Methods of mRNA delivery and compositions thereof

Abstract

The present disclosure provides lipid nanoparticles for extrahepatic delivery of mRNA, the lipid nanoparticles comprising: (i) an mRNA load; (ii) a phosphatidylcholine lipid content of from 30 mol% to 70 mol%; (iii) an ionizable cationic lipid content of from 5 mol% to 50 mol%; (iv) a sterol selected from cholesterol or a derivative thereof; and (v) a hydrophilic polymer-lipid conjugate present at a lipid content of from 0.5 mol% to 5 mol. Also provided are lipid nanoparticles comprising an encapsulated mRNA and 20 to 70 mol% of a phosphatidylcholine lipid, an ionizable lipid; and at least one of a sterol and a hydrophilic polymer-lipid conjugate, the lipid nanoparticles exhibiting at least 10% increase in mRNA gene expression in vivo measured in one or more extrahepatic organs or tissues. Methods of administering such lipid nanoparticles are also provided.

Description

Incorporation by reference into the priority application

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 362,345, filed on April 1, 2022, which is hereby expressly incorporated by reference in its entirety. Technical Field

[0002] The present disclosure relates to methods of delivering mRNA and compositions thereof for delivery to extrahepatic tissues. Background Art

[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 TM .Onpattro TM It is a lipid nanoparticle-based short interfering RNA (siRNA) drug formulation for the treatment of polyneuropathy induced by hereditary transthyretin amyloidosis. The success of this LNP delivery system paves the way for the clinical development of the leading LNP-based COVID-19 mRNA vaccine.

[0004] Onpattro TM The LNP formulation consists of four major lipid components (i.e., ionizable amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and polyethylene glycol-conjugated lipid (PEG-lipid)) in respective molar amounts of 50 / 10 / 38.5 / 1.5. TM It is still considered the gold standard for comparison in LNP-mediated efficacy studies, and current LNP design methods show little deviation from the four-component system.

[0005] Of these four components, ionizable lipids make up Onpattro TM The majority of the preparation (50mol%), and is considered to be important for the in vitro and in vivo activity of LNP. Therefore, most of the work in this area is mainly focused on improving this lipid component. Ionizable lipids are usually amino lipids, which are carefully designed to be charged at low pH values ​​and close to neutral at physiological pH values. This allows electrostatic interactions between lipids and negatively charged nucleic acids during initial formulation. Since ionizable lipids are close to neutral at physiological pH values, toxicity and renal clearance are reduced. After cellular uptake by endocytosis, the acidic environment of the endosome causes the net positive charge of the ionizable amino lipid to increase, thereby promoting fusion with the anionic lipids of the endosomal membrane and subsequent membrane instability and releasing nucleic acid-based therapeutic agents into the cytoplasm to exert their effects.

[0006] Regarding the remaining three lipid components, PEG-lipids are well known for improving the circulation life of LNPs and stabilizing the cholesterol function of the particles. However, in general, relatively little attention has been devoted to studying DSPC, except for its role as a structural lipid.

[0007] Although great progress has been made in the research related to LNP-mediated nucleic acid delivery, it is well known that Onpattro TM Preparations are mainly accumulated in liver (hepatic) tissue. The ability of LNP to accumulate in organs and tissues other than the liver will greatly expand the clinical practicality of these delivery systems. In order to improve the delivery of nucleic acid cargo to extrahepatic tissues (such as bone marrow and spleen), particles should show enhanced circulation life. Traditional methods to achieve this goal rely on optimizing the level of PEG lipids in LNP, but including PEG-lipids in LNPs usually leads to low transfection efficacy or adverse immune responses.

[0008] In vivo studies have been conducted to investigate the four-component Onpattro TM In particular, Onpattro TM The results showed that 10 mol% DSPC Onpattro TM The sample formulation has similar liver accumulation and blood circulation life span as the 40mol% DSPC formulation (MC3 / Chol / DSPC / PEG-DMG; 18.5 / 40 / 40 / 1.5mol%). In addition, in bone marrow gene silencing, the 40mol% DSPC siRNA-LNP only performed comparable to the 10mol% DSPC formulation. Therefore, previous studies have shown that adjusting DSPC levels to improve extrahepatic delivery of nucleic acid payloads has no obvious benefit.

[0009] The above-mentioned studies investigating extrahepatic delivery are limited to LNPs with siRNA payloads. However, messenger RNA (mRNA) therapy is gradually becoming an important tool for treating diseases, and delivering mRNA to extrahepatic tissues will expand the clinical application of mRNA therapy beyond the liver. Similar to siRNA, mRNA molecules degrade rapidly in the body, so LNPs are used to reduce this degradation. Nevertheless, there are inherent chemical and structural differences between mRNA and siRNA in terms of nucleic acid length, stability and charge density. (Kauffman et al., 2015, NanoLetters, 15(11): 7300-7306). Therefore, siRNA-LNP studies may not provide information for LNPs designed for mRNA delivery. In addition, current work on LNP mRNA systems for intravenous administration focuses primarily on the development of improved ionizable cationic lipids. (Semple et al., Nat Biotechnol 2010, 28: 172). In addition, these systems use Onpattro TM The lipid composition (see above) and has a short circulation life, with most of the cargo accumulating in the liver within 30 minutes. (Akinc et al., 2019, Nat Nanotechnol., 14: 1084).

[0010] Therefore, there is a need in the art for improved extrahepatic delivery of nucleic acids (eg, mRNA) in vivo using lipid nanoparticles. Summary of the invention

[0011] The present disclosure solves one or more of the aforementioned problems in the prior art and / or provides useful alternatives to known compositions for delivering mRNA.

[0012] The present disclosure is based on the following discovery: TM Compared to benchmark LNPs, ionizable LNP formulations including elevated levels of phosphatidylcholine lipids (referred to herein as "lcLNPs") can significantly improve in vivo extrahepatic delivery of mRNA. In some embodiments, the present disclosure provides mRNA-LNP formulations that are compatible with Onpattro TM Compared to LNP, it has increased in vivo expression of mRNA in extrahepatic tissues as measured in immune cell subsets in the bone marrow and spleen in animal models at predetermined times after administration (e.g., at 1 hour and 3 hours after administration).

[0013] In one aspect, the present disclosure provides lipid nanoparticles for extrahepatic delivery of mRNA, the lipid nanoparticles comprising: (i) mRNA; (ii) a phosphatidylcholine lipid content of 30 to 70 mol %; (iii) an ionizable content of 5 to 50 mol %; (iv) a sterol selected from cholesterol or a derivative thereof; and (v) a hydrophilic polymer-lipid conjugate present in a lipid content of 0.5 to 5 mol %, wherein each lipid content is measured relative to the total lipid content of the lipid nanoparticle.

[0014] In another aspect, the present disclosure provides lipid nanoparticles comprising encapsulated mRNA and 20 to 70 mol % of phosphatidylcholine lipids, ionizable lipids, relative to the total lipids present in the lipid nanoparticles; and at least one of a sterol and a hydrophilic polymer-lipid conjugate, the lipid nanoparticles exhibiting at least a 10% increase in gene expression of the mRNA in vivo measured in a cell subpopulation in the bone marrow or spleen 24 hours and / or 3 days after injection, the cells being selected from macrophages, monocytes and / or T cells, compared to an Onpattro-type formulation of MC3 / DSPC / cholesterol / PEG-lipids at 50 / 10 / 38.5 / 1.5 mol:mol for encapsulating the mRNA, but otherwise measured under the same conditions, wherein the gene expression is quantified in an animal model by detecting cells positive for green fluorescent protein (GFP) using flow cytometry.

[0015] In one embodiment, the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), or dipalmitoylphosphatidylcholine (DPPC).

[0016] In another embodiment, the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC).

[0017] In yet another embodiment, the phosphatidylcholine content is between 40mol% and 60mol%. In another embodiment, the phosphatidylcholine content is between 42mol% and 58mol%. In yet another embodiment, the phosphatidylcholine content is between 45mol% and 55mol%.

[0018] In another embodiment, the ionizable lipid is a cationic lipid. In yet another embodiment, the ionizable cationic lipid is an amino lipid.

[0019] In yet another embodiment, the content of ionizable lipids is less than 40 mol %.

[0020] In another embodiment, the hydrophilic polymer-lipid conjugate is a polyethylene glycol-lipid conjugate. In yet another embodiment, the sterol is present at 15 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle. In another embodiment, the sterol is present at 18 mol% to 40 mol% based on the total lipid present in the lipid nanoparticle.

[0021] In yet another embodiment, the lipid nanoparticles exhibit at least a 10% increase in gene expression of an in vivo mRNA measured in a cell subpopulation selected from macrophages, monocytes and / or T cells in the bone marrow or spleen 24 hours and / or 3 days after injection compared to an Onpattro-type formulation of MC3 / DSPC / cholesterol / PEG-lipid at 50 / 10 / 38.5 / 1.5 mol:mol encapsulating the mRNA, but otherwise measured under the same conditions, wherein gene expression is quantified in an animal model using flow cytometry by detecting cells positive for green fluorescent protein (GFP). In yet another embodiment, green fluorescent protein is measured in a population of macrophages / monocytes isolated from the spleen or liver of a mouse after injection, and wherein the increase in expression is determined by measuring the percentage of cells in the cell population that are eGFP positive.

[0022] In another embodiment, green fluorescent protein is measured 3 days after injection, and wherein the animal model is a mouse.

[0023] In yet another embodiment, in vivo expression of green fluorescent protein in macrophage / monocyte populations isolated from mouse bone marrow or spleen following injection is increased by at least 50% compared to LNPs containing sphingomyelin in which sphingomyelin is used instead of DSPC but otherwise has the same lipid composition as the lipid nanoparticles.

[0024] In another aspect, a method for delivering mRNA to a mammalian subject in vivo is provided, the method comprising: administering to the mammalian subject the lipid nanoparticle as described in any embodiment or aspect above.

[0025] In yet another embodiment, the mRNA accumulates in the spleen or bone marrow of the subject for at least one day after administration.

[0026] In another embodiment, the disease or condition is an autoimmune disorder. In yet another embodiment, the disease or condition is an infectious disease. In yet another embodiment, the disease or condition is cancer.

[0027] In yet another embodiment, the use of lipid nanoparticles for delivering and expressing mRNA into mammalian cells in vivo or in vitro is provided.

[0028] In yet another embodiment, the lipid nanoparticles exhibit at least a 10% increase in gene expression of mRNA in vivo as measured in an extrahepatic tissue or organ.

[0029] In another embodiment, the extrahepatic tissue or organ is spleen, bone marrow, lung, kidney, heart, abdominal skin, dorsal skin, and / or ear.

[0030] A method for delivering mRNA to a mammalian subject in vivo, the method comprising: administering to the mammalian subject the lipid nanoparticles of any one of the preceding embodiments.

[0031] In yet another embodiment, a method is provided for delivering mRNA for in vivo production of a protein or peptide in an extrahepatic tissue or organ of a subject, the method comprising administering to the subject a lipid nanoparticle having at least 20 mol% distearoylphosphatidylcholine (DSPC) and / or dipalmitoylphosphatidylcholine (DPPC) and an ionizable cationic lipid, wherein the mRNA is encapsulated within the lipid nanoparticle, and wherein administration of the lipid nanoparticle results in extrahepatic tissue or organ expression of the protein or peptide encoded by the mRNA, wherein the lipid nanoparticle has at least a 10% increase in expression of the protein or peptide encoded by the mRNA in the extrahepatic tissue or organ relative to an Onpattro-type formulation of MC3 / DSPC / cholesterol / PEG-lipid encapsulating the mRNA at 50 / 10 / 38.5 / 1.5 mol:mol, but otherwise measured under the same conditions.

