Methods for mRNA delivery and compositions thereof

Abstract

The present disclosure provides lipid nanoparticles for extrahepatic delivery of mRNA, the lipid nanoparticles comprising: (i) an mRNA cargo; (ii) a phosphatidylcholine lipid at a content of 30 mol% to 70 mol%; (iii) an ionizable cationic lipid at a 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%. Further provided are lipid nanoparticles comprising encapsulated mRNA and an ionizable lipid, phosphatidylcholine lipid at 20 to 70 mol%, and at least one of a sterol and a hydrophilic polymer lipid conjugate, the lipid nanoparticles increasing gene expression of the mRNA in vivo by at least 10% as measured in one or more extrahepatic organs or tissues. Further provided are methods of administration of such lipid nanoparticles.

Description

[Technical field]

[0001] (Incorporation by reference to priority application) This application claims priority to U.S. Provisional Application No. 63 / 362,345, filed April 1, 2022, which is expressly incorporated by reference herein in its entirety.

[0002] The present disclosure relates to methods of delivering mRNA and compositions for delivering it to extrahepatic tissues. [Background technology]

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

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

[0005] Of these four components, ionizable lipids make up the majority (50 mol%) of the Onpattro™ formulation and are believed to be important for the in vitro and in vivo activity of LNPs. Thus, most research in this field has focused primarily on improving this lipid component. Ionizable lipids are typically amino lipids, carefully designed to be charged at low pH and nearly neutral at physiological pH. This allows electrostatic interactions between the lipid and the negatively charged nucleic acid upon initial formulation. Because ionizable lipids are nearly neutral at physiological pH, toxicity and renal clearance are reduced. After intracellular uptake by endocytosis, the acidic environment of the endosome increases the net positive charge of the ionizable amino lipids, which promotes fusion with anionic lipids in the endosomal membrane, which then destabilizes the membrane and releases the nucleic acid-based therapeutic into the cytoplasm to exert its effects.

[0006] With regard to the remaining three lipid components, it is well known that PEG-lipids improve the circulation lifetime of LNPs and cholesterol functions to stabilize the particles, but in general, relatively little attention has been paid to studying DSPC beyond its role as a structural lipid.

[0007] Although there have been great advances in the study of nucleic acid delivery via LNPs, it is widely known that the Onpattro™ formulation accumulates primarily in liver tissue. The ability of LNPs to accumulate in organs and tissues other than the liver would greatly expand the clinical utility of these delivery systems. To improve the delivery of nucleic acid cargo to extrahepatic tissues such as the bone marrow and spleen, it is necessary to extend the circulatory lifetime of the particles. Traditional approaches to achieve this rely on optimizing the levels of PEG-lipids within the LNPs, but the inclusion of PEG-lipids in LNPs often results in lower transfection efficacy or unfavorable immune responses.

[0008] In vivo studies were conducted to investigate the ability of four-component Onpattro™-type formulations to deliver siRNA beyond the liver. In particular, siRNA gene silencing outside the liver was investigated with Onpattro™-type LNPs incorporating 10 mol% and 40 mol% DSPC (MC3 / Chol / DSPC / PEG-DMG) (Ordobadi, Lipid Nanoparticles for Delivery of Bioactive Molecules, 2019, A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy, The University of British Columbia). The 10 mol% DSPC Onpattro™-like formulation was shown to have similar liver accumulation and blood circulation lifetime as the 40 mol% DSPC formulation (MC3 / Chol / DSPC / PEG-DMG; 18.5 / 40 / 40 / 1.5 mol%). Furthermore, 40 mol% DSPC siRNA-LNPs performed comparably to 10 mol% DSPC formulations in myeloid gene silencing. Thus, these previous studies did not demonstrate a clear advantage gained by adjusting the level of DSPC to improve extrahepatic delivery of nucleic acid cargo.

[0009] The above studies that investigated extrahepatic delivery were limited to LNPs with siRNA cargo. However, messenger RNA (mRNA) therapy is becoming an increasingly important tool in the treatment of diseases, and delivery of mRNA to extrahepatic tissues would expand the clinical utility of mRNA therapy beyond the liver. Similar to siRNA, mRNA molecules are rapidly degraded in the body, and LNPs are used to suppress such 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). As a result, siRNA-LNP studies may not be useful for designing LNPs for mRNA delivery. Furthermore, current research on LNP-mRNA systems for intravenous administration has focused primarily on the development of improved ionizable cationic lipids. (Semple et al., Nat Biotechnol 2010, 28:172). Furthermore, these systems use the Onpattro™ lipid composition (see above), which has a short circulatory lifetime, with most cargo accumulating in the liver within 30 minutes (Akinc et al., 2019, Nat Nanotechnol., 14:1084).

[0010] Thus, there is a need in the art for improved in vivo extrahepatic delivery of nucleic acids, such as mRNA, using lipid nanoparticles. Summary of the Invention

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

[0012] The present disclosure is based on the discovery that ionizable LNP formulations (hereinafter "lcLNPs") with increased levels of phosphatidylcholine lipids compared to Onpattro™ benchmark LNPs can significantly improve extrahepatic delivery of mRNA in vivo. In some embodiments, the present disclosure provides mRNA-LNP formulations that have improved in vivo expression of mRNA in extrahepatic tissues compared to Onpattro™ LNPs, as measured in subsets of immune cells in bone marrow and spleen in animal models at predetermined times post-administration, such as 1 hour and 3 hours post-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 in a content of 30-70 mol%; (iii) an ionizable lipid in a content of 5 mol%-50 mol%; (iv) a sterol selected from cholesterol or its derivatives; and (v) a hydrophilic polymer-lipid conjugate in a content of 0.5 mol%-5 mol of lipid; each lipid content being measured relative to the total lipid content of the lipid nanoparticle.

[0014] In another aspect, the disclosure provides lipid nanoparticles comprising: an encapsulated mRNA; and 20-70 mol% phosphatidylcholine lipid relative to the total lipid present in the lipid nanoparticle; an ionizable lipid; and at least one of a sterol and a hydrophilic polymer lipid conjugate, wherein the lipid nanoparticles have at least a 10% increase in in vivo gene expression of the mRNA measured in a subset of bone marrow or spleen cells selected from macrophages, monocytes, and / or T cells at 24 hours and / or 3 days post-injection compared to a 50 / 10 / 38.5 / 1.5 mol:mol Onpattro formulation of MC3 / DSPC / cholesterol / PEG-lipid encapsulating the mRNA, but measured under otherwise identical conditions, and wherein said gene expression is quantified in an animal model by detecting green fluorescent protein (GFP) positive cells 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 a further embodiment, the phosphatidylcholine content is 40 mol% to 60 mol%. In another embodiment, the phosphatidylcholine content is 42 mol% to 58 mol%. In yet a further embodiment, the phosphatidylcholine content is 45 mol% to 55 mol%.

