionizable cationic lipid content, similarly showing similar luminescence intensities for formulations with different ionizable cationic lipid contents. Notably, formulations with a low ionizable cationic lipid content of 15 mol% and a cholesterol content of 83.5 mol% showed nearly equivalent luminescence intensity to Onpattro™ in liver and spleen. This is somewhat surprising since the latter MC3 / cholesterol / PEG-DMG formulation contains 35 mol% less MC3 ionizable cationic lipid than the Onpattro™ formulation and is devoid of phospholipids. Moreover, in both cases, LNPs showed higher expression of mRNA luciferase in the liver compared to the spleen.

[0167] Example 4: PEG lipid content above 3 mol% reduces in vivo luminescence intensity We next investigated the effect of increasing the PEG lipid content above 1.5 mol% on the luminescence intensity of LNPs containing Luc mRNA in the liver and spleen. The LNPs investigated contained MC3 / cholesterol / PEG-DMG at 35 / 63.5 / 1.5 mol% and 35 / 61.7 / 3.3 mol% and were prepared as described in Example 2. In vivo studies to assess mRNA luminescence intensity were performed using the methods described in Example 3 above.

[0168] Figure 6A shows that the luminescence intensity of LNPs containing 1.5 mol% PEG lipids was approximately two-fold higher than that of LNPs containing 3.3 mol% PEG lipids in the liver, and Figure 6B shows that the luminescence intensity of LNPs containing 1.5 mol% PEG lipids was approximately three-fold higher than that of LNPs containing 3.3 mol% PEG lipids in the spleen.

[0169] These results indicate that in some embodiments, it is most advantageous to have a PEG lipid content of less than 3 mol%. Furthermore, for each formulation examined, LNPs had higher mRNA luciferase expression in the liver compared to the spleen (see Figures 6A and 6B). Figure 6A shows that the luminescence intensity of LNPs containing 1.5 mol% PEG lipid was nearly two times higher than that of LNPs containing 3.3 mol% PEG lipid in the liver. Figure 6B shows that the luminescence intensity of LNPs containing 1.5 mol% PEG lipid was nearly three times higher than that of LNPs containing 3.3 mol% PEG lipid in the spleen. These results indicate that in some embodiments, it is most advantageous to have a PEG lipid content of less than 3 mol%. Furthermore, for each formulation examined, LNPs had higher mRNA luciferase expression in the liver compared to the spleen (see Figures 6A and 6B).

[0170] Example 5: Sterol-rich LNP formulations target the liver rather than the spleen As mentioned above, Examples 3 and 4 above suggest that sterol-rich mRNA LNPs increase in vivo mRNA expression levels in the liver compared to the spleen (see Figures 5A-D, Figures 6A and 6B). Comparing Figures 5A and 5B, the luminescence intensity / mg liver (Figure 5A) for each high cholesterol LNP formulation (cholesterol 53.5 mol%-83.5%) was up to 1,000-fold higher than the luminescence intensity / mg spleen (Figure 5B) for the same formulation. A similar trend of liver targeting was observed for LNP formulations containing ionizable cationic lipid / cholesterol / PEG-DMG ratios of 35 / 63.5 / 1.5 mol% and 35 / 61.7 / 3.3 mol%, respectively (Figures 6A vs. 6B).

[0171] In this and subsequent examples, the effect of varying the lipid composition of sterol-rich LNPs to enhance mRNA expression in the liver compared to the spleen was investigated in more detail. LNPs were prepared as described above in Example 2, and in vivo studies to assess mRNA luminescence intensity in the liver and spleen were performed using the procedures previously described in Example 3. The liver and spleen were harvested and assessed for mRNA expression (luminescence intensity) 4 hours after injection as described above.

[0172] We first investigated the in vivo expression of mRNA with varying levels of DSPC and SPC-cholesterol conjugates in which one acyl chain of DSPC is replaced by cholesterol (see the structure of H-0001 above). In particular, the cholesterol-rich LNP formulations investigated included a three-component LNP with MC3, 56.5 mol% cholesterol, and 1.5 mol% PEG-DMG, and two four-component cholesterol-rich LNPs that included 3 mol% DSPC or SPC-chol conjugates in the formulation at the expense of cholesterol. [Table 1]

[0173] The formulation characteristics, including size (nm), PDI, and encapsulation rate of each LNP, were evaluated as described in Example 1, and the results are shown in Figure 7A.