[0032] In yet another embodiment, there is provided a method as defined above, wherein the lipid nanoparticles have at least 30 mol % distearoylphosphatidylcholine (DSPC) and / or dipalmitoylphosphatidylcholine (DPPC) and less than 40 mol % ionizable cationic lipids.

[0033] In another embodiment, the extrahepatic tissue or organ is spleen, bone marrow, lung, kidney, heart, abdominal skin, dorsal skin, and / or ear.

[0034] In another embodiment, the mRNA accumulates in the spleen or bone marrow of the subject for at least one day after administration.

[0035] In another embodiment, the lipid nanoparticles are used to treat a disease or disorder, wherein the disease or disorder is an autoimmune disorder.

[0036] In another embodiment, the lipid nanoparticles are used to treat a disease or condition, which is an infectious disease.

[0037] According to yet another embodiment, the lipid nanoparticles are used to treat a disease or condition, wherein the disease or condition is cancer.

[0038] In another embodiment, there is provided the use of lipid nanoparticles as described above in the preparation of a medicament for delivering mRNA to mammalian cells in vivo or in vitro. In some embodiments, the mRNA is used for targeting autoimmune disorders in vivo. In yet another embodiment, the mRNA is used for targeting infectious diseases in vivo.

[0039] In yet another embodiment, the mRNA is used to target cancer in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1A Shown are the percentages of enhanced green fluorescent protein (eGFP)-positive cells (% eGFP) in the indicated immune cell subsets (monocytes / macrophages, neutrophils, B cells, CD4 T cells, and CD8 T cells) in the spleen 4 hours after injection of phosphate-buffered saline (PBS); and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3: DSPC: Chol: PEG-lipid (50:10:38.5:1.5) ("Onpattro TM ”); or 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)). MF019 and nMC3 are ionizable cationic lipids described herein (Example 1).

[0041] Figure 1B Shown are the percentages of eGFP-positive cells (% eGFP) in the indicated immune cell subsets (monocytes / macrophages, neutrophils, B cells, CD4 T cells, and CD8 T cells) in the spleen 24 hours after injection of phosphate-buffered saline (PBS); and eGFP-mRNA LNPs into mice. + ), the eGFP-mRNA LNP has 10 mol% DSPC (nMC3: DSPC: Chol: PEG-lipid (50: 10: 38.5: 1.5) ("Onpattro TM ”); or 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)).

[0042] Figure 1CThe percentage of eGFP-positive cells (% eGFP) in the indicated immune cell subsets (monocytes / macrophages, neutrophils, B cells, CD4 T cells, and CD8 T cells) in the spleen 3 days after injection of phosphate-buffered saline (PBS); and eGFP-mRNA LNPs into mice is shown. + ), the eGFP-mRNA LNP has 10 mol% DSPC (nMC3: DSPC: Chol: PEG-lipid (50: 10: 38.5: 1.5) ("Onpattro TM ”); or 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)).

[0043] Figure 1D Shown are the mean fluorescence intensities (MFI) of the indicated immune cell subsets (monocytes / macrophages, neutrophils, B cells, CD4 T cells, and CD8 T cells) in the spleen 4 hours after injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro TM ”); or 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)).

[0044] Figure 1E Shown are the MFI of the indicated immune cell subsets (monocytes / macrophages, neutrophils, B cells, CD4 T cells, and CD8 T cells) in the spleen 24 hours after injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro TM ”); or 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)).

[0045] Figure 1FShown are the MFI of the indicated immune cell subsets (monocytes / macrophages, neutrophils, B cells, CD4 T cells, and CD8 T cells) in the spleen 3 days after injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro TM ”); or 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)).

[0046] Figure 2A Representative dot plots depicting eGFP in spleen 24 hours after injection of phosphate buffered saline (PBS) into mice are shown. + Percentage of monocytes / macrophages.

[0047] Figure 2B Representative dot plots depicting eGFP in the spleen 24 hours after injection of eGFP-mRNA LNPs into mice are shown. + The percentage of monocytes / macrophages, the eGFP-mRNA LNP with 10 mol% DSPC (nMC3: DSPC: Chol: PEG-lipid (50:10:38.5:1.5) ("Onpattro TM ”)).

[0048] Figure 2C Representative dot plots depicting eGFP in the spleen 24 hours after injection of eGFP-mRNA LNPs into mice are shown. + Percentage of monocytes / macrophages, the eGFP-mRNA LNP with 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) ("lcLNP TM ”)).

[0049] Figure 3A Shown are the percentages of eGFP-positive cells (% eGFP) in the indicated immune cell subsets (erythroid, B cells, T cells, monocytes / macrophages, and neutrophils) in the bone marrow 4 hours after injection of phosphate-buffered saline (PBS); and eGFP-mRNA LNPs into mice. +), the eGFP-mRNA LNP has 10 mol% DSPC (nMC3: DSPC: Chol: PEG-lipid (50: 10: 38.5: 1.5) ("Onpattro TM ”); or 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)).

[0050] Figure 3B The percentage of eGFP-positive cells (% eGFP) in the indicated immune cell subsets (erythroid cells, B cells, T cells, monocytes / macrophages, and neutrophils) in the bone marrow 24 hours after injection of phosphate-buffered saline (PBS); and eGFP-mRNA LNPs into mice is shown. + ), the eGFP-mRNA LNP has 10 mol% DSPC (nMC3: DSPC: Chol: PEG-lipid (50: 10: 38.5: 1.5) ("Onpattro TM ”); or 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)).

[0051] Figure 3C The percentage of eGFP-positive cells (% eGFP) in the bone marrow of the indicated immune cell subsets (erythroid cells, B cells, T cells, monocytes / macrophages, and neutrophils) 3 days after injection of phosphate-buffered saline (PBS); and eGFP-mRNA LNPs into mice is shown. + ), the eGFP-mRNA LNP has 10 mol% DSPC (nMC3: DSPC: Chol: PEG-lipid (50: 10: 38.5: 1.5) ("Onpattro TM ”); or 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)).

[0052] Figure 3DShown are the mean fluorescence intensities (MFI) of immune cell subsets (erythroid cells, B cells, T cells, monocytes / macrophages, and neutrophils) shown in the bone marrow 4 hours after injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3: DSPC: Chol: PEG-lipid (50:10:38.5:1.5) ("Onpattro TM ”); or 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)).

[0053] Figure 3E Shown are the mean fluorescence intensities (MFI) of immune cell subsets (erythroid cells, B cells, T cells, monocytes / macrophages, and neutrophils) shown in the bone marrow 24 hours after injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3: DSPC: Chol: PEG-lipid (50:10:38.5:1.5) ("Onpattro TM ”); or 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)).

[0054] Figure 3F Shown are the MFI of the indicated immune cell subsets (erythroid cells, B cells, T cells, monocytes / macrophages, and neutrophils) in the bone marrow 3 days after injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro TM ”); or 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)).

[0055] Figure 4A Representative scatter plots depicting eGFP in the bone marrow 24 hours after injection of phosphate-buffered saline (PBS) into mice are shown. + The percentage of T cells.

[0056] Figure 4B Representative dot plots depicting eGFP in the bone marrow 24 hours after injection of eGFP-mRNA LNPs into mice are shown. + The percentage of T cells, the eGFP-mRNA LNP with 10 mol% DSPC (nMC3: DSPC: Chol: PEG-lipid (50:10:38.5:1.5) ("Onpattro TM ”)).

[0057] Figure 4C Representative dot plots depicting eGFP in the bone marrow 24 hours after injection of eGFP-mRNA LNPs into mice are shown. + The percentage of T cells, the eGFP-mRNA LNP with 50 mol% DSPC (MF019: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) ("lcLNP TM ”)).

[0058] Figure 5A Figure 2 shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in the bone marrow of the indicated immune cell subsets (erythroid cells, B cells, T cells, monocytes / macrophages, and neutrophils) after injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs into mice 24 hours later. + ), the eGFP-mRNA LNP has 10 mol% DSPC (MC3: DSPC: Chol: PEG-lipid (50: 10: 38.5: 1.5) ("Onpattro TM ”); or 50 mol% DSPC (MC3: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)). MC3 is an ionizable cationic amino lipid described herein.

[0059] Figure 5B Shown are the mean fluorescence intensities (MFI) of immune cell subsets (erythroid cells, B cells, T cells, monocytes / macrophages, and neutrophils) shown in the bone marrow 24 hours after injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs with 10 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro TM”); or 50 mol% DSPC (MC3: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)).

[0060] Fig. 6A Representative scatter plots depicting eGFP in the bone marrow 24 hours after injection of phosphate-buffered saline into mice are shown. + The percentage of T cells.

[0061] Figure 6B Representative dot plots depicting eGFP in the bone marrow 24 hours after injection of eGFP-mRNA LNPs into mice are shown. + The percentage of T cells, the eGFP-mRNA LNP with 10 mol% DSPC (MC3: DSPC: Chol: PEG-lipid (50: 10: 38.5: 1.5) ("Onpattro TM ”)).

[0062] Figure 6C Representative dot plots depicting eGFP in the bone marrow 24 hours after injection of eGFP-mRNA LNPs into mice are shown. + The percentage of T cells, the eGFP-mRNA LNP with 50 mol% DSPC (MC3: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) ("lcLNP TM ”)).

[0063] Figure 7 Figure 2 shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in monocytes / macrophages in the spleen 24 hours after injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs with 10 mol% DSPC (MC3: DSPC: Chol: PEG-lipid (50: 10: 38.5: 1.5 mol: mol) ("Onpattro TM ”); or 50 mol% DSPC (MC3: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) (“lcLNP TM ”)).

[0064] Fig. 8A Representative dot plots depicting eGFP in spleen 24 hours after injection of phosphate buffered saline (PBS) into mice are shown. + Percentage of monocytes / macrophages.

[0065] Figure 8B Representative dot plots depicting eGFP in the spleen 24 hours after injection of eGFP-mRNA LNPs into mice are shown. + The percentage of monocytes / macrophages, the eGFP-mRNA LNP with 10 mol% DSPC (MC3: DSPC: Chol: PEG-lipid (50:10:38.5:1.5) ("Onpattro TM ”)).

[0066] Figure 8C Representative dot plots depicting eGFP in the spleen 24 hours after injection of eGFP-mRNA LNPs into mice are shown. + Percentage of monocytes / macrophages, the eGFP-mRNA LNP with 50 mol% DSPC (MC3: DSPC: Chol: PEG-lipid (27.4: 50: 21.1: 1.5 mol: mol) ("lcLNP TM ”)).

[0067] Fig. 9A Figure 2 shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in the bone marrow of the indicated immune cell subsets (erythroid cells, B cells, T cells, monocytes / macrophages, and neutrophils) after injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs into mice 24 hours later. + ), the eGFP-mRNA LNP had 50 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5) ("lcLNP")); or had 50 mol% egg sphingomyelin (ESM) (MC3:ESM:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol)).

[0068] Fig. 9BShown are mean fluorescence intensities (MFI) of the indicated immune cell subsets (erythroid, B cells, T cells, monocytes / macrophages, and neutrophils) in the bone marrow 24 hours after injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs with 50 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP")); or with 50 mol% hen's egg sphingomyelin (ESM) (MC3:ESM:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol)) into mice.

[0069] Fig. 10A Representative dot plots depicting eGFP in the bone marrow 24 hours after injection of eGFP-mRNA LNPs into mice are shown. + The percentage of T cells, the eGFP-mRNA LNP with 50 mol% DSPC (MC3: DSPC: Chol: PEG-lipid (50: 10: 38.5: 1.5) ("lcLNP TM ”)).

[0070] Fig. 10B Representative dot plots depicting eGFP in the bone marrow 24 hours after injection of eGFP-mRNA LNPs into mice are shown. + The percentage of T cells, the eGFP-mRNA LNPs had 50 mol% ESM (MC3:ESM:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) (“ESM”; Fig. 10B )).