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

[0019] In a further embodiment, the ionizable lipid is present at less than 40 mol %.

[0020] In another embodiment, the hydrophilic polymer lipid conjugate is a polyethylene glycol lipid conjugate. In a further 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 a further embodiment, the lipid nanoparticles provide at least 10% increased in vivo gene expression of said mRNA measured in a subset of bone marrow or spleen cells selected from macrophages, monocytes, and / or T cells 24 hours and / or 3 days after injection, compared to a 50 / 10 / 38.5 / 1.5 mol:mol Onpattro-type formulation of MC3 / DSPC / cholesterol / PEG-lipid encapsulating said mRNA, but measured under otherwise identical conditions, and gene expression is quantified in an animal model by detecting green fluorescent protein (GFP) positive cells using flow cytometry. In a further embodiment, green fluorescent protein is measured in macrophage / monocyte cell populations isolated from the spleen or liver of mice after injection, and increased expression is determined by measuring the percentage of eGFP positive cells in the cell population.

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

[0023] In a further embodiment, in vivo expression of green fluorescent protein in macrophage / monocyte cell populations isolated from the bone marrow or spleen of mice following injection is increased by at least 50% compared to sphingomyelin-containing LNPs that use sphingomyelin instead of DSPC but otherwise have the same lipid composition as the lipid nanoparticles.

[0024] In another aspect, there is provided a method for in vivo delivery of mRNA to a mammalian subject, the method comprising administering to the mammalian subject a lipid nanoparticle as described in any of the above embodiments or aspects.

[0025] In a further embodiment, the mRNA accumulates in the spleen or bone marrow of said subject at least one day after administration.

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

[0027] In a further embodiment, there is provided the use of lipid nanoparticles for in vivo or in vitro delivery and expression of mRNA in mammalian cells.

[0028] In a further embodiment, the lipid nanoparticles increase mRNA gene expression in vivo by at least 10% when measured in extrahepatic tissues or organs.

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

[0030] A method for in vivo delivery of mRNA to a mammalian subject, the method comprising administering to the mammalian subject a lipid nanoparticle as described in any of the embodiments above.

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

[0032] In a further 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 the spleen, bone marrow, lung, kidney, heart, abdominal skin, dorsal skin and / or ear.

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

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

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

[0037] According to a further embodiment, the lipid nanoparticles are used to treat a disease or disorder that is cancer.

[0038] In another embodiment, the use of the lipid nanoparticles described above is provided for the manufacture of a medicament for in vivo or in vitro delivery of mRNA to mammalian cells.In some embodiments, the mRNA is used to target autoimmune disease in vivo.In further embodiments, the mRNA is used to target infectious disease in vivo.

[0039] In a further embodiment, the mRNA is used to target cancer in vivo. [Brief description of the drawings]

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

[0041] FIG. 1B shows the percentage of eGFP positive cells (%eGFP) in the spleen of immune cell subpopulations (monocytes / macrophages, neutrophils, B cells, CD4 T cells, and CD8 T cells) 24 hours after injection of mice with phosphate buffered saline (PBS) and eGFP-mRNA LNPs containing 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro™")) or eGFP-mRNA LNPs containing 50 mol% DSPC (MF019:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP™")). + ) is shown.

[0042] FIG. 1C shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (%eGFP) in the spleen of immune cell subpopulations (monocytes / macrophages, neutrophils, B cells, CD4 T cells, and CD8 T cells) 3 days after injection of mice with phosphate buffered saline (PBS) and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro™")) or eGFP-mRNA LNPs with 50 mol% DSPC (MF019:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP™")). + ) is shown.

[0043] [Fig. 1D-F] FIG. 1D shows the mean fluorescence intensity (MFI) in the spleen of immune cell subpopulations (monocytes / macrophages, neutrophils, B cells, CD4 T cells, and CD8 T cells) 4 hours after injection of mice with phosphate buffered saline (PBS) and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro™")) or eGFP-mRNA LNPs with 50 mol% DSPC (MF019:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP™")).

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

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

[0046] [Diagram 2] FIG. 2A shows a representative scatter plot showing the percentage of eGFP+ monocytes / macrophages in the spleen 24 hours after injection of mice with phosphate-buffered saline (PBS).

[0047] FIG. 2B shows the level of eGFP in the spleen 24 hours after injecting mice with eGFP-mRNA LNPs containing 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro™"). + Representative scatter plots showing monocyte / macrophage ratios are shown.

[0048] FIG. 2C shows eGFP in the spleen 24 hours after injection of mice with eGFP-mRNA LNP 50 mol% DSPC (MF019:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP™")). + Representative scatter plots showing monocyte / macrophage ratios are shown.

[0049] [Figure 3A-C]FIG. 3A shows the percentage of eGFP positive cells (%eGFP+) in bone marrow for the indicated immune cell subpopulations (erythrocytes, B cells, T cells, monocytes / macrophages, and neutrophils) 4 hours after injection of mice with phosphate buffered saline (PBS) and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro™")) or eGFP-mRNA LNPs with 50 mol% DSPC (MF019:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP™")).

[0050] FIG. 3B shows the percentage of eGFP positive cells (%eGFP) in bone marrow for the indicated immune cell subpopulations (erythrocytes, B cells, T cells, monocytes / macrophages, and neutrophils) 24 hours after injection of mice with phosphate buffered saline (PBS) and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro™")) or eGFP-mRNA LNPs with 50 mol% DSPC (MF019:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP™")). + ) is shown.

[0051] FIG. 3C shows the percentage of eGFP positive cells (%eGFP) in the bone marrow for the indicated immune cell subpopulations (erythrocytes, B cells, T cells, monocytes / macrophages, and neutrophils) 3 days after injection of mice with phosphate buffered saline (PBS) and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro™")) or eGFP-mRNA LNPs with 50 mol% DSPC (MF019:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP™")). + ) is shown.