[0174] The high sterol formulations (samples B-D) have comparable mRNA expression to Onpattro™ (sample A), but surprisingly, compared to Onpattro™, the formulations of the present invention are more liver-tropic in that lower mRNA expression levels are observed in the spleen compared to the liver (see Figures 7B and 7C). Notably, the inclusion of 3 mol% phospholipids (DSPC and SPC-Chol conjugates) in the high sterol LNPs further reduces splenic activity compared to sample B, which does not contain phospholipids (samples C and D above).

[0175] Example 6: Increasing N / P in a high sterol formulation does not affect liver activity and maintains hepatotropism To investigate the effect of varying the amine to phosphate ratio (N / P) on liver targeting, a 42 / 3 / 53.5 / 1.5 mol% MC3 / SPC-Chol conjugate / Chol / PEG-DMG high sterol mRNA-LNP formulation (formulation D in Table 1) was prepared with N / P values ​​of 5, 6, 7, 8, and 9. LNPs were prepared as described in Example 2 above, and in vivo studies to assess mRNA emission intensity in the liver and spleen were performed using the procedures previously described in Example 3.

[0176] The formulation properties of each LNP, including size (nm), PDI, and encapsulation rate of luciferase-encoding mRNA, were evaluated as described in Example 1, and the results are shown in Figure 8A.

[0177] As shown in Figures 8B and 8C, increasing the N / P of the high sterol formulation (42 / 3 / 53.5 / 1.5MC3 / SPC-Chol / Chol / PEG-DMG (mol:mol)) did not significantly affect activity (luciferase mRNA expression) in the liver or spleen. Thus, the formulation can encapsulate a wide range of N / P without affecting liver tropism.

[0178] Example 7: Reducing PEG lipids in sterol-rich formulations improves liver targeting The inventors investigated in vivo expression of luciferase mRNA in both liver and spleen using the LNP compositions in Table 2 below with varying levels of PEG lipid. In particular, the high sterol LNP formulations investigated included four-component LNPs with MC3 / SPC-Chol / Chol / PEG-DMG at 42 / 55 to X / 3 / X mol%, where X is 1.0, 1.5, or 2.0 mol%. [Table 2]

[0179] LNPs were prepared as described in Example 2 above, and in vivo studies to assess mRNA emission intensity in the liver and spleen were performed using the procedures previously described in Example 3.

[0180] The formulation characteristics, including size (nm), PDI, and encapsulation rate of each LNP, were evaluated as described in Example 1, and the results are shown in Figure 9A.

[0181] Decreasing the PEG lipid mol% in the high sterol formulation (samples BD in Table 2) increased liver activity (see FIG. 9B). Surprisingly, formulations containing 1.0 mol% and 1.5 mol% PEG lipid (samples B and C in Table 2) showed higher levels of mRNA expression compared to Onpattro™ (sample A in Table 2).

[0182] Example 8: Various ionizable cationic lipids can be formulated into sterol-rich mRNA lipid nanoparticles to improve liver tropism To investigate whether various ionizable cationic lipids could be formulated into high sterol LNPs, a formulation of ionizable cationic lipid / SPC-Chol / Chol / PEG-DMG was prepared at 42 / 2.7 / 54.3 / 1.0 mol% using the following ionizable cationic lipids with amino ionizable head groups: norMC3, C108, C109, C123, C124, C136, C137, and C142. The ability of the ionizable cationic lipids to improve liver activity compared to Onpattro™ was also investigated. The control was Onpattro™ (see Table 1 above).

[0183] The ionizable cationic lipids are listed in Table 3 below. [Table 3]

[0184] LNPs were prepared as described in Example 2 above, and in vivo studies to assess mRNA emission intensity in the liver and spleen were performed using the procedures previously described in Example 3.

[0185] The formulation characteristics, including size (nm), PDI, and encapsulation rate of each LNP were evaluated as described in Example 1, and the results are shown in Figure 10A.