[0071] Fig.11A Shown are the percentages of enhanced green fluorescent protein (eGFP)-positive cells (% eGFP) in the indicated immune cell subsets (monocytes / macrophages, neutrophils, B cells, CD4 T cells, and CD8 T cells) in the spleen 24 hours after injection of phosphate-buffered saline (PBS); and eGFP-mRNA LNPs into mice. + ), the eGFP-mRNA LNP had 50 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP")); or had 50 mol% egg sphingomyelin (ESM) (MC3:ESM:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol)).

[0072] Fig. 11BShown are the mean fluorescence intensities (MFI) of the indicated immune cell subsets (monocytes / macrophages, neutrophils, B cells, CD4 T cells, and CD8 T cells) in the spleen 24 hours after injection of phosphate-buffered saline (PBS); and eGFP-mRNA LNPs with 50 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) (“lcLNP”)); or with 50 mol% hen egg sphingomyelin (ESM) (MC3:ESM:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol)) into mice.

[0073] Fig. 12A Representative dot plots depicting eGFP in the spleen 24 hours after injection of eGFP-mRNA LNPs into mice are shown. + The percentage of macrophages / monocytes, the eGFP-mRNA LNP with 50 mol% DSPC (MC3: DSPC: Chol: PEG-lipid (50: 10: 38.5: 1.5) ("lcLNP TM ”)).

[0074] Fig. 12B Representative dot plots depicting eGFP in the spleen 24 hours after injection of eGFP-mRNA LNPs into mice are shown. + Percentage of macrophages / monocytes, the eGFP-mRNA LNPs with 50 mol% ESM (MC3:ESM:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("ESM")).

[0075] Fig.13A mRNA-LNP MF019:DSPC:Chol:PEG is shown 2000 -DMG (27.4:50:21.1:1.5 mol:mol) and NTx-C16:DSPC:Chol:PEG 2000 -DMG (27.4:50:21.1:1.5 mol:mol) compared to Onpattro TM nMC3:DSPC:Chol:PEG 2000 -Luminescence fold change of DMG (50:10:38.5:1.5 mol:mol) administered to CD-1 mice at a dose of 1 mg / kg. mRNA expression was quantified in liver, spleen, lung, kidney, heart, bone marrow (BM), abdomen, back and ear 24 hours after injection by measuring luminescence.

[0076] Fig. 13B mRNA-LNP MF019:DSPC:Chol:PEG is shown 2000 -DMG (27.4:50:21.1:1.5 mol:mol) and NTx-C16:DSPC:Chol:PEG 2000 -DMG (27.4:50:21.1:1.5 mol:mol) compared to Onpattro TM nMC3:DSPC:Chol:PEG 2000 -Luminescence fold change of DMG (50:10:38.5:1.5 mol:mol) administered to CD-1 mice at a dose of 5 mg / kg. mRNA expression was quantified in liver, spleen, lung, kidney, heart, bone marrow (BM), abdomen, back and ear 24 hours after injection by measuring luminescence.

[0077] Fig. 13C Serum activities (U / L) of alanine aminotransferase (ALT) (left) and aspartate aminotransferase (AST) (right) in CD-1 mice 24 hours after injection of 5 mg / kg of LNP-mRNA encoding luciferase. TM (nMC3:DSPC:Chol:PEG 2000 -DMG(50:10:38.5:1.5mol:mol)) and mRNA-LNP MF019:DSPC:Chol:PEG 2000 -DMG (27.4:50:21.1:1.5 mol:mol) and NTx-C16:DSPC:Chol:PEG 2000 -DMG(27.4:50:21.1:1.5mol:mol).

[0078] Fig.14A Shows Onpattro TM mRNA capture %, particle size (nm) and polydispersity index (PDI) of LNPs (LNP A); LNPs with 50 mol% of phosphatidylcholine lipids DSPC, DOPC, POPC and DPPC (LNP BF); LNPs with 40:10, 30:20, 20:30 and 10:40 mol:mol DSPC:DOPC (GJ). LNP formulations AJ are provided in Table 7 of Example 5, and mRNA encodes luciferase.

[0079] Fig. 14B Shows Onpattro TM(LNP A); LNPs with 50 mol% of phosphatidylcholine lipids DSPC, DOPC, POPC and DPPC (LNP BF); LNPs with 40:10, 30:20, 20:30 and 10:40 mol:mol DSPC:DOPC (GJ) of CD-1 mice luminescence intensity / mg liver. LNP formulations AJ are provided in Table 7 of Example 5, and mRNA encodes luciferase. mRNA expression was quantified by measuring luminescence 24 hours after injection.

[0080] Fig. 14C Shows Onpattro TM (LNP A); LNPs with 50 mol% of phosphatidylcholine lipids DSPC, DOPC, POPC and DPPC (LNP BF); LNPs with 40:10, 30:20, 20:30 and 10:40 mol:mol DSPC:DOPC (GJ) of CD-1 mice luminescence intensity / mg spleen. LNP formulations AJ are provided in Table 7 of Example 5, and mRNA encodes luciferase. mRNA expression was quantified by measuring luminescence 24 hours after injection.

[0081] Fig.14D Shows Onpattro TM (LNP A); LNPs with 50 mol% of phosphatidylcholine lipids DSPC, DOPC, POPC and DPPC (LNP BF); LNPs with 40:10, 30:20, 20:30 and 10:40 mol:mol DSPC:DOPC (GJ) of CD-1 mice luminescence intensity / mg heart. LNP formulations AJ are provided in Table 7 of Example 5, and mRNA encodes luciferase. mRNA expression was quantified by measuring luminescence 24 hours after injection.

[0082] Fig.14E Shows Onpattro TM (LNP A); LNPs with 50 mol% of phosphatidylcholine lipids DSPC, DOPC, POPC and DPPC (LNP BF); LNPs with 40:10, 30:20, 20:30 and 10:40 mol:mol DSPC:DOPC (GJ) of CD-1 mice luminescence intensity / mg lung. LNP formulations AJ are provided in Table 7 of Example 5, and mRNA encodes luciferase. mRNA expression was quantified by measuring luminescence 24 hours after injection.

[0083] Fig.14F Shows Onpattro TM(LNP A); LNPs with 50 mol% of phosphatidylcholine lipids DSPC, DOPC, POPC and DPPC (LNP BF); LNPs with 40:10, 30:20, 20:30 and 10:40 mol:mol DSPC:DOPC (GJ) of CD-1 mice luminescence intensity / mg kidney. LNP formulations AJ are provided in Table 7 of Example 5, and mRNA encodes luciferase. mRNA expression was quantified by measuring luminescence 24 hours after injection.

[0084] Figure 14G Shows Onpattro TM (LNP A); LNPs with 50 mol% of phosphatidylcholine lipids DSPC, DOPC, POPC and DPPC (LNP BF); LNPs with 40:10, 30:20, 20:30 and 10:40 mol:mol DSPC:DOPC (GJ) of CD-1 mice luminescence intensity / mg muscle tissue. LNP formulations AJ are provided in Table 7 of Example 5, and mRNA encodes luciferase. mRNA expression was quantified by measuring luminescence 24 hours after injection.

[0085] Fig.14H Shows Onpattro TM (LNP A); LNPs with 50 mol% of phosphatidylcholine lipids DSPC, DOPC, POPC and DPPC (LNP BF); LNPs with 40:10, 30:20, 20:30 and 10:40 mol:mol DSPC:DOPC (GJ) of CD-1 mice luminescence intensity / mg bone marrow (BM). LNP formulations AJ are provided in Table 7 of Example 5, and mRNA encodes luciferase. mRNA expression was quantified by measuring luminescence 24 hours after injection.

[0086] Fig.14I Shows Onpattro TM (LNP A); LNPs with 50 mol% of phosphatidylcholine lipids DSPC, DOPC, POPC and DPPC (LNP BF); LNPs with 40:10, 30:20, 20:30 and 10:40 mol:mol DSPC:DOPC (GJ) of CD-1 mice luminescence intensity / mg ear. LNP formulations AJ are provided in Table 7 of Example 5, and mRNA encodes luciferase. mRNA expression was quantified by measuring luminescence 24 hours after injection.

[0087] Fig.15AThe luminescence intensity / mg bone marrow (BM) of the LNP groups having different levels of DSPC and ionizable cationic lipids shown in Table 8 of Example 6 in CD-1 mice is shown.

[0088] Fig. 15B The luminescence intensity / mg spleen of the LNP groups having different levels of DSPC and ionizable cationic lipids shown in Table 8 of Example 6 in CD-1 mice is shown.

[0089] Fig. 15C The luminescence intensity / mg skin of the LNP groups with different levels of DSPC and ionizable cationic lipids shown in Table 8 of Example 6 in CD-1 mice is shown.

[0090] Fig.15D The luminescence intensity / mg heart of the LNP groups with different levels of DSPC and ionizable cationic lipids shown in Table 8 of Example 6 in CD-1 mice is shown.

[0091] Fig.15E The luminescence intensity / mg lung of the LNP groups with different levels of DSPC and ionizable cationic lipids shown in Table 8 of Example 6 in CD-1 mice is shown.

[0092] Fig.15F The luminescence intensity / mg small intestine of the LNP groups having different levels of DSPC and ionizable cationic lipids shown in Table 8 of Example 6 in CD-1 mice is shown.

[0093] Figure 15G The luminescence intensity / mg cecum of the LNP groups having different levels of DSPC and ionizable cationic lipids shown in Table 8 of Example 6 in CD-1 mice is shown.

[0094] Fig.15H The luminescence intensity / mg colon of the LNP groups having different levels of DSPC and ionizable cationic lipids shown in Table 8 of Example 6 in CD-1 mice is shown.

[0095] Fig.16 Shown is a cryo-TEM image of lipid nanoparticles composed of MF019 / DSPC / Chol / PEG-DMG (27.4 / 50 / 21.1 / 1.5 mol:mol) encapsulating mRNA encoding luciferase. DETAILED DESCRIPTION

[0096] The lipid nanoparticles described herein are an improvement over conventional four-component LNPs (referred to herein as Onpattro TM) is an improvement. In certain advantageous embodiments, the LNP is an improved formulation comprising an ionizable lipid, a phosphatidylcholine lipid; cholesterol and a hydrophilic polymer-lipid conjugate, and wherein the phosphatidylcholine lipid is present in a mol% of at least 20 mol% or at least 30 mol%, and the ionizable lipid is present in a mol% of less than 40 mol%. In this article, relative to the benchmark Onpattro TM formulations, with elevated phosphatidylcholine mol % over the mol % used in conventional nucleic acid delivery formulations, provide a surprising improvement in mRNA delivery to extrahepatic tissues.

[0097] The lipid nanoparticles described herein include a payload as messenger RNA. The term "messenger RNA" or "mRNA" used herein refers to a polynucleotide encoding and expressing at least one peptide, polypeptide or protein. The term includes but is not limited to small activation RNA (saRNA) and trans-amplification RNA (taRNA).

[0098] As used herein, referring to incorporating mRNA into lipid nanoparticles, the term "encapsulation" refers to any association of mRNA with any lipid component or compartment of the lipid nanoparticle. In one example of the present disclosure, the mRNA is present in the core of the LNP.

[0099] The concentration of mRNA in the LNP can be between 0.01 and 20 mg / mL, or between 0.01 and 10 mg / mL, or between 0.05 and 5 mg / mL, or between 0.075 and 4 mg / mL.

[0100] mRNA used herein includes both modified and unmodified mRNA. In one embodiment, mRNA comprises one or more coding regions and non-coding regions. Can be purified from natural sources, produced using a recombinant expression system and purified alternatively, or can be synthesized by chemosynthesis mRNA.