[0052] [Fig. 3D-F] FIG. 3D shows mean fluorescence intensity (MFI) in bone marrow of the indicated immune cell subpopulations (erythrocytes, B cells, T cells, monocytes / macrophages, and neutrophils) 4 hours after injection of mice with phosphate buffered saline (PBS) and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro™")) or eGFP-mRNA LNPs with 50 mol% DSPC (MF019:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP™")).

[0053] FIG. 3E shows the mean fluorescence intensity (MFI) in bone marrow of the indicated immune cell subpopulations (erythrocytes, B cells, T cells, monocytes / macrophages, and neutrophils) 24 hours after injection of mice with phosphate buffered saline (PBS) and eGFP-mRNA LNPs with 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro™")) or eGFP-mRNA LNPs with 50 mol% DSPC (MF019:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP™")).

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

[0055] [Figure 4] FIG. 4A is a representative scatter plot showing the percentage of eGFP+ T cells in the bone marrow 24 hours after injection of mice with phosphate buffered saline (PBS).

[0056] FIG. 4B shows the levels of eGFP in bone marrow 24 hours after mice were injected with eGFP-mRNA LNPs containing 10 mol% DSPC (nMC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) (Onpattro™). + Representative scatter plots showing the percentage of T cells are shown.

[0057] FIG. 4C shows eGFP expression in bone marrow 24 hours after mice were injected with eGFP-mRNA LNPs containing 50 mol% DSPC (MF019:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP™"). + Representative scatter plots showing the percentage of T cells are shown.

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

[0059] FIG. 5B shows mean fluorescence intensity (MFI) in bone marrow of the indicated immune cell subpopulations (erythrocytes, B cells, T cells, monocytes / macrophages, and neutrophils) 24 hours after injection of mice with phosphate buffered saline (PBS) and eGFP-mRNA LNPs with 10 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro™")) or eGFP-mRNA LNPs with 50 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP™")).

[0060] [Figure 6] FIG. 6A is a representative scatter plot showing the percentage of eGFP+ T cells in the bone marrow 24 hours after injection of mice with phosphate buffered saline.

[0061] FIG. 6B shows the levels of eGFP in bone marrow 24 hours after mice were injected with eGFP-mRNA LNPs containing 10 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) (Onpattro™). + Representative scatter plots showing the percentage of T cells are shown.

[0062] FIG. 6C shows the concentration of eGFP in bone marrow 24 hours after injecting mice with eGFP-mRNA LNPs containing 50 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP™"). + Representative scatter plots showing the percentage of T cells are shown.

[0063] [Figure 7]1 shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (%eGFP+) in spleens of monocytes / macrophages 24 hours after injection of mice with 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™")) or eGFP-mRNA LNPs with 50 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP")).

[0064] [Figure 8] FIG. 8A shows a representative scatter plot showing the percentage of eGFP+ monocytes / macrophages in the spleen 24 hours after injection of mice with phosphate-buffered saline (PBS).

[0065] FIG. 8B shows eGFP expression in the spleen 24 hours after mice were injected with eGFP-mRNA LNPs containing 10 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("Onpattro™")). + Representative scatter plots showing monocyte / macrophage ratios are shown.

[0066] FIG. 8C shows eGFP expression in the spleen 24 hours after injecting mice with eGFP-mRNA LNPs containing 50 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNP™")). + Representative scatter plots showing monocyte / macrophage ratios are shown.

[0067] [Figure 9]FIG. 9A shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (%eGFP+) in bone marrow for the indicated immune cell subpopulations (erythrocytes, B cells, T cells, monocytes / macrophages, and neutrophils) 24 hours after injection of mice with phosphate buffered saline (PBS) and eGFP-mRNA LNPs containing 50 mol% DSPC (MC3:DSPC:Chol:PEG-lipids (27.4:50:21.1:1.5 mol:mol) (“lcLNPs”) or eGFP-mRNA LNPs containing 50 mol% egg sphingomyelin (ESM) (MC3:ESM:Chol:PEG-lipids (27.4:50:21.1:1.5 mol:mol).

[0068] FIG. 9B shows mean fluorescence intensity (MFI) in bone marrow of the indicated immune cell subpopulations (erythrocytes, B cells, T cells, monocytes / macrophages, and neutrophils) 24 hours after injection of mice with phosphate buffered saline (PBS) and eGFP-mRNA LNPs containing 50 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("lcLNPs")) or eGFP-mRNA LNPs containing 50 mol% egg sphingomyelin (ESM) (MC3:ESM:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol)).

[0069] [Figure 10] FIG. 10A shows a representative scatter plot showing the percentage of eGFP+ T cells in bone marrow 24 hours after injecting mice with eGFP-mRNA LNPs containing 50 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("lcLNP™")).

[0070] FIG. 10B shows the eGFP mRNA expression level in the bone marrow 24 hours after injecting mice with eGFP-mRNA LNPs containing 50 mol% ESM (MC3:ESM:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("ESM"; FIG. 10B)). +Representative scatter plots showing the percentage of T cells are shown.

[0071] [Figure 11] FIG. 11A shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (%eGFP+) of the indicated immune cell subpopulations (monocytes / macrophages, neutrophils, B cells, CD4 T cells, and CD8 T cells) in the spleen 24 hours after injection of mice with phosphate buffered saline (PBS) and eGFP-mRNA LNPs with 50 mol% DSPC (MC3:DSPC:Chol:PEG-lipids (27.4:50:21.1:1.5 mol:mol) (“lcLNPs”) or eGFP-mRNA LNPs with 50 mol% egg sphingomyelin (ESM) (MC3:ESM:Chol:PEG-lipids (27.4:50:21.1:1.5 mol:mol).

[0072] FIG. 11B shows the mean fluorescence intensity (MFI) of the indicated immune cell subpopulations (monocytes / macrophages, neutrophils, B cells, CD4 T cells, and CD8 T cells) in the spleen 24 hours after injection of mice with 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) ("lcLNPs")) or eGFP-mRNA LNPs with 50 mol% egg sphingomyelin (ESM) (MC3:ESM:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol)).

[0073] [Figure 12] FIG. 12A shows a representative scatter plot showing the percentage of eGFP+ macrophages / monocytes in the spleen 24 hours after injecting mice with eGFP-mRNA LNPs containing 50 mol% DSPC (MC3:DSPC:Chol:PEG-lipid (50:10:38.5:1.5) ("lcLNP™").