[0186] Surprisingly, formulations containing ionizable cationic lipids with sulfur atoms in the lipophilic chain showed higher levels of mRNA expression compared to Onpattro™ (Sample A in Table 1). C123, 124, 136, and 143 ionizable cationic lipids showed 4.2-6.8-fold improved liver activity (see Figure 10B), while maintaining liver tropism as evidenced by low expression in the spleen (Figure 10C).

[0187] Example 9: Various ionizable lipids can encapsulate plasmid DNA in sterol-rich formulations and improve in vitro transfection The ability to encapsulate plasmid DNA in various high sterol LNPs containing various ionizable cationic lipids was investigated. In particular, a formulation containing ionizable cationic lipid / SPC-Chol / Chol / PEG-DMG at 42 / 2.7 / 54.3 / 1.0 mol% was prepared using the following ionizable cationic lipids with amino ionizable head groups: nMC3, C123, C124, and C136 (see Table 3 in Example 8 above for structures). The ability of the ionizable cationic lipids to improve in vitro transfection compared to Onpattro™ was also investigated. The control was Onpattro™ (see Table 1 above).

[0188] LNPs were prepared as described in Example 2 above, except that Aldevron Gwiz-Luciferase™ plasmid was used instead of luciferase mRNA, and in vitro studies to assess luminescence intensity in Huh7 cells were performed using the procedures previously described in Example 2.

[0189] The formulation characteristics, including size (nm), PDI, and encapsulation rate of each LNP were evaluated as described in Example 1, and the results are shown in Figure 11A.

[0190] Surprisingly, formulations containing ionizable cationic lipids with a sulfur atom in the lipophilic chain showed higher levels of luciferase expression compared to Onpattro™ (Sample A in Table 1). For nMC3, C123, 124, and 136 ionizable cationic lipids, the in vitro transfection activity was improved by 1.1-14.7-fold at the same dose (see Figure 11B).

[0191] Example 10: Various ionizable cationic lipids can encapsulate siRNA in sterol-rich formulations The ability to encapsulate siRNA in sterol-rich LNPs formulated with various ionizable cationic lipids was investigated. A 42 / 2.7 / 54.3 / 1.0 mol% ionizable cationic lipid / SPC-Chol / Chol / PEG-DMG LNP formulation was prepared using the following ionizable cationic lipids with amino ionizable head groups: nMC3, C123, C124, C136 (see Table 3 in Example 8 for structures).

[0192] LNPs were prepared as described in Example 2 above, except that luciferase siRNA from Integrated DNA Technologies™ (IDT) was used instead of luciferase mRNA.

[0193] The formulation characteristics, including size (nm), PDI, and encapsulation rate of each LNP were evaluated as described in Example 1, and the results are shown in FIG.

[0194] Example 11: Sterol-rich LNPs exhibit similar hepatocyte specificity to Onpattro™ but are more liver-tropic To determine the cell specificity of high sterol LNPs, mCherry mRNA was encapsulated within a high sterol LNP formulation of 42 / 2.7 / 54.3 / 1.0 mol% nMC3 / SPC-Chol / Chol / PEG-DMG and compared to Onpattro™ (nMC3), injected at 1 mg / kg into CD-1 mice, and mice were euthanized at the 24-hour endpoint. Mice were euthanized and perfused with 15 mL of cold 1x phosphate-buffered saline (PBS). Livers were harvested in 5 mL of RPMI supplemented with 5% heat-inactivated fetal bovine serum (HI-FBS). Livers were then minced and digested in RPMI containing 0.5 mg / mL collagenase type IV (Sigma-Aldrich™) and 2 mg / mL DNase I (Sigma-Aldrich™) for 30 min at 37°C with shaking at 200 rpm. The digested liver was passed through a 70 μm sieve to obtain a single cell suspension. The single cell suspension was then centrifuged at 50 g for 3 min at 4° C. to pellet and collect the hepatocytes. The supernatant containing the non-parenchymal cell fraction was then collected. The hepatocytes were then washed two more times with 10 mL of flow cytometry staining buffer (FACS) buffer. The liver non-parenchymal fraction was then separated using a 20% OptiPrep™ gradient (Stem Cell Technologies™, see manufacturer's protocol). The cells were washed with FACS buffer and analyzed by flow cytometry (see below).