[0101] In those embodiments in which the mRNA is chemically synthesized, the mRNA may comprise nucleoside analogs, such as analogs with chemically modified bases or sugars and / or backbone modifications. In some embodiments, the mRNA is or comprises 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-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine); , 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0102] The mRNA disclosed herein can be synthesized according to any of a variety of known methods. For example, in certain embodiments, the mRNA can be synthesized by in vitro transcription (IVT). In short, IVT is generally performed using a linear or circular DNA template containing a promoter, a ribonucleotide triphosphate pool, 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 inhibitor.

[0103] In some embodiments, in vitro synthesized mRNA can be purified prior to encapsulation to remove unwanted impurities, including various enzymes and other reagents used during mRNA synthesis.

[0104] The present disclosure can be used to prepare mRNA of various lengths. In certain embodiments, the present disclosure can be used to prepare and encapsulate in vitro synthesized mRNA with a length range of about 250bp to 20kb, about 500bp to 20kb, about 1 to 15kb, about 1 to 10kb, about 5 to 20kb, about 5 to 15kb, about 5 to 12kb, about 5 to 10kb, about 8 to 20kb, or about 8 to 15kb.

[0105] Typically, mRNA synthesis involves the addition of a "cap" on the 5' end and a "tail" on the 3' end. The presence of the cap provides resistance to nucleases found in most eukaryotic cells. The presence of the "tail" helps protect the mRNA from exonuclease degradation.

[0106] 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 an iron response element. In some embodiments, the length of the 5' untranslated region can be between about 50 and 500 nucleotides.

[0107] In some embodiments, the 3' untranslated region includes one or more of the following: a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA at a location in the cell, or one or more binding sites for a miRNA. In some embodiments, the length of the 3' untranslated region may be between 50 and 500 nucleotides or longer.

[0108] Although, in certain embodiments, mRNA provided from an in vitro transcription reaction may be desirable, other sources of mRNA are contemplated, such as mRNA produced by bacteria, fungi, plants, and / or animals.

[0109] The mRNA sequence may contain a reporter gene sequence, although inclusion of a reporter gene sequence in a pharmaceutical formulation for administration is optional. These sequences can be incorporated into mRNA for in vivo studies in animal models to assess biodistribution. Phosphatidylcholine lipids

[0110] LNPs generally include one or more structural lipids, meaning amphiphilic lipids that allow particle formation and that generally have no net charge at physiological pH. The term includes zwitterionic lipids that have little charge at physiological pH, and includes phospholipids.

[0111] In certain embodiments, structured lipid is a phosphatidylcholine lipid selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dipalmitoylphosphatidylcholine (DPPC). The structured lipid content can include a mixture of two or more different structured lipids. In one embodiment, the phosphatidylcholine lipid content is a mixture of DSPC and DOPC and POPC. In such embodiments, the DSPC content of the mixture can be at least 20 or 30mol%.

[0112] In some embodiments, the structural lipid content is greater than 20mol%, greater than 25mol%, greater than 30mol%, greater than 32mol%, greater than 34mol%, greater than 36mol%, greater than 38mol%, greater than 40mol%, greater than 42mol%, greater than 44mol%, greater than 46mol%, greater than 48mol% or greater than 50mol%. In some embodiments, the upper limit of the auxiliary lipid content is 70mol%, 65mol%, 60mol%, 55mol%, 50mol% or 45mol%. The present disclosure also includes the sub-range of any combination of the aforementioned upper and lower numerical limits.

[0113] For example, in certain embodiments, the phosphatidylcholine lipid content is 20 mol% to 80 mol%, or 25 mol% to 60 mol%, or 30 mol% to 60 mol%, or 35 mol% to 60 mol%, or 40 mol% to 60 mol%, or 42 mol% to 58 mol%, or 43 mol% to 57 mol%, or 44 mol% to 56 mol%, or 45 mol% to 55 mol% of the total lipids present in the lipid nanoparticles.

[0114] In one embodiment, structural lipid is DSPC.For example, in certain embodiments, DSPC lipid content is 20mol% to 80mol%, or 25mol% to 60mol%, or 30mol% to 60mol%, or 35mol% to 60mol%, or 40mol% to 60mol%, or 42mol% to 58mol%, or 43mol% to 57mol%, or 44mol% to 56mol%, or 45mol% to 55mol% of the total lipid present in the lipid nanoparticle.

[0115] In certain embodiments, including sphingomyelin is undesirable (e.g., see Example 2). In certain embodiments, the sphingomyelin content of the lipid nanoparticle is less than 5mol%, less than 4mol%, less than 3mol%, less than 2mol%, less than 1mol%, less than 0.75mol%, or less than 0.5mol%. In certain embodiments, the LNP is "sphingomyelin-free", which means that there is no sphingomyelin or substantially no sphingomyelin in the LNP, which means that the sphingomyelin content is less than 5mol%.

[0116] In addition to the phosphatidylcholine lipids, LNPs may include additional structural lipids. For example, LNPs may include structural lipids with a net positive or negative charge at physiological pH. In general, the content of such lipids is less than 10 mol% or less than 5 mol%.

[0117] The structural lipid content was determined based on the total amount of lipids (including sterols) in the lipid nanoparticles. Ionizable lipids

[0118] LNP of the present disclosure has ionizable lipid.Ionizable lipid can be charged at low pH value, and does not have net charge substantially at physiological pH value.This allows electrostatic interaction between lipid and negatively charged nucleic acid payload during initial preparation.Since ionizable lipid is close to neutrality at physiological pH value, toxicity and renal clearance are reduced.After the cell uptake by endocytosis, the acidic environment of endosome causes the net positive charge of ionizable amino lipid to increase, thereby promoting the fusion of anionic lipid with endosomal membrane and subsequent membrane instability and releasing the therapeutic agent based on nucleic acid into cytoplasm to play its role.

[0119] In some embodiments, it is desirable to include less than 50 mol% ionizable lipids. That is, 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%.

[0120] In certain embodiments, the ionizable lipid content is 5 mol% to 50 mol%, or 8 mol% to 47 mol%, or 10 mol% to 50 mol%, or 15 mol% to 45 mol%, or 15 mol% to 35 mol% of the total lipid present in the lipid nanoparticle.

[0121] As used herein, the term "cationic lipid" refers to a lipid that is electrostatically neutral at a given pH value (e.g., physiological pH) and can accept or donate protons to be electrostatically positively charged, and the electrostatically neutral form has a calculated logarithm of the partition coefficient between water and 1-octanol (i.e., a cLogP) greater than 8. In some embodiments, the pKa of the cationic lipid is between 5.0 and 8.0.

[0122] In certain embodiments, the cationic lipid has an amino group. In some embodiments, the cationic lipid includes a protonable tertiary amine (e.g., pH titratable) head group, a C16 to C18 alkyl chain, a linkage between the head group and the alkyl chain, and 0 to 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 and KC2 type lipids, which are well known to those skilled in the art. In another embodiment, the ionizable lipid is selected from one or more lipids specified in the following: WO 2022 / 246555; WO 2022 / 246568; WO2022 / 24657; WO2022 / 155728; PCT / CA2023 / 050129 filed on January 31, 2023; U.S. Provisional Application No. 63 / 340,687 filed on May 11, 2022; U.S. Provisional Application No. 63 / 410,281 filed on September 27, 2022; U.S. Provisional Application No. 63 / 410,261 filed on September 27, 2022; U.S. Provisional Application No. 63 / 434,506 filed on December 22, 2022; U.S. Provisional Application No. 63 / 410,273 filed on September 27, 2022; and U.S. Provisional Application No. 63 / 445,854 filed on February 15, 2023, each of which is incorporated herein by reference.

[0123] 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 distal branching and / or one or more cyclic groups. Examples of ionizable cationic lipids comprising an ionizable amino head group and two lipophilic chains (at least one chain comprising one or more sulfur atoms and / or ester groups) are described in co-owned and co-pending U.S. Provisional Application No. 63 / 340,687, filed May 11, 2022; U.S. Provisional Application No. 63 / 410,281, filed September 27, 2022; U.S. Provisional Application No. 63 / 410,261, filed September 27, 2022; U.S. Provisional Application No. 63 / 434,506, filed December 22, 2022; and U.S. Provisional Application No. 63 / 410,273, filed September 27, 2022, each of which is incorporated herein by reference. Functional groups comprising one or more heteroatoms are capable of biodegradation in vivo.

[0124] In one embodiment, the ionizable cationic lipid has a protonatable amino head group; at least two lipophilic moieties, wherein the amino head group has a central nitrogen or carbon atom, each of the two lipophilic moieties being directly bonded to the central nitrogen or carbon atom; each lipophilic chain has a total of 15 to 40 carbon atoms; and wherein the lipid has (i) a pK between 6 and 7.5; a ; and (ii) a logP of at least 11.

[0125] Optionally, at least one lipophilic moiety bonded to the head group has a biodegradable group. In a non-limiting example, at least one lipophilic moiety has a formula of:

[0126] In one embodiment, R 1 and R 2 is independently a linear, cyclic or branched optionally substituted C3-C 20 alkyl, and optionally with varying degrees of unsaturation; and n is 4 to 8.

[0127] In certain embodiments, it is desirable to include less than 50mol% ionizable cationic lipids in LNP. That is, the ionizable cationic lipid content can be less than 50mol%, less than 45mol%, less than 40mol%, less than 35mol%, less than 30mol%, less than 25mol%, less than 20mol%, less than 15mol%, less than 10mol% or less than 5mol%.

[0128] In certain embodiments, the ionizable cationic lipid content is 5 mol% to 50 mol%, or 8 mol% to 47 mol%, or 10 mol% to 50 mol%, or 15 mol% to 45 mol%, or 15 mol% to 35 mol% of the total lipid present in the lipid nanoparticle.

[0129] The ionizable lipid component can include an ionizable anionic lipid as part of the ionizable lipid content. An example of such a lipid is cholesterol hemisuccinate (CHEMS). More examples of ionizable anionic lipids are described in the co-pending and co-owned U.S. Provisional Patent Application No. 63 / 453,766, filed on March 22, 2023, entitled "Ionizable Anionic Lipids", which is incorporated herein by reference in its entirety. Sterols

[0130] In some embodiments, the LNP further comprises a sterol.The term "sterol" refers to a naturally occurring or synthetic compound having a gonane backbone and having a hydroxyl moiety attached to one of its rings (usually the A ring).

[0131] Examples of sterols include cholesterol or cholesterol derivatives, the latter being cholesterol molecules having a sterane structure and one or more additional functional groups.

[0132] Examples of derivatives include β-sitosterol, 3-sitosterol, campesterol, stigmasterol, fucoxanthol, or stigmasterol, dihydrocholesterol, an enantiomer of cholesterol (ent-cholesterol), epi-cholesterol, streptosterol, 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-oxa-cholesterol, 23-oxa-cholesterol, 24-oxa-cholesterol, Cycloartenol, 22-ketosterol, 20-hydroxycholesterol, 7-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, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, photosterol, sitolcalciferol, calcipotriol, fecosterol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, yeast sterol, diosgenin, fucosterol, fecosterol or their salts or esters.

[0133] In one embodiment, the sterol is present at 15 mol % to 50 mol %, 18 mol % to 45 mol %, 20 mol % to 45 mol %, 25 mol % to 45 mol %, or 30 mol % to 45 mol % based on the total lipids present in the lipid nanoparticle.

[0134] In another embodiment, the sterol is cholesterol and is present at 15 mol % to 50 mol %, 18 mol % to 45 mol %, 20 mol % to 45 mol %, 25 mol % to 45 mol %, or 30 mol % to 45 mol % based on the total lipids present in the lipid nanoparticles.

[0135] In another embodiment, the sterol is a cholesterol derivative, and the cholesterol derivative is present at 15 mol % to 50 mol %, 18 mol % to 45 mol %, 20 mol % to 45 mol %, 25 mol % to 45 mol %, or 30 mol % to 45 mol % based on the total lipids present in the lipid nanoparticles.