[0074] FIG. 12B shows eGFP expression in the spleen 24 hours after mice were injected with eGFP-mRNA LNPs containing 50 mol% ESM (MC3:ESM:Chol:PEG-lipid (27.4:50:21.1:1.5 mol:mol) ("ESM")). + Representative scatter plots showing macrophage / monocyte ratios are shown.

[0075] [Figure 13A-B] Figure 13A shows the fold change in luminescence of mRNA-LNP MF019:DSPC:Chol:PEG2000-DMG (27.4:50:21.1:1.5 mol:mol) and NTx-C16:DSPC:Chol:PEG2000-DMG (27.4:50:21.1:1.5 mol:mol) vs. Onpattro™ nMC3:DSPC:Chol:PEG2000-DMG (50:10:38.5:1.5 mol:mol) administered at a dose of 1 mg / kg to CD-1 mice. mRNA expression 24 hours post-injection was quantified in liver, spleen, lung, kidney, heart, bone marrow (BM), abdomen, back, and ear by measuring luminescence.

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

[0077] [Figure 13C]Serum activity (U / L) of alanine transaminase (ALT) (left) and aspartate transaminase (AST) (right) 24 hours after injection of CD-1 mice with a dose of 5 mg / kg of LNP-mRNA encoding luciferase. LNPs were Onpattro™ (nMC3:DSPC:Chol:PEG2000-DMG 50:10:38.5:1.5 mol:mol) and mRNA-LNPs MF019:DSPC:Chol:PEG2000-DMG (27.4:50:21.1:1.5 mol:mol) and NTx-C16:DSPC:Chol:PEG2000-DMG (27.4:50:21.1:1.5 mol:mol).

[0078] [Figure 14A] Shown are the mRNA capture rates, particle sizes (nm), and polydispersity index (PDI) for Onpattro™ (LNP A), LNPs containing 50 mol% of the phosphatidylcholine lipids DSPC, DOPC, POPC, and DPPC (LNP BF), and LNPs with 40:10, 30:20, 20:30, and 10:40 mol:mol DSPC:DOPC (GJ). LNP formulations A-J are shown in Table 7 of Example 5, where the mRNA encodes luciferase.

[0079] [Figure 14B-C] FIG. 14B shows the luminescence intensity per mg of liver in CD-1 mice for Onpattro™ (LNP A), LNPs containing 50 mol% of the phosphatidylcholine lipids DSPC, DOPC, POPC, and DPPC (LNP BF), and LNPs with 40:10, 30:20, 20:30, and 10:40 mol:mol DSPC:DOPC (GJ). LNP formulations A-J are shown in Table 7 of Example 5, with the mRNA encoding luciferase. Expression of the mRNA was quantified by measuring luminescence 24 hours after injection.

[0080] FIG. 14C shows the luminescence intensity per mg of spleen from CD-1 mice for Onpattro™ (LNP A), LNPs containing 50 mol% of the phosphatidylcholine lipids DSPC, DOPC, POPC, and DPPC (LNP BF), and LNPs with 40:10, 30:20, 20:30, and 10:40 mol:mol DSPC:DOPC (GJ). LNP formulations A-J are shown in Table 7 in Example 5, and the mRNA encodes luciferase. Expression of the mRNA was quantified by measuring luminescence 24 hours after injection.

[0081] [Fig. 14D-E] FIG. 14D shows the luminescence intensity per mg of CD-1 mouse heart for Onpattro™ (LNP A), LNPs containing 50 mol% of the phosphatidylcholine lipids DSPC, DOPC, POPC, and DPPC (LNP BF), and LNPs with 40:10, 30:20, 20:30, and 10:40 mol:mol DSPC:DOPC (GJ). LNP formulations A-J are shown in Table 7 of Example 5, with the mRNA encoding luciferase. Expression of the mRNA was quantified by measuring luminescence 24 hours after injection.

[0082] FIG. 14E shows the luminescence intensity per mg of lung in CD-1 mice for Onpattro™ (LNP A), LNPs containing 50 mol% of the phosphatidylcholine lipids DSPC, DOPC, POPC, and DPPC (LNP BF), and LNPs with 40:10, 30:20, 20:30, and 10:40 mol:mol DSPC:DOPC (GJ). LNP formulations A-J are shown in Table 7 in Example 5, with the mRNA encoding luciferase. Expression of the mRNA was quantified by measuring luminescence 24 hours after injection.

[0083] [Fig. 14F-G]FIG. 14F shows the luminescence intensity per mg of liver in CD-1 mice for Onpattro™ (LNP A), LNPs containing 50 mol% of the phosphatidylcholine lipids DSPC, DOPC, POPC, and DPPC (LNP BF), and LNPs with 40:10, 30:20, 20:30, and 10:40 mol:mol DSPC:DOPC (GJ). LNP formulations A-J are shown in Table 7 in Example 5, with the mRNA encoding luciferase. Expression of the mRNA was quantified by measuring luminescence 24 hours after injection.

[0084] FIG. 14G shows the luminescence intensity per mg of muscle tissue from CD-1 mice for Onpattro™ (LNP A), LNPs containing 50 mol% of the phosphatidylcholine lipids DSPC, DOPC, POPC, and DPPC (LNP BF), and LNPs with 40:10, 30:20, 20:30, and 10:40 mol:mol DSPC:DOPC (GJ). LNP formulations A-J are shown in Table 7 of Example 5, and the mRNA encodes luciferase. Expression of the mRNA was quantified by measuring luminescence 24 hours after injection.

[0085] [Fig. 14H-I] FIG. 14H shows the luminescence intensity per mg of bone marrow (BM) from CD-1 mice for Onpattro™ (LNP A), LNPs containing 50 mol% of the phosphatidylcholine lipids DSPC, DOPC, POPC, and DPPC (LNP BF), and LNPs with 40:10, 30:20, 20:30, and 10:40 mol:mol DSPC:DOPC (GJ). LNP formulations A-J are shown in Table 7 of Example 5, and the mRNA encodes luciferase. Expression of the mRNA was quantified by measuring luminescence 24 hours after injection.

[0086] FIG. 14I shows the luminescence intensity per mg in CD-1 mouse ears for Onpattro™ (LNP A), LNPs containing 50 mol% of the phosphatidylcholine lipids DSPC, DOPC, POPC, and DPPC (LNP BF), and LNPs with 40:10, 30:20, 20:30, and 10:40 mol:mol DSPC:DOPC (GJ). LNP formulations A-J are shown in Table 7 in Example 5, where the mRNA encodes luciferase. Expression of the mRNA was quantified by measuring luminescence 24 hours after injection.