[0195] Surface staining for flow cytometry Hepatocytes were resuspended in PBS containing 5% HI-FBS, 0.05% NaN3, and 2.5 mM EDTA (FACS buffer) and incubated with anti-FcgRII / RIII (2.4G2) for at least 15 min on ice, after which cells were stained with one of the following fluorochromes conjugated to CD11b [M1 / 70], CD26 [H94-112], CD31 [MEC13.3], CD38

[90] , panCD45 [I3 / 2], CD45R (B220) [RA3-6B2], Clec4F [3E3F9], Ly6G [IA8], or TCRβ [Η57-597] (Thermo Fisher™, BD bioscience, BioLegend) for 45 min on ice in the dark. To exclude dead cells, cells were stained with Fixable Viability Dye (Thermo Fisher™) according to the manufacturer's instructions prior to flow cytometry acquisition.

[0196] Flow cytometry data acquisition and analysis A Cytoflex LX™ was used to acquire flow cytometry data and analysis was performed using FlowJo™ (TreeStar™).

[0197] LNPs were prepared as described in Example 2 above, except that mCherry mRNA was used instead of luciferase mRNA.

[0198] The formulation characteristics, including size (nm), PDI, and encapsulation rate of each LNP were evaluated as described in Example 1, and the results are shown in Figure 13A.

[0199] Surprisingly, the high sterol formulation showed comparable mCherry expression to Onpattro™ (including nMC3) in all cell types evaluated (Figures 13B and 13C). Combined with the data shown in Figures 10B and 10C (Example 8), the results show a formulation comparable to Onpattro™ with the same ionizable lipids while increasing cell tropism to the liver.

Claims

1. Lipid nanoparticles Nucleic acid cargo molecules and; Sterols or their derivatives present in a content of 49 mol% to 85 mol%; It contains virtually no phospholipids; Ionizable lipids and; It contains a hydrophilic polymer lipid conjugate present in a content of 0.5 mol% to 3 mol%; Each mol% content is relative to the total lipids present in the lipid nanoparticles, wherein the lipid nanoparticles are lipid nanoparticles.

2. The lipid nanoparticles according to claim 1, wherein the phospholipid content is less than 1 mol%.

3. The lipid nanoparticles according to claim 1, wherein the phospholipid content is less than 0.5 mol%.

4. The lipid nanoparticles according to claim 1, wherein the content of the hydrophilic polymer lipid is less than 2.5 mol%.

5. The lipid nanoparticle according to claim 1, wherein the cargo molecule is siRNA, mRNA, vector nucleic acid, antisense oligonucleotide, or nucleic acid-protein or peptide complex.

6. The lipid nanoparticle according to claim 5, wherein the cargo molecule is siRNA, a vector nucleic acid, or an antisense oligonucleotide.

7. The lipid nanoparticle according to claim 1, wherein the ionizable lipid is an aminolipid.

8. The lipid nanoparticle according to claim 1, wherein the sterol derivative is a noncationic lipid.

9. The lipid nanoparticles according to claim 1, wherein the lipid nanoparticles have a core with high electron density that can be visualized by cryo-TEM.

10. The lipid nanoparticles according to claim 1, wherein the lipid nanoparticles are substantially free of neutral lipids.

11. The lipid nanoparticle according to claim 1, wherein the sterol is cholesterol, or the sterol derivative is a cholesterol derivative.

12. The lipid nanoparticles according to claim 1, further comprising tocopherol.

13. The lipid nanoparticles according to claim 12, wherein the tocopherol is present in an amount of 0.5 to 15 mol%.

14. The lipid nanoparticles according to claim 1, wherein the phospholipid is present in an amount of 0.5 to 3 mol%.

15. The lipid nanoparticle according to claim 14, wherein the phospholipid is a phospholipid-cholesterol conjugate.

16. The lipid nanoparticle according to claim 15, wherein the phospholipid-cholesterol conjugate is a phosphatidylcholine-cholesterol conjugate.

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