[0136] In one embodiment, the combined (i) sterol content (e.g., cholesterol or a cholesterol derivative thereof); and (ii) neutral lipid content is at least 50 mol%, at least 55 mol%, at least 60 mol%, at least 65 mol%, at least 70 mol%, at least 75 mol%, at least 80 mol% or at least 85 mol%, based on the total lipid present in the lipid nanoparticle. Hydrophilic polymer-lipid conjugates

[0137] In one embodiment, lipid nanoparticles include a hydrophilic polymer lipid conjugate that can be incorporated into the LNP. The conjugate includes a lipid that forms a vesicle with a polar head group, and a hydrophilic polymer chain that is covalently attached to the head group. The example of a hydrophilic polymer includes polyethylene glycol (PEG), polyvinyl pyrrolidone, polyvinyl methyl ether, polyhydroxypropyl methacrylate, polyhydroxypropyl methacrylamide, polyhydroxyethyl acrylate, polymethacrylamide, polydimethylacrylamide, polymethyl oxazoline, polyethyl oxazoline, polyhydroxyethyl oxazoline, polyhydroxypropyl oxazoline, polysarcosine and polyaspartamide (polyaspartamide). In another embodiment, the hydrophilic polymer-lipid conjugate is a PEG-lipid conjugate. The hydrophilic polymer lipid conjugate can also be a naturally occurring or synthetic molecule containing oligosaccharides, such as monosialoganglioside (G M1 ). The ability of a given hydrophilic polymer lipid conjugate to enhance the circulation life of the LNPs herein can be readily determined by one skilled in the art using known methods.

[0138] The hydrophilic polymer lipid conjugate can be present in the nanoparticle at 0.5mol% to 5mol%, or 0.5mol% to 3mol%, or 0.5mol% to 2.5mol%, or 0.5mol% to 2.0mol%, or 0.5mol% to 1.8mol% of the total lipid. In certain embodiments, the hydrophilic polymer lipid conjugate can be present in the nanoparticle at 0mol% to 5mol%, or 0mol% to 3mol%, or 0mol% to 2.5mol%, or 0mol% to 2.0mol%, or 0mol% to 1.8mol% of the total lipid.

[0139] In another embodiment, the PEG-lipid conjugate can be present in the nanoparticle with 0.5mol% to 5mol%, or 0.5mol% to 3mol%, or 0.5mol% to 2.5mol%, or 0.5mol% to 2.0mol%, or 0.5mol% to 1.8mol% of the total lipid. In certain embodiments, the PEG-lipid conjugate can be present in the nanoparticle with 0mol% to 5mol%, or 0mol% to 3mol%, or 0mol% to 2.5mol%, or 0mol% to 2.0mol%, or 0mol% to 1.8mol% of the total lipid. Nanoparticle formulation and morphology

[0140] Delivery vectors incorporating mRNA can be prepared using various suitable methods (e.g., rapid mixing / ethanol dilution method). 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, each of which is incorporated herein by reference in its entirety.

[0141] Without being bound by theory, the mechanism by which lipid nanoparticles containing encapsulated mRNA can be formed using the rapid mixing / ethanol dilution method can be assumed to start from the formation of a dense region of a hydrophobic mRNA-ionizable lipid core at low pH (e.g., pH 4), which is surrounded by a monolayer of auxiliary lipids / cholesterol, and as ionizable cationic lipids are converted to neutral forms, the pH value rises, and auxiliary lipids / cholesterol fuse with smaller empty vesicles. As the ratio of bilayer auxiliary lipids increases, bilayer lipids gradually form bubbles, and ionizable lipids migrate to the internal hydrophobic core. At sufficiently high auxiliary lipid content, the outer bilayer of the preference auxiliary lipid can form a complete lipid layer (e.g., continuous or discontinuous bilayer) around the internal trapped volume (trapped volume).

[0142] LNP can comprise " core " area. Surprisingly, it has been observed that the core is non-uniform because it includes electron-dense area and aqueous part or compartment visualized by cryo-EM microscopy. In some embodiments, the core can be characterized as non-solid. Without limitation, as observed by cryo-EM, the electron-dense area in the core can be partially surrounded by aqueous part or compartment in the closed space. The aqueous part can form a distinct aqueous region or compartment in the lipid nanoparticle. In other words, it is believed that the aqueous part or compartment is not just a hydration layer. Such non-solid core particles are described in co-owned and co-pending WO 2022 / 251959, the contents of which are incorporated herein by reference.

[0143] In one embodiment, at least about one-fifth of the core (capture volume) contains an aqueous portion or compartment as qualitatively determined by cryo-EM, and wherein the electron-dense region within the core is partially continuous with the lipid layer comprising the bilayer. In one embodiment, at least about one-quarter of the core contains an aqueous portion or compartment as qualitatively determined by cryo-EM, and wherein the electron-dense region within the core is also partially continuous with the lipid layer comprising the bilayer. In one embodiment, at least about one-third of the core contains an aqueous portion or compartment as qualitatively determined by cryo-EM, and wherein the electron-dense region within the core is also partially continuous with the lipid layer comprising the bilayer. In another embodiment, at least about one-half of the core contains an aqueous portion or compartment as qualitatively determined by cryo-EM, and wherein the electron-dense core is partially continuous with the lipid layer comprising the bilayer.

[0144] In another embodiment, the electron dense region of the LNP surprisingly appears to be completely surrounded by the aqueous portion of the core as visualized by cryo-EM. This morphology is observed in a single plane, and a portion of the observed electron dense region is continuous with the lipid layer (e.g., bilayer), but cannot be seen because the portion is not in the plane that can be visualized.

[0145] In one embodiment, the electron dense region is generally spherical in shape. In another embodiment, the electron dense region is hydrophobic.

[0146] The lipid nanoparticles herein can exhibit particularly high capture efficiency of mRNA. Thus, in one embodiment, the capture efficiency is at least 50, 55, 60, 65, 70, 75, 80, 85 or 90%.

[0147] In one embodiment, the mRNA is at least partially encapsulated in an electron-dense region. For example, in one embodiment, at least 50, 60, 70 or 80 mol% of the mRNA is encapsulated in an electron-dense region. In another embodiment, at least 50, 60, 70 or 80 mol% of the ionizable lipids are in an electron-dense region.

[0148] In another embodiment, the mRNA and the cationic lipid are present in the electron dense region. In yet another embodiment, the helper lipid is present in the lipid layer comprising the bilayer.

[0149] In some embodiments, the lipid nanoparticle may comprise a single bilayer, or may be a combination of bilayers and monolayers. In one embodiment, the lipid layer is a continuous bilayer surrounding the core.

[0150] In certain embodiments, the electron dense region of the core is separated from the lipid layer comprising the bilayer by an aqueous portion or compartment. For example, the present disclosure provides a lipid nanoparticle formulation comprising a plurality of lipid nanoparticles, in which at least 20%, 30%, 40%, 50%, 60% or 70% of the particles contain a core having an electron dense region and an aqueous portion or compartment as determined by cryo-EM microscopy, and wherein the aqueous portion or compartment is partially surrounded by a lipid layer comprising a bilayer, as visualized by cryo-EM microscopy.

[0151] In another embodiment, without limitation, the present disclosure provides a lipid nanoparticle formulation comprising a plurality of lipid nanoparticles in which generally at least 10%, 20%, 30%, 40%, 50%, 60% or 70% of the particles have an elongated shape (e.g., generally elliptical) as qualitatively determined by cryo-EM microscopy. In the latter embodiment, the electron dense region of the nucleus may be partially surrounded by an aqueous space as visualized by cryo-EM microscopy.

[0152] In one embodiment, the lipid nanoparticle is part of a lipid nanoparticle formulation, and wherein at least 20% of the electron dense region of the lipid nanoparticle is (i) encapsulated by the aqueous portion, or (ii) partially surrounded by the aqueous portion, and wherein a portion of the periphery of the electron dense region is continuous with the lipid layer, as visualized in a single plane by cryo-EM microscopy.

[0153] In certain embodiments, the present disclosure provides lipid nanoparticle preparations comprising a plurality of lipid nanoparticles, wherein typically at least 10%, 20%, 30%, 40%, 50%, 60% or 70% of the particles have a core comprising an electron-dense region as determined by cryo-EM microscopy, which is continuous with a lipid layer comprising a bilayer as visualized by cryo-EM microscopy.

[0154] In another embodiment, without limitation, the present disclosure provides a lipid nanoparticle preparation comprising a plurality of lipid nanoparticles, wherein generally at least 10%, 20%, 30%, 40%, 50%, 60% or 70% of the particles have a core comprising an electron-dense region that appears to be surrounded or encapsulated by a continuous aqueous space disposed between a lipid layer (e.g., a bilayer) and the electron-dense region, as visualized in one plane by cryo-EM microscopy.

[0155] As described in the Examples section below, LNPs were visualized by cryo-TEM.

[0156] In another embodiment, the polydispersity index (PDI) of the LNP formulation is less than 0.2, 0.15, 0.12 or 0.10.

[0157] In another embodiment, the particle size distribution is such that at least 90% of the particles in the LNP formulations of the present disclosure have a diameter between 40 nm and 200 nm, between 45 and 150 nm, or between 50 and 140 nm.

[0158] Lipid nanoparticles herein can show particularly high nucleic acid encapsulation efficiency.As used herein, mentioning that nucleic acid is incorporated into lipid nanoparticles, term "encapsulation" refers to any association of nucleic acid with any lipid component or compartment (including lipophilic or aqueous part) of lipid nanoparticles.In one embodiment, nucleic acid is present at least in the core of LNP.

[0159] In one embodiment, the encapsulation efficiency is at least 50, 55, 60, 65, 70, 75, 80, 85, 90% or 92%.The encapsulation efficiency of nucleic acids is determined as described in the Materials and Methods section of the Examples herein.

[0160] The disclosed embodiments also provide lipid nanoparticles, which are described according to the molar ratio between the positively charged amine groups (N) and the negatively charged phosphate groups (P) of the amino lipids of the oligonucleotide to be encapsulated. This can be represented mathematically by the equation N / P. In one embodiment, the N / P ratio of the lipid nanoparticle is between 4 and 15, or between 4.5 and 10, or between 5 and 10, or between 5.5 and 8.

[0161] In one embodiment, the N / P ratio of the lipid nanoparticle is at least 4, 4.25, 4.50, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0 or 6.25. The upper limit can be 15, 14, 13, 12, 11, 10, 9 or 8. The present disclosure also includes any two combinations of the upper and lower limits.

[0162] In one embodiment, the weight nucleic acid / micromole of total lipid of the lipid nanoparticle is 0.05: 1 to 1: 1. In one embodiment, the lower limit is 0.06: 1, 0.08: 1, 0.10: 1, 0.12: 1, 0.14: 1, 0.16: 1, 0.18: 1, 0.20: 1, 0.22: 1, 0.24: 1, 0.26: 1, 0.28: 1, 0.30: 1, 0.32: 1, 0.34: 1, 0.36: 1, 0.38: 1 or 0.40: 1 weight nucleic acid / micromole of total lipid. In another embodiment, the upper limit is micromoles of nucleic acid weight / total lipid of 0.80: 1, 0.82: 1, 0.84: 1, 0.86: 1, 0.88: 1, 0.90: 1, 0.92: 1, 0.94: 1, 0.96: 1, or 0.98: 1. The present disclosure also includes combinations of any two of the upper and lower limits.

[0163] In one embodiment, the mRNA copy number / LNP is 1-10 or 4-8. Improved gene expression in extrahepatic organs or tissues

[0164] As used herein, "expression" of mRNA refers to the translation of the mRNA into a peptide (e.g., an antigen), polypeptide, or protein (e.g., an enzyme), and may also include post-translational modifications of the peptide, polypeptide, or fully assembled protein (e.g., an enzyme) as indicated in the context.