[0087] [Figure 15A-C] FIG. 15A shows the luminescence intensity per mg of bone marrow (BM) in CD-1 mice for a panel of LNPs containing various levels of DSPC and ionizable cationic lipids, as shown in Table 8 of Example 6.

[0088] FIG. 15B shows the luminescence intensity per mg of spleen in CD-1 mice for a panel of LNPs containing various levels of DSPC and ionizable cationic lipids, as shown in Table 8 of Example 6.

[0089] FIG. 15C shows the luminescence intensity per mg of skin in CD-1 mice for a panel of LNPs containing various levels of DSPC and ionizable cationic lipids, as shown in Table 8 of Example 6.

[0090] [Fig. 15D-F] FIG. 15D shows the luminescence intensity per mg of heart in CD-1 mice for a panel of LNPs containing various levels of DSPC and ionizable cationic lipids, as shown in Table 8 of Example 6.

[0091] FIG. 15E shows the luminescence intensity per mg of lung in CD-1 mice for a panel of LNPs containing various levels of DSPC and ionizable cationic lipids, as shown in Table 8 of Example 6.

[0092] FIG. 15F shows the luminescence intensity per mg of small intestine in CD-1 mice for a panel of LNPs containing various levels of DSPC and ionizable cationic lipids, as shown in Table 8 of Example 6.

[0093] [Fig. 15G-H] FIG. 15G shows the luminescence intensity per mg of cecum in CD-1 mice for a panel of LNPs containing various levels of DSPC and ionizable cationic lipids, as shown in Table 8 of Example 6.

[0094] FIG. 15H shows the luminescence intensity per mg of colon in CD-1 mice for a panel of LNPs containing various levels of DSPC and ionizable cationic lipids, as shown in Table 8 of Example 6.

[0095] [Figure 16] Cryo-TEM images of lipid nanoparticles composed of MF019 / DSPC / Chol / PEG-DMG (27.4 / 50 / 21.1 / 1.5 mol:mol) encapsulating mRNA encoding luciferase are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0096] (Detailed Description) The lipid nanoparticles described herein are an improvement over the conventional four-component LNPs used for siRNA delivery, and are referred to herein as Onpattro™. In certain advantageous embodiments, the LNPs are improved formulations that include ionizable lipids, phosphatidylcholine lipids, cholesterol, and hydrophilic polymer-lipid conjugates, where the phosphatidylcholine lipids are present at a mol% of at least 20 mol% or at least 30 mol%, and the ionizable lipids are present at less than 40 mol%. As described herein, the higher mol% of phosphatidylcholine beyond that used in conventional formulations for nucleic acid delivery surprisingly increases mRNA delivery to extrahepatic tissues compared to the benchmark Onpattro™ formulation.

[0097] The lipid nanoparticles described herein comprise a cargo that is messenger RNA.As used herein, the term "messenger RNA" or "mRNA" refers to a polynucleotide that encodes and expresses at least one peptide, polypeptide, or protein.This term is intended to include, but is not limited to, small activating RNA (saRNA) and trans-amplifying RNA (taRNA).

[0098] As used herein, the term "encapsulation" with respect to incorporating mRNA within a lipid nanoparticle refers to any association of the mRNA with any lipid component or compartment of the lipid nanoparticle. In one embodiment of the present disclosure, the mRNA is present within the core of the LNP.

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

[0100] As used herein, mRNA includes both modified and unmodified mRNA.In one embodiment, mRNA comprises one or more coding regions and non-coding regions.MRNA can be purified from natural sources, or produced using recombinant expression systems and optionally purified, or chemically synthesized.

[0101] In those embodiments in which the mRNA is chemically synthesized, the mRNA may include nucleoside analogs, such as analogs with chemically modified bases or sugars, and / or backbone modifications. In some embodiments, the mRNA may include nucleoside analogs such as natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deoxyur ... are or contain: zaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0102] The mRNA of the present invention can be synthesized according to any of a variety of known methods.For example, the mRNA in certain embodiments can be synthesized by in vitro transcription (IVT).Briefly, IVT is usually carried out using a linear or circular DNA template that contains a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.

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

[0104] The present disclosure may be used to formulate mRNAs of various lengths. In some embodiments, the present disclosure may be used to formulate and encapsulate in vitro synthesized mRNAs ranging in length from about 250 bp to 20 kb, about 500 bp to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about 8 to 15 kb.

[0105] Typically, mRNA synthesis involves the addition of a "cap" to the 5' end and a "tail" to the 3' end. The presence of the cap may confer resistance to nucleases found in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from degradation by exonucleases.

[0106] In some embodiments, the mRNA comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect the stability or translation of the mRNA (e.g., an iron-responsive element). In some embodiments, the 5' untranslated region can be about 50-500 nucleotides in length.

[0107] In some embodiments, the 3' untranslated region includes a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location in the cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be 50 to 500 nucleotides in length or more.

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

[0109] The mRNA sequence may include a reporter gene sequence, although including a reporter gene sequence in the pharmaceutical formulation for administration is optional. Such sequences are incorporated into the mRNA for in vivo studies in animal models to assess biodistribution.

[0110] Phosphatidylcholine lipids LNPs typically include one or more structural lipids, which refers to amphipathic lipids that allow for particle formation and typically have no net charge at physiological pH, which term includes zwitterionic lipids, which have substantially no charge at physiological pH, and phospholipids.

[0111] In some embodiments, the structured lipid is a phosphatidylcholine lipid selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and 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, DOPC, and POPC. In such an embodiment, the mixture can have a DSPC content of at least 20 or 30 mol%.

[0112] In some embodiments, the structured lipid content is more than 20mol%, more than 25mol%, more than 30mol%, more than 32mol%, more than 34mol%, more than 36mol%, more than 38mol%, more than 40mol%, more than 42mol%, more than 44mol%, more than 46mol%, more than 48mol%, or more than 50mol%. In some embodiments, the upper limit of the helper lipid content is 70mol%, 65mol%, 60mol%, 55mol%, 50mol% or 45mol%. The present disclosure also includes any combination of upper and lower limit subranges of the above numerical values.