[0165] In one embodiment, the lipid nanoparticles exhibit at least a 10% increase in gene expression of mRNA in vivo, as measured in an extrahepatic tissue or organ.

[0166] In another embodiment, the extrahepatic tissue or organ is spleen, bone marrow, lung, kidney, heart, abdominal skin, dorsal skin, and / or ear.

[0167] As described in Example 1, the previous Onpattro for mRNA delivery TM Compared with the preparation, LNP of the present disclosure can provide improved mRNA delivery to a wider range of tissues, including but not limited to delivery to the spleen and / or bone marrow. Whether lipid particles show this enhanced delivery to a given tissue or organ can be determined by biodistribution studies in an in vivo mouse model. In such embodiments, enhanced green fluorescent protein (eGFP) can be used to detect mRNA expression in a given tissue or organ. In particular, according to such embodiments, an LNP mRNA system encapsulating mRNA encoding eGFP is prepared, and biodistribution and GFP expression in cell populations in the spleen and bone marrow are evaluated using flow cytometry after systemic administration.

[0168] To evaluate whether a given lipid nanoparticle exhibits increased gene expression in relevant tissues or organs 12 hours, 24 hours, 48 ​​hours, or 3 days after injection, the mRNA-LNP of the present disclosure was compared with the Onpattro TM The two LNPs being compared were subjected to the same experimental methods and materials to determine the in vivo expression specified in Example 1.

[0169] In one embodiment, relative to Onpattro® using a mixture of nor-MC3 / DSPC / cholesterol / PEG-lipid at 50 / 10 / 38.5 / 1.5 (mol:mol), TM The lipid nanoparticles of the type formulation encapsulate eGFP-mRNA, as measured in vivo in spleen and / or bone marrow immune cell population subsets at 24 hours and / or 3 days after injection, the lipid nanoparticles exhibit at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290% or 300% increase in gene expression of the encapsulated mRNA encoding enhanced GFP (eGFP). Measured using flow cytometry and gene expression, determined by quantifying the % positive cell count in a given immune cell subset as shown in Example 1. By comparing the percentage of positive cells (i.e., detection of eGFP) in a given cell type in the spleen or bone marrow and comparing this percentage to that of Onpattro injected using otherwise identical materials and methods. TM The percentage increase was determined by the percentage of positive cells produced by the preparation.

[0170] In one embodiment, the selected immune cell subset is from the spleen and is selected from one or more of monocytes / macrophages, neutrophils, B cells, CD4 T cells and / or CD8 T cells. In one embodiment, the immune cell subsets for quantitative eGFP expression are from the spleen and are monocytes / macrophages (one or two cell types), and the increased expression is measured by measuring the positive cell count (% eGFP positive) by flow cytometry 24 hours or 3 days after injection. In another embodiment, the immune cell subsets in the spleen are monocytes / macrophages, and the positive cell count (% eGFP positive) is measured 3 days after injection.

[0171] In one embodiment, the selected immune cell subsets are from bone marrow and are selected from one or more of erythroid cells, B cells, T cells, monocytes / macrophages and neutrophils. In one embodiment, the immune cell subsets quantitatively expressed are from bone marrow and are monocytes / macrophages or T cells, and the increased expression is measured by measuring the positive cell counts (% eGFP positive) by flow cytometry 24 hours or 3 days after injection. In another embodiment, the immune cell subsets in the spleen are monocytes / macrophages or T cells, and the positive cell counts (% eGFP positive) are measured 3 days after injection. Clinical and nonclinical applications of LNP in this article

[0172] In some embodiments, the lipid nanoparticles comprising mRNA are part of a pharmaceutical composition and are used to treat and / or prevent disease conditions. Treatment can provide preventive (preventive), ameliorative or therapeutic benefits. The pharmaceutical composition will be administered at any suitable dosage.

[0173] The LNPs described herein can be used to treat and / or prevent any disease, disorder or condition in a mammalian subject. This includes diseases, disorders or conditions such as cancer, infectious diseases (e.g., bacterial, viral, fungal or parasitic infections), inflammatory diseases and / or autoimmune diseases, including the induction of immune tolerance and the treatment of cardiovascular diseases (e.g., hypertension, arrhythmias and restenosis).

[0174] Examples of cancer include lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, stomach cancer (stomach / gastric cancer), esophageal cancer; gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer, kidney cancer (e.g., renal cell carcinoma), central nervous system cancer, glioblastoma, skin cancer, lymphoma, choriocarcinoma, head and neck cancer, osteogenic sarcoma, and blood cancer. Non-limiting examples of specific types of liver cancer include hepatocellular carcinoma (HCC), secondary liver cancer (e.g., caused by metastasis of other non-liver cancer cell types), and hepatoblastoma.

[0175] Non-limiting examples of other diseases, disorders, or conditions that can be treated by the mRNA-LNPs herein and that can be attributable, at least in part, to immune diseases include colitis, Crohn's disease, allergic encephalitis, allogeneic transplant / graft vs. host disease (GVHD), diabetes, and multiple sclerosis.

[0176] In addition to treating and / or preventing diseases or conditions, the LNPs herein can also be used in other applications. LNPs can be used to treat aging, preventive medicine, and / or as part of a personalized medicine regimen. In yet another embodiment, LNPs are used for diagnostic applications.

[0177] In one embodiment, the LNP is part of a pharmaceutical composition that is administered parenterally (i.e., intra-arterially, intravenously, subcutaneously, or intramuscularly). In yet another embodiment, the pharmaceutical composition is for intratumoral administration. In another embodiment, the pharmaceutical composition is administered intranasally, intravitreally, subretinally, intrathecally, or by other topical routes.

[0178] The pharmaceutical composition includes a pharmaceutically acceptable salt and / or an excipient.

[0179] The compositions described herein can be administered to a patient. The term patient as used herein includes human or non-human subjects.

[0180] The following examples are intended to illustrate the preparation of specific lipid nanoparticle mRNA formulations and their properties, but are in no way intended to limit the scope of the invention. Example Materials and methods LNP formulation

[0181] LNPs were prepared by dissolving mRNA in 25 mM sodium acetate (pH 4.0), while the lipid components at the specified mole % were dissolved in anhydrous ethanol. Lipids in ethanol and eGFP mRNA in buffer were mixed in a volume ratio of 1:3 using a T-junction with a dual syringe. The solution was pushed through the T-junction at a combined flow rate of 20 mL / min (5 mL / min for the syringe containing lipids and 15 mL / min for the syringe containing mRNA). The mixture was then dialyzed overnight in approximately 100 volumes of 1× phosphate-buffered saline (pH 7.4) using a Spectro / Por dialysis membrane (molecular weight cutoff 12000-14000 Da). Dialysis was performed as needed using an Amicon Ultra TM The LNP was concentrated in a 10000 MWCO (molecular weight cut-off) regenerated cellulose concentrator.

[0182] By measuring the i ) Add RiboGreen TM The unencapsulated mRNA content was determined by fluorescence after PCR and compared with that of 2% Triton X-100 (F t ) The total mRNA content obtained after cleavage of LNPs was used to calculate the encapsulation efficiency: % encapsulation = (F t –F i ) / F t ×100.

[0183] Using the ZetasizerNano ZS TM Characterize particle size and polydispersity index (PDI). Flow cytometry in vivo studies

[0184] LNPs with an eGFP mRNA concentration of 0.1 mg / mL were injected intravenously (iv) into mice in one volume using the mouse's formula weight (unit: gram)*10 μL. Spleen and bone marrow were harvested 4 hours, 1 day, and 3 days after LNP injection.

[0185] Harvest spleen and bone marrow and process into single cell suspension.Particularly, use 5% isoflurane anesthesia mice until reflex loss, then expose to CO containing 1% air 2 in.Separate spleen and femur.By homogenization and by 70 μ m sieve spleen is processed into single cell suspension.By centrifugal skeleton 30s when 3810g, separate bone marrow from femur, and be resuspended in FACS buffer (1x sterile PBS (pH 7.4), 2.5mM ethylenediaminetetraacetic acid (EDTA), 0.05% (w / v) sodium azide (NaN 3 ), 2% (v / v) bovine serum albumin (BSA)).

[0186] After separation, spleen and bone marrow cells were stained using the ThermoFisher Countess II TM One million cells were counted and added to the wells of a 96-well round-bottom plate and the volume in each well was increased to 200 μL using FACS buffer. The cells were centrifuged at 484 g for 5 minutes at 4°C and the liquid was discarded. Subsequently, the cells were incubated with a solution containing the antibody for 45 minutes. The volume was increased to 200 μL and the cells were centrifuged at 484 g for 5 minutes at 4°C and the liquid was discarded. A volume of 100 μL of propidium iodide (PI) was added at a 1:5,000 dilution (1 mg / mL stock solution) and the stained single cells were introduced into a flow cytometer (Cytoflex TM , Becman Coulter TM ). Generate a compensation matrix that is applied to all samples using the single color setting.

[0187] Using FlowJo TM Version 10 (Becton Dickension TM & Company (BD)) analyzed flow cytometry data. The corresponding immune cell subsets were identified based on the gating schemes shown in Tables 2 and 3 above. Prism TM Version 8 (GraphPad TM ) software to generate eGFP positive (eGFP+ ) cells and bar graphs of the mean fluorescence intensity (MFI) of eGFP. The gating schemes for spleen and bone marrow are shown in Tables 1 and 2 below. Tissue homogenate assay

[0188] Tissues were removed from mice and placed in 2 mL tubes and snap frozen in liquid nitrogen. Tissues were then stored at -80°C. An appropriate volume of Promega TM GLO TM Lysis buffer, ensuring that the sample remains frozen before adding the lysis buffer. Place the sample in the FastPrep TM The homogenizer was run at a speed of 6 m / s for 20 seconds and repeated 2 times for a total of three rounds. The homogenized sample was rotated at 12,000 rpm for 10 minutes at room temperature, and then 50 μL of the homogenate was added to a black plate in duplicate. The plate was transferred to a plate reader and the fluorescence was read at 640 nm excitation / 720 nm emission. By adding 50 μL of Steady Glo TM The luciferase signal is read using a luciferase substrate to measure luminescence. Example 1: Significant increase in extrahepatic mRNA gene expression in immune cell subsets using high DSPC LNPs

[0189] The effect of increasing the amount of DSPC from 10 mol% to 50 mol% in a four-component LNP containing an enhanced green fluorescent protein mRNA ("eGFP mRNA") loading was evaluated in vivo. TM formulation, LNPs containing elevated levels of DSPC relative to Onpattro TM The formulations showed significant increases (>>10%) in gene expression in macrophages, monocytes and / or T cells harvested from the spleen and / or bone marrow harvested at 24 hours and 3 days after injection.

[0190] In particular, in this example, the following two four-component eGFP mRNA preparations (reported in mol %) containing ionizable lipid, DSPC, cholesterol and PEG-lipid were compared. Table 1: Formulations containing eGFP mRNA examined in vivo

[0191] lcLNP of the present invention in Table 1 TM The DSPC content of Onpattro was increased to 50 mol% at the expense of both ionizable lipids and cholesterol. TMThe ionizable lipid included in the LNPs is nor-MC3 (nMC3) described in co-owned and co-pending WO2022 / 246571, and the ionizable lipid in the formulation of the invention comprising 50 mol% DSPC LNPs is MF019 described in WO 2022 / 155728A1 (each of which is incorporated herein by reference). 10 mol% DSPC (Onpattro TM 50 mol% DSPC preparation of the present invention (lcLNP) TM ) has a nitrogen and phosphate ratio (N / P) of 6 and 9 respectively.

[0192] Onpattro TM The size of the lcLNP was 38.4 nm, the polydispersity index (PDI) was 0.087, and the capture efficiency was 88.4%. The formulation of the present invention (lcLNP) containing 50 mol% DSPC TM ) size is 78.9 nm, PDI is 0.052, and capture efficiency is 64.4%.