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

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

[0115] In some embodiments, the inclusion of sphingomyelin is undesirable (see, e.g., Example 2). In some embodiments, the sphingomyelin content of the lipid nanoparticles is less than 5 mol%, less than 4 mol%, less than 3 mol%, less than 2 mol%, less than 1 mol%, less than 0.75 mol%, or less than 0.5 mol%. In some embodiments, the LNPs are "sphingomyelin-free", meaning that the LNPs are free of sphingomyelin or substantially free of sphingomyelin, meaning that they contain less than 5 mol% sphingomyelin.

[0116] In addition to phosphatidylcholine lipids, LNPs may contain additional structural lipids. For example, LNPs may contain structural lipids that have a net positive or negative charge at physiological pH. Typically, such lipids are present at less than 10 mol% or less than 5 mol%.

[0117] The structural lipid content is determined based on the total amount of lipid in the lipid nanoparticles including sterol.

[0118] Ionizable lipids The LNPs of the present disclosure have ionizable lipids. The ionizable lipids are charged at low pH and may have substantially no net charge at physiological pH. This allows electrostatic interactions between the lipids and the negatively charged nucleic acid cargo upon initial formulation. The ionizable lipids are close to neutral at physiological pH, reducing toxicity and renal clearance. After intracellular uptake via endocytosis, the acidic environment of the endosome increases the net positive charge of the ionizable amino lipids, which promotes fusion with anionic lipids in the endosomal membrane, followed by membrane destabilization and release of the nucleic acid-based therapeutic into the cytoplasm to exert its effects.

[0119] In some embodiments, it is desirable to have less than 50 mol% ionizable lipids, i.e., the ionizable lipid content can be less than 50 mol%, less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol%, or less than 5 mol%.

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

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

[0122] In some embodiments, the cationic lipid has an amino group. In some embodiments, the cationic lipid comprises a protonatable tertiary amine (e.g., pH-titratable) head group, a C16-C18 alkyl chain, an ether bond between the head group and the alkyl chain, and 0-3 double bonds. Such lipids include, but are not limited to, sulfur lipids such as MF019 and DODMA described herein. Other lipids that may be used in the practice of the present disclosure include MC3-type lipids and KC2-type lipids that are well known to those skilled in the art. In further embodiments, the ionizable lipid is selected from the group consisting of WO2022 / 246555; WO2022 / 246568; WO2022 / 24657; WO2022 / 155728; PCT / CA2023 / 050129, filed January 31, 2023; U.S. Provisional Application No. 63 / 340,687, filed May 11, 2022; U.S. Provisional Application No. 63 / 340,687, filed September 27, 2022; and U.S. Provisional Application No. 63 / 340,687, filed May 11, 2022; each of which is incorporated herein by reference. No. 63 / 410,281, filed on September 27, 2022; U.S. Provisional Application No. 63 / 410,261, filed on December 22, 2022; U.S. Provisional Application No. 63 / 434,506, filed on September 27, 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.

[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 contains a heteroatom, such as an ester or one or more sulfur atoms. In some embodiments, at least one lipophilic group comprises a distal branch and / or one or more cyclic groups. Examples of ionizable cationic lipids comprising an ionizable amino head group and two lipophilic chains (at least one chain containing one or more sulfur atoms and / or ester groups) are described in commonly 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 containing one or more heteroatoms may be biodegradable in vivo.

[0124] In one embodiment, the ionizable cationic lipid has a protonizable amino head group, at least two lipophilic moieties, the amino head group having a central nitrogen or carbon atom to which each of the two lipophilic moieties is directly attached, each lipophilic chain having a total of 15-40 carbon atoms, and the lipid has (i) a pK of 6-7.5. a and (ii) has a logP of at least 11.

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

[0126] In one embodiment, R 1 and R 2 are independently selected from linear, cyclic or branched, optionally substituted, C3-C6 alkyl groups having various degrees of unsaturation. 20alkyl, and n is 4 to 8.

[0127] In some embodiments, it is desirable to include less than 50 mol% ionizable cationic lipids in the LNPs, i.e., the ionizable cationic 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%.

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

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

[0130] Sterols In some embodiments, the LNP further comprises a sterol. The term "sterol" refers to a natural or synthetic compound that has a gonane backbone with a hydroxyl moiety attached to one of the rings, usually the A ring.

[0131] Examples of sterols include cholesterol, or cholesterol derivatives, the latter of which refers to a cholesterol molecule with a gonane structure and one or more additional functional groups.

[0132] Cholesterol derivatives include β-sitosterol, 3-sitosterol, campesterol, stigmasterol, fucosterol or stigmastanol, dihydrocholesterol, ent-cholesterol, epi-cholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 3β[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 1 ... Including cytocholesterol, 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, lumisterol, cytocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroegocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fucosterol, fecosterol, or a salt or ester thereof.

[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 lipid 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 lipid present in the lipid nanoparticle.

[0135] In another embodiment, the sterol is a cholesterol derivative 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 lipid present in the lipid nanoparticle.

[0136] In one embodiment, the sum of (i) the sterol content (e.g., cholesterol or a cholesterol derivative thereof); and (ii) the 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.

[0137] Hydrophilic polymer lipid conjugates In one embodiment, the lipid nanoparticle comprises a hydrophilic polymer lipid conjugate that can be incorporated into the LNP. The conjugate comprises a vesicle-forming lipid with a polar head group and a hydrophilic polymer chain covalently attached to the head group. Examples of hydrophilic polymers include polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinylmethylether, polyhydroxypropylmethacrylate, polyhydroxypropylmethacrylamide, polyhydroxyethylacrylate, polymethacrylamide, polydimethylacrylamide, polymethyloxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polysarcosine, and polyaspartamide. In one embodiment, the hydrophilic polymer lipid conjugate is a PEG-lipid conjugate. The hydrophilic polymer lipid conjugate is a monosialoganglioside (G M1The ability of a particular hydrophilic polymer-lipid conjugate to extend the circulatory life of the LNPs described herein can be readily determined by one of skill in the art using known methodologies.

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

[0139] In another embodiment, the PEG-lipid conjugate is present in the nanoparticle at 0.5 mol% to 5 mol%, or 0.5 mol% to 3 mol%, or 0.5 mol% to 2.5 mol%, or 0.5 mol% to 2.0 mol%, or 0.5 mol% to 1.8 mol% of the total lipid. In certain embodiments, the PEG-lipid conjugate may be present in the nanoparticle at 0 mol% to 5 mol%, or 0 mol% to 3 mol%, or 0 mol% to 2.5 mol%, or 0 mol% to 2.0 mol%, or 0 mol% to 1.8 mol% of the total lipid.

[0140] Nanoparticle preparation and morphology Delivery vehicles incorporating mRNA can be prepared using various suitable methods, such as rapid mixing / ethanol dilution process.Examples of preparation methods are disclosed in Jeffs, LB, et al., Pharm Res, 2005, 22(3):362-72; and Leung, AK, et al., The Journal of Physical Chemistry. C, Nanomaterials and Interfaces, 2012, 116(34): 18440-18450, each of which is incorporated herein by reference in its entirety.

[0141] Without being bound by theory, it can be hypothesized that the mechanism by which lipid nanoparticles containing encapsulated mRNA can be formed using the rapid mixing / ethanol dilution process begins with the formation of a dense region of hydrophobic mRNA-ionizable lipid core at low pH (e.g., pH 4), surrounded by a monolayer of helper lipid / cholesterol that fuses with smaller empty vesicles as the pH increases due to the conversion of ionizable cationic lipids to neutral forms. As the proportion of bilayer helper lipid increases, the bilayer lipids gradually form blebs, and the ionizable lipids migrate to the interior hydrophobic core. If the helper lipid content is high enough, the helper lipids that prefer the outer bilayer can form a complete lipid layer, such as a continuous or discontinuous bilayer, around the interior trapped volume.

[0142] LNPs may include a "core" region. Surprisingly, it has been observed that the core visualized by cryo-electron microscopy contains both electron-dense regions and aqueous portions or compartments, and thus the core is not homogeneous. In some embodiments, the core may be characterized as non-solid. Without being limited thereto, as observed by cryo-TEM, the electron-dense regions in the core may be partially surrounded by aqueous portions or compartments in a closed space. The aqueous portions may form distinct aqueous regions or compartments within the lipid nanoparticle. In other words, it is believed that the aqueous portions or compartments are not simply hydration layers. Such non-solid core particles are described in co-owned and co-pending WO2022 / 251959, the contents of which are incorporated herein by reference.

[0143] In one embodiment, at least about one-fifth of the core (trapped volume) comprises an aqueous portion or compartment, and the electron-dense regions within the core are partially adjacent to a lipid layer comprising a bilayer, as determined qualitatively by cryo-electron microscopy (cryo-EM). In another embodiment, at least about one-quarter of the core comprises an aqueous portion or compartment, and the electron-dense cores are each partially adjacent to a lipid layer comprising a bilayer, as determined qualitatively by cryo-electron microscopy. In a further embodiment, at least about one-third of the core comprises an aqueous portion or compartment, and the electron-dense regions are each partially adjacent to a lipid layer comprising a bilayer, as determined qualitatively by cryo-electron microscopy. In another embodiment, at least about one-half of the core comprises an aqueous portion or compartment, and the electron-dense cores are each partially adjacent to a lipid layer comprising a bilayer, as determined qualitatively by cryo-electron microscopy.

[0144] In another embodiment, the electron-dense regions of the LNPs surprisingly appear to be completely surrounded by the aqueous portion of the core as visualized by cryo-TEM microscopy, and this morphology is observed in a single plane, with some of the observed electron-dense regions adjacent to a lipid layer (e.g., bilayer) that cannot be seen because it is not in the plane that can be visualized.

[0145] In one embodiment, the electron-dense regions are approximately spherical, hi another embodiment, the electron-dense regions are hydrophobic.

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

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

[0148] In another embodiment, the mRNA and cationic lipids are present in electron dense regions. In a further embodiment, the helper lipids are present in a lipid layer that comprises the bilayer.

[0149] The lipid nanoparticles may comprise a single bilayer, or in some embodiments, a combination of bilayers and monolayers, hi one embodiment, the lipid layer is a continuous bilayer surrounding a 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 preparation comprising a plurality of lipid nanoparticles, wherein at least 20%, 30%, 40%, 50%, 60%, or 70% of the particles as determined by cryo-electron microscopy have a core with an electron-dense region and an aqueous portion or compartment, and the aqueous portion or compartment is partially surrounded by a lipid layer comprising a bilayer as visualized by cryo-electron microscopy.

[0151] In another embodiment, but not by way of limitation, the present disclosure provides a lipid nanoparticle preparation comprising a plurality of lipid nanoparticles, typically at least 10%, 20%, 30%, 40%, 50%, 60% or 70% of the particles having an elongated shape (e.g., generally elliptical) as qualitatively determined by cryo-electron microscopy. In this latter embodiment, the core electron-dense region may be partially surrounded by aqueous space as visualized by cryo-electron microscopy.

[0152] In one embodiment, the lipid nanoparticle is part of a lipid nanoparticle preparation, wherein at least 20% of the electron-dense regions of the lipid nanoparticle are (i) enveloped by aqueous moieties or (ii) partially surrounded by aqueous moieties, and a portion of the periphery of the electron-dense regions is adjacent to the lipid layer, as visualized by cryo-electron microscopy in a single plane.

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

[0154] In another embodiment, without limitation, the present disclosure provides a lipid nanoparticle preparation comprising a plurality of lipid nanoparticles, typically 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 enveloped by an adjacent aqueous space disposed between the lipid layer (e.g., bilayer) and the electron-dense region, as visualized in a single plane by cryo-electron microscopy.

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

[0156] In another embodiment, the polydispersity index (PDI) of the LNP preparation 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 an LNP preparation 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] The lipid nanoparticles of the present invention can show particularly high encapsulation efficiency of nucleic acid.As used herein, the term "encapsulation" with respect to incorporating nucleic acid in lipid nanoparticles refers to any association between any lipid component or compartment (including lipophilic part or aqueous part) of lipid nanoparticle and nucleic acid.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 present disclosure also provides lipid nanoparticles described according to the molar ratio of the positively charged amine groups (N) of the amine lipids (N) to the negatively charged phosphate groups (P) of the encapsulated oligonucleotides. This can be mathematically expressed by the formula N / P. In one embodiment, the lipid nanoparticles have an N / P ratio of 4-15, or 4.5-10, or 5-10, or 5.5-8.

[0161] In one embodiment, the lipid nanoparticles have an N / P ratio of 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 is 15, 14, 13, 12, 11, 10, 9, or 8. The disclosure also includes any two combinations of the upper and lower limits.

[0162] In one embodiment, the lipid nanoparticles have a nucleic acid weight / total lipid micromoles ratio of 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 nucleic acid weight / total lipid micromoles. In another embodiment, the upper limit is 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 weight of nucleic acid / micromoles of total lipid. The present disclosure also includes any two combinations of the upper and lower limits.