[0193] In this example, flow cytometry was used to investigate the expression of eGFP mRNA in immune cell subsets in the bone marrow and spleen of C57BL / 6J mice at different time points after injection. The gating scheme for flow cytometry of spleen and bone marrow cells is shown in Tables 2 and 3 below. Table 2: Gating scheme for spleen

[0194] Live immune (CD45 + )Gating on cells: Groups in the spleen Markers Monocytes / macrophages <![CDATA[CD19 - TCRb - CD11b + Ly6G - ]]> Neutrophils <![CDATA[CD19 - TCRb - CD11b + Ly6G + ]]> B cells <![CDATA[CD19 + ]]> CD4 T cells <![CDATA[TCRb + CD4 + ]]> CD8 T cells <![CDATA[TCRb + CD8 + ]]> Table 3: Gating scheme for bone marrow

[0195] Gating on singlets: Groups in the bone marrow Markers Erythroid cells* <![CDATA[Ter119 + ]]> B cells <![CDATA[Ter119 - CD45 + CD19 + ]]> T cells <![CDATA[Ter119 - CD45 + TCRb + ]]> Monocytes / macrophages <![CDATA[Ter119 - CD45 + CD19 - TCRb - CD11b + Ly6G - ]]> Neutrophils <![CDATA[Ter119 - CD45 + CD19 - TCRb - CD11b + Ly6G + ]]> *After gating on erythroid cells, live immune cells were gated as CD45 + PI - cell.

[0196] The results of mRNA expression detected in spleen subcellular populations are as follows Figure 1A -F and Figure 2A -C.

[0197] Results of mRNA expression in bone marrow Figure 3A -F as shown.

[0198] It can be seen that Figure 1AThe results in Figure 4-C show that at 4 hours, 24 hours and 3 days, the Onpattro TM Formulation, using lcLNPs containing GFP mRNA with 50 mol% DSPC TM Monocyte and macrophage subsets in the spleen of treated mice showed an increase of 20% or more in GFP-positive cells. Similar results were observed at the same time points when the data were plotted using the measured MFI of GFP ( Figure 1D -F).

[0199] Figures 2A-2C Shows Figure 1B and 1E Representative scatter plots of the data shown depict eGFP expression levels at single-cell resolution in the monocyte / macrophage population in the spleen 24 hours after injection. For better visualization, mRNA-lcLNPs containing 50 mol% DSPC TM With significantly more mRNA-Onpattro TM eGFP + Cells. mRNA-lcLNP TM eGFP + Cellular mRNA-Onpattro TM Twice as much. TM The number of eGFP-positive cells was 18.5%, and the lcLNP TM is 39.3%).

[0200] Similarly, Figure 3A -C shows that in the bone marrow 24 hours and 3 days after injection, the expression of Onpattro TM Preparation, monocytes and macrophage subsets of mice treated with mRNA-lcLNPs with 50 mol% DSPC showed an increase of 20% or more in green fluorescent protein-positive cells (GFP + When the data were plotted using the measured MFI of GFP, similar results were observed at 24 hours and 3 days ( Figure 3D -F). In addition, relative to Onpattro TM The T cell subsets in the bone marrow of mice treated with mRNA-lcLNPs containing 50 mol% DSPC showed an increase of 20% or more in green fluorescent protein (GFP) at time points greater than 24 hours. + Cells) positive cells ( Figure 3B , 3C , 3E and 3F).

[0201] Figures 4A-4C Shows Figure 3B and 3E A representative scatter plot of the data shown depicts the level of eGFP expression at single-cell resolution in a population of bone marrow T cells 24 hours after injection. It can be better visualized that mRNA-lcLNPs are expressed in a population of T cells at 24 hours post-injection. TM eGFP + Cells relative to mRNA-Onpattro TM 5 times more. (Onpattro TM The number of eGFP-positive cells was 2.98%, and the lcLNP with 50 mol% DSPC TM is 19.5%). Example 2: Significant increase in mRNA gene expression in immune cell subsets using high DSPC LNPs incorporated with various ionizable lipids

[0202] Example 1 shows that increasing the DSPC content in a four-component mRNA-LNP system containing the ionizable sulfur lipid MF019 leads to a significant increase in extrahepatic mRNA expression. To examine the effect of increasing DSPC in a four-component LNP system containing different ionizable lipids, the same formulation as in Example 1 was examined for mRNA gene expression in the spleen and bone marrow, but instead the ionizable lipid MC3 was incorporated (DLin-MC3-DMA).

[0203] As discussed below, similar trends in mRNA expression in bone marrow and spleen were observed with increasing levels of DSPC, but the ionizable lipid in the preparation was MC3.

[0204] Two four-component eGFP mRNA formulations containing MC3 ionizable lipid, DSPC, cholesterol and PEG-lipid were compared. Table 4: MC3 ionizable lipid formulations containing eGFP mRNA examined in vivo *MC3 is an ionizable lipid DLin-MC3-DMA

[0205] As can be seen in the bar graph in Figure 5, at 24 hours, the TM Formulation, using lcLNPs containing GFP mRNA with 50 mol% DSPC TM T cells, monocyte and macrophage subsets, and neutrophils in the bone marrow of treated mice showed an increase of 20% or more in GFP-positive cells. The same general trend was observed when the data were plotted using the measured MFI of GFP ( Figure 5B ).

[0206] Figures 6A-6C Shows Figure 5A and 5B Representative scatter plots of the data shown depict eGFP expression levels of T cell populations in the bone marrow at single-cell resolution. For better visualization, mRNA-lcLNPs containing 50 mol% DSPC TM ( Figure 6C ) has significantly more mRNA-Onpattro TM ( Figure 6B ) eGFP-positive cells. mRNA-lcLNP TM eGFP-positive cells are mRNA-Onpattro TM At least four times ( Figure 6B shows that only 4.75% of cells were positive for eGFP, whereas Figure 6C 18.3% in the survey).

[0207] Figure 7 is a bar graph showing the difference in the expression of Onpattro with only 10 mol% DSPC at 24 hours. TM Formulation, using lcLNPs with 50 mol% DSPC containing GFP mRNA TM The monocyte and macrophage (mono / macs) subsets in the spleen of treated mice showed an increase of about 2-fold or more in GFP-positive cells.

[0208] Figures 8A-8C Shows Figure 7 Representative scatter plots of the data shown depict eGFP expression levels at single-cell resolution in the monocyte / macrophage population in the spleen. mRNA-lcLNPs containing 50 mol% DSPC TM ( Figure 8C ) has significantly more mRNA-Onpattro TM ( Figure 8B ) eGFP-positive cells. mRNA-lcLNP TM The number of eGFP-positive cells was higher than that of mRNA-Onpattro TM At least twice as much. Figure 8B In the , 5.78% of cells were positive, while in Figure 8C 12.4% of cells were eGFP positive). Example 3: Use of high DSPC LNPs but not sphingomyelin significantly increases mRNA gene expression in immune cell subsets

[0209] The effect of increasing the content of another neutral lipid, egg sphingomyelin (ESM), on mRNA expression in vivo was evaluated using the same formulation as in Examples 1 and 2 (but using ESM instead of DSPC). Surprisingly, the increase in mRNA expression in extrahepatic tissues with high DSPCLNPs was not observed with LNPs with elevated levels of ESM.

[0210] The formulations examined are shown in Table 5 below. Table 5: Formulations containing eGFP mRNA examined in vivo

[0211] It can be seen that Fig. 9A The bar graph in Figure 2 shows that lcLNPs containing GFP mRNA with 50 mol% DSPC increased 24 hours after injection relative to the same formulation with egg sphingomyelin (ESM) as the neutral lipid. TM Cell subsets in the bone marrow of treated mice (erythroid cells, B cells, T cells, monocytes / macrophages, and neutrophils) showed an increase of >>20% or more in GFP-positive cells in most cell types measured. A similar overall trend was observed when the data were plotted using the measured MFI of GFP ( Fig. 9B ).

[0212] Fig. 10A and 10B Shows Fig. 9A and 9B Representative scatter plots of the data shown depict eGFP expression levels of T cell populations in the bone marrow at single-cell resolution. For better visualization, mRNA-lcLNPs containing 50 mol% DSPC TM ( Fig. 10A ) of eGFP-positive cells than those of egg sphingomyelin (ESM) preparations ( Fig. 10B ) about 4 times more (eGFP in lcLNPTM + is 18.3, while ESM LNP is only 4.26).

[0213] Fig.11A is a bar graph showing the effects of lcLNPs containing GFP mRNA with 50 mol% DSPC relative to an otherwise identical formulation containing ESM at 24 hours. TM Various subpopulations in the spleen of treated mice showed an increase in GFP-positive cells.

[0214] Compared with ESM LNP, DSPC LNP increased the number of EGF mRNA-positive cells in monocytes and macrophages by about 6 times ( Fig.11A). The mean fluorescence intensity (MFI; Fig. 11B ), the same general trend was observed.

[0215] Fig. 12A and 12B A representative scatter plot of the data shown in Figure 11 is shown, which depicts the eGFP expression level of the monocyte / macrophage population in the spleen at single cell resolution. mRNA-lcLNP containing 50 mol% DSPC TM ( Fig. 12A ) showed significantly more eGFP-positive cells than the same preparation in which egg sphingomyelin was used instead of DSPC ( Fig. 12B ). mRNA-lcLNP containing DSPC TM The number of eGFP-positive cells was at least 20-fold greater than that of ESM LNPs. Example 4: Significant increase in mRNA gene expression and reduction in liver toxicity using high DSPC LNPs in multiple tissues and organs

[0216] This example shows that at 24 hours after injection, the TM Type preparations with elevated levels of DSPC showed increased expression of mRNA in spleen, lung, kidney, heart, bone marrow, abdomen, back, and ear.

[0217] The LNPs in Table 6 below were examined. Table 6: Relative to Onpattro TM The expression of LNPs in the liver, spleen, lung, kidney, heart, bone marrow, abdomen, back, and ear was examined.

[0218] The N / P of each preparation is 9, and the control is phosphate buffered saline (PBS). mRNA encodes luciferase, LNP is administered to CD-1 mice at 1 mg / kg and 5 mg / kg, and luciferase is quantified in tissue homogenate 24 hours after administration. The ionizable cationic lipid MF019 is described in WO 2022 / 155728A1. The ionizable cationic lipid NTx-C16 has the following structure:

[0219] Fig.13A and 13B lcLNPs with greater than 50 mol% DSPC in each extrahepatic organ examined at doses of 1 mg / kg and 5 mg / kg, respectively TM Samples were mixed with Onpattro with 10 mol% DSPC TMThe fold change of the type preparation was greater than 1.

[0220] Fig. 13C The lcLNPs shown in Table 6 above TM Samples and Onpattro TM Serum activities (U / L) of enzymes related to liver toxicity. In particular, the graph shows the levels (U / L) of liver serum enzymes alanine aminotransferase (ALT) (left) and aspartate aminotransferase (AST) (right) 24 hours after injection of CD-1 mice at a dose of 5 mg / kg of LNP-mRNA encoding luciferase. TM The ALT and AST activities (U / L) in serum increased after injection of 50 mol% DSPC in two lcLNPs. TM After the sample, their activity was relatively low. ALT and AST are markers of liver toxicity, so it indicates that relative to Onpattro TM , LNPs with elevated phosphatidylcholine showed reduced toxicity in the liver. Example 5: mRNA gene expression of LNPs with elevated levels of different phosphatidylcholine lipids

[0221] The following in vivo data show that, compared with Onpattro TM Formulations of this type, LNPs with elevated levels of different phosphatidylcholine lipids showed increased mRNA expression in spleen, heart, lung, kidney, muscle tissue, bone marrow (BM), and ear.

[0222] The following mRNA-LNPs were prepared with increasing levels of DSPC, DOPC, POPC, DPPC, and a combination of DSPC and DOPC in the molar ratios shown in Table 7 below. Table 7: LNPs prepared using different phosphatidylcholine lipids for examination of extrahepatic expression

[0223] The mRNA encoding luciferase and the N / P of each preparation are as follows. As previously mentioned, the tissue homogenate luciferase levels of CD-1 mice were measured 24 hours after injection. The ionizable cationic lipid nMC3 is described in co-owned and co-pending WO 2022 / 246571, and the C-109 ionizable lipid is described in U.S. Provisional Application No. 63 / 410,261 (Compound 7 with acetal head group) filed on September 27, 2022, which is incorporated herein by reference.

[0224] The particle size and polydispersity index (PDI) of each formulation (AJ in Table 7 above) and the % capture of mRNA were Fig.14A as shown in .

[0225] Fig. 14B Shows Onpattro TM The luminescence intensity / mg liver of the LNP-A formulation (LNP A) was elevated, while the rest of the LNPs with different levels of 50 mol % DSPC, DOPC, POPC, DPPC or a combination of DSPC and DOPC (combined content 50 mol %) were close to the baseline level.

[0226] Figures 14C-14I The luminescence intensity / mg organ or tissue of spleen, heart, lung, kidney, muscle, bone marrow (BM) and ear 24 hours after injection is shown. TM , LNPs B and C with 50 mol% DSPC showed the highest luminescence intensity / mg organ or tissue, although LNP F with 50 mol% DPPC also had acceptable luminescence intensity in many organs / tissues examined (spleen, heart, kidney, bone marrow (BM), and ear). Dioleoylphosphatidylcholine (DOPC and POPC) LNPs (LNP D and E) showed lower luminescence intensity in each organ / tissue in the liver, but LNPs containing a mixture of dioleoylphosphatidylcholine (DOPC) and DSPC (LNP G to J) showed higher luminescence intensity in many organs / tissues examined in the study (see Fig. 14C -I). These results suggest that LNPs with elevated levels of phosphatidylcholine lipids with unsaturated chains and with phase transition temperatures above physiological temperature (DSPC and DPPC) improve delivery of mRNA outside the liver relative to LNPs with unsaturated chains (DOPC or POPC) and phase transition temperatures below physiological temperatures. However, combinations of phosphatidylcholine lipids with high (e.g., DSPC or DPPC) and low (e.g., DOPC or POPC) phase transition temperatures may also provide improved extrahepatic delivery (e.g., as Fig. 14C , 14D LNPs G, H, and I shown). Example 6: Multiple LNPs with elevated phosphatidylcholine content (30 mol% and higher) and different ionizable lipids exhibit enhanced extrahepatic mRNA expression in vivo

[0227] The following in vivo data demonstrate that LNPs with elevated levels of phosphatidylcholine lipids (e.g., 30-50 mol% DSPC) and formulated with various ionizable cationic lipids exhibit enhanced mRNA expression in bone marrow (BM), spleen, skin, heart, lung, small intestine (SI), cecum, and colon.

[0228] The groups of mRNA LNPs shown in Table 8 were prepared with different PC contents, different ionizable cationic lipids and N / P. The encapsulated mRNA encodes luciferase. As previously described, luciferase levels in tissue homogenates of CD-1 mice were measured 24 hours after injection. Table 8: LNP groups with different DSPC contents; different ionizable cationic lipids and N / P contents

[0229] The ionizable cationic lipids used in the LNP group are shown in Table 9 below. Table 9: Ionizable Cationic Lipids for the High DSPC Formulation Group

[0230] Figures 15A-15H In vivo data for tissue homogenates in liver, spleen, bone marrow (BM), abdominal skin, heart, lung, small intestine (SI), cecum, and colon are shown for the LNP groups with elevated DSPC (greater than 30 mol%) in Table 8. Example 7: LNPs with elevated phosphatidylcholine content have unique morphology

[0231] A cryo-TEM image of lipid nanoparticles composed of MF019 / DSPC / Chol / PEG-DMG (27.4 / 50 / 21.1 / 1.5 mol:mol) encapsulating mRNA encoding luciferase was obtained. Fig.16 shown.

[0232] Images of lipid nanoparticles with mRNA encapsulated using high levels of phosphatidylcholine lipids have a morphology in which electron-dense regions exist within the bilayer. In turn, the core is surrounded by structures consistent with a lipid bilayer, such as Fig.16 This morphology is unique to LNPs with elevated phosphatidylcholine lipids and may provide the LNPs with improved in vivo delivery properties to extrahepatic (non-liver) target sites, as observed in the previous examples.

[0233] The examples are intended to illustrate the preparation of specific lipid nanoparticle mRNA formulations and their properties, but are in no way intended to limit the scope of the invention.

[0234] As used herein, the articles "a" and "an" shall include both the singular and the plural, unless otherwise stated.

Claims

1. A lipid nanoparticle for extrahepatic delivery of mRNA, the lipid nanoparticle comprising: (i) mRNA; (ii) a phosphatidylcholine lipid content of 30 mol % to 70 mol %; (iii) an ionizable cationic lipid content of 5 mol % to 50 mol %; (iv) a sterol selected from cholesterol or its derivatives; and (v) a hydrophilic polymer-lipid conjugate present at a lipid content of 0.5 mol % to 5 mol %, Each lipid content is relative to the total lipid content of the lipid nanoparticles.

2. A lipid nanoparticle comprising encapsulated mRNA and 20 to 70 mol % of phosphatidylcholine lipids, ionizable lipids, relative to the total lipids present in the lipid nanoparticle; and at least one of a sterol and a hydrophilic polymer-lipid conjugate, wherein the lipid nanoparticles exhibit at least a 10% increase in gene expression of the mRNA in vivo measured in a cell subpopulation in the bone marrow or spleen 24 hours and / or 3 days after injection, the cells being selected from macrophages, monocytes and / or T cells, compared to an Onpattro-type formulation of MC3 / DSPC / cholesterol / PEG-lipid at 50 / 10 / 38.5 / 1.5 mol:mol of the mRNA encapsulated, but otherwise measured under the same conditions, wherein the gene expression is quantified in an animal model by detecting cells positive for green fluorescent protein (GFP) using flow cytometry.

3. The lipid nanoparticle of claim 1, wherein the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dipalmitoylphosphatidylcholine (DPPC).

4. The lipid nanoparticle of claim 1, wherein the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC) or dipalmitoylphosphatidylcholine (DPPC). The lipid nanoparticle according to claim 1 , wherein the phosphatidylcholine lipid is a mixture of two phosphatidylcholine lipids.

6. The lipid nanoparticle of claim 5, wherein the mixture comprises distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylcholine (DOPC).

7. The lipid nanoparticle according to any one of claims 1 to 6, wherein the phosphatidylcholine content is between 40 mol% and 60 mol%.

8. The lipid nanoparticle according to claim 7, wherein the phosphatidylcholine content is between 42 mol% and 58 mol%.

9. The lipid nanoparticle according to claim 8, wherein the phosphatidylcholine content is between 45 mol% and 55 mol%.

10. The lipid nanoparticle according to any one of claims 1 to 9, wherein the cationic lipid is an amino lipid.

11. The lipid nanoparticle according to any one of claims 1 to 10, wherein the ionizable cationic lipid is present in less than 40 mol%.

12. The lipid nanoparticle according to any one of claims 1 to 11, wherein the hydrophilic polymer-lipid conjugate is a polyethylene glycol-lipid conjugate.

13. The lipid nanoparticle according to any one of claims 1 to 12, wherein the sterol is present at 15 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle.

14. The lipid nanoparticle according to any one of claims 1 to 12, wherein the sterol is present at 18 mol% to 40 mol% based on the total lipid present in the lipid nanoparticle.

15. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle exhibits at least a 10% increase in gene expression of mRNA in vivo measured in a cell subpopulation in the bone marrow or spleen 24 hours and / or 3 days after injection, the cells being selected from macrophages, monocytes and / or T cells, compared to an Onpattro-type formulation of MC3 / DSPC / cholesterol / PEG-lipid encapsulating mRNA at 50 / 10 / 38.5 / 1.5 mol:mol, but otherwise measured under identical conditions, wherein gene expression is quantified in an animal model by detecting cells positive for green fluorescent protein (GFP) using flow cytometry.

16. The lipid nanoparticle of claim 2 or 15, wherein green fluorescent protein is measured in a macrophage / monocyte population isolated from the spleen or liver of a mouse after injection, and wherein the increase in expression is determined by measuring the percentage of cells in the cell population that are positive for eGFP.

17. The lipid nanoparticle of claim 16, wherein green fluorescent protein is measured 3 days after injection, and wherein the animal model is a mouse.

18. The lipid nanoparticle of claim 15, wherein the in vivo expression of green fluorescent protein in a macrophage / monocyte population isolated from mouse bone marrow or spleen after injection is increased by at least 50% compared to a sphingomyelin-containing LNP that uses sphingomyelin instead of DSPC but otherwise has the same lipid composition as the lipid nanoparticle.

19. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle exhibits at least a 10% increase in gene expression of mRNA in vivo as measured in extrahepatic tissues or organs.

20. The lipid nanoparticle of claim 1, wherein the extrahepatic tissue or organ is spleen, bone marrow, lung, kidney, heart, abdominal skin, back skin and / or ear.

21. A method for in vivo delivery of mRNA to a mammalian subject, the method comprising: Administering the lipid nanoparticle of any one of claims 1 to 20 to a mammalian subject.

22. A method for delivering mRNA for in vivo production of a protein or peptide in an extrahepatic tissue or organ of a subject, the method comprising administering to the subject a lipid nanoparticle having at least 20 mol% distearoylphosphatidylcholine (DSPC) and / or dipalmitoylphosphatidylcholine (DPPC) and an ionizable cationic lipid, wherein the mRNA is encapsulated within the lipid nanoparticle, and wherein administration of the lipid nanoparticle results in extrahepatic tissue or organ expression of the protein or peptide encoded by the mRNA, wherein the lipid nanoparticle has at least a 10% increase in expression of the protein or peptide encoded by the mRNA in the extrahepatic tissue or organ relative to an Onpattro-type formulation of 50 / 10 / 38.5 / 1.5 mol:mol MC3 / DSPC / cholesterol / PEG lipids encapsulating the mRNA, but otherwise measured under the same conditions.

23. The method of claim 22, wherein the lipid nanoparticles have at least 30 mol% distearoylphosphatidylcholine (DSPC) and / or dipalmitoylphosphatidylcholine (DPPC) and less than 40 mol% ionizable cationic lipids.

24. The method of claim 22, wherein the extrahepatic tissue or organ is spleen, bone marrow, lung, kidney, heart, abdominal skin, back skin and / or ear.

25. The method of claim 22, wherein the mRNA accumulates in the spleen or bone marrow of the subject for at least one day after administration.

26. The method of claim 22, wherein the lipid nanoparticles are used to treat a disease or disorder, and the disease or disorder is an autoimmune disorder.

27. The method of claim 22, wherein the lipid nanoparticles are used to treat a disease or condition, and the disease or condition is an infectious disease.

28. The method of claim 22, wherein the lipid nanoparticles are used to treat a disease or condition, and the disease or condition is cancer.

29. Use of the lipid nanoparticles of any one of claims 1 to 20 for delivering and expressing mRNA to mammalian cells in vivo or in vitro.

30. Use of the lipid nanoparticles of any one of claims 1 to 20 in the preparation of a medicament for delivering mRNA to mammalian cells in vivo or in vitro.

31. Use of the lipid nanoparticles according to claim 29 or 30, wherein the mRNA is used to target autoimmune disorders in vivo.

32. Use of the lipid nanoparticles according to claim 29 or 30, wherein the mRNA is used to target infectious diseases in vivo.

33. Use of the lipid nanoparticles according to claim 29 or 30, wherein the mRNA is used to target cancer in vivo.

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