[0163] In one embodiment, the mRNA copy number / LNP is 1-10 or 4-8.

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

[0165] In one embodiment, the lipid nanoparticles increase mRNA gene expression in vivo by at least 10% when measured in extrahepatic tissues or organs.

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

[0167] As described in Example 1, the LNPs of the present disclosure may provide improved mRNA delivery to a broader range of tissues than previous Onpattro™ formulations for mRNA delivery, including but not limited to delivery to the spleen and / or bone marrow. Whether the lipid particles exhibit such enhanced delivery to specific tissues or organs may be determined by biodistribution studies in in vivo mouse models. In such embodiments, enhanced green fluorescent protein (eGFP) may be used to detect mRNA expression in specific tissues or organs. In particular, according to such embodiments, LNP mRNA systems encapsulating mRNA encoding eGFP are prepared and biodistribution and GFP expression in spleen and bone marrow cell populations are evaluated using flow cytometry after systemic administration.

[0168] To evaluate whether a particular lipid nanoparticle exhibits increased gene expression in relevant tissues or organs at 12 hours, 24 hours, 48 ​​hours, or 3 days after injection, the mRNA-LNPs of the present disclosure are compared to the Onpattro™-type formulation of Example 1. The two LNPs being compared are used to determine in vivo expression using the same experimental methods and materials as described in Example 1.

[0169] In one embodiment, the lipid nanoparticles showed in splenic and / or bone marrow immune cell population subsets at 24 hours and / or 3 days after injection when compared to lipid nanoparticles encapsulating eGFP-mRNA using an Onpattro™ type formulation of 50 / 10 / 38.5 / 1.5; mol:mol nor-MC3 / DSPC / cholesterol / PEG-lipid. exhibiting at least a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 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 encapsulated mRNA encoding enhanced GFP (eGFP) as measured in vivo, where gene expression is measured in a mouse model by detecting eGFP translated from the mRNA. Measurements are performed using flow cytometry, and gene expression is determined by quantifying the % of positive cell numbers within specific immune cell subsets, as described in Example 1. The increase is determined by comparing the percentage of positive cells (i.e., detection of eGFP) in a particular cell type in the spleen or bone marrow and comparing this percentage to the percentage of positive cells resulting from injection of the Onpattro™ formulation using otherwise identical materials and methods.

[0170] In one embodiment, the immune cell subset selected 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 subset quantified for eGFP expression is from the spleen and is monocytes / macrophages (either or both cell types), and the increase in expression is measured by determining the number of positive cells (eGFP positivity) by flow cytometry 24 hours or 3 days after injection. In another embodiment, the immune cell subset in the spleen is monocytes / macrophages, and the number of positive cells (eGFP positivity) is determined 3 days after injection.

[0171] In one embodiment, the immune cell subset selected is from bone marrow and is selected from one or more of erythrocytes, B cells, T cells, monocytes / macrophages, and neutrophils.In one embodiment, the immune cell subset quantified for expression is from bone marrow and is monocytes / macrophages or T cells, and the increase in expression is measured by determining the number of positive cells (eGFP positivity) by flow cytometry 24 hours or 3 days after injection.In another embodiment, the immune cell subset in the spleen is monocytes / macrophages or T cells, and the number of positive cells (eGFP positivity) is determined 3 days after injection.

[0172] Clinical and Non-Clinical Uses of LNPs Herein In some embodiments, the lipid nanoparticles containing mRNA are part of a pharmaceutical composition and are administered to treat and / or prevent a disease state. Treatment can have a preventive, ameliorative, or therapeutic effect. The pharmaceutical composition is administered in any suitable dose.

[0173] The LNPs described herein may be used to treat and / or prevent any disease, disorder, or condition in a mammal, including cancer, infectious diseases such as bacterial, viral, fungal, or parasitic infections, inflammatory and / or autoimmune diseases (including treatments that induce immune tolerance), and cardiovascular diseases such as hypertension, arrhythmias, and restenosis.

[0174] Examples of cancer include lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, stomach (gastric) cancer, esophageal cancer, gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer (e.g., renal cell carcinoma), cancer of the central nervous system, glioblastoma, skin cancer, lymphoma, choriocarcinoma, head and neck cancer, osteosarcoma, 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] Other non-limiting examples of diseases, disorders or conditions treatable by the mRNA-LNPs herein and which may be due, at least in part, to immune disease include colitis, Crohn's disease, allergic encephalitis, allograft / graft-versus-host disease (GVHD), diabetes, and multiple sclerosis.

[0176] The LNPs herein may be used for other applications besides the treatment and / or prevention of a disease or disorder. The LNPs may be used to treat conditions such as aging, preventive medicine, and / or as part of a personalized medicine plan. In further embodiments, the LNPs are used for diagnostic applications.

[0177] In one embodiment, the LNP is part of a pharmaceutical composition that is administered parenterally, i.e., intraarterially, 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, subretinal, intrathecal, or via other local routes.

[0178] The pharmaceutical composition comprises a pharma- ceutically acceptable salt and / or excipient.

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

[0180] The following examples are intended to illustrate the preparation of particular lipid nanoparticle mRNA preparations and their properties and are not intended to limit the scope of the invention. EXAMPLES

[0181] Materials and Methods Preparation of LNPs LNPs were prepared by dissolving mRNA in 25 mM sodium acetate (pH 4.0) and the lipid components at the indicated mol % were dissolved in absolute ethanol. Lipids in ethanol and eGFP mRNA in buffer were mixed in a 1:3 volume ratio using a dual-syringe T-junction. The solution was extruded through the T-junction at a total flow rate of 20 mL / min (5 mL / min for the lipid-containing syringe and 15 mL / min for the mRNA-containing syringe). The mixture was subsequently dialyzed overnight against approximately 100x volume of 1x phosphate-buffered saline (pH 7.4) using a Spectro / Por dialysis membrane (molecular weight cut-off 12,000–14,000 Da). LNPs were concentrated as required using Amicon Ultra™ 10 000 MWCO (molecular weight cut-off) regenerated cellulose concentrators.

[0182] The encapsulation efficiency was calculated using the mRNA-LNP(F i The unencapsulated mRNA content was determined by measuring the fluorescence upon addition of RiboGreen™ to the 2% Triton X-100 (F t ) was calculated relative to the total mRNA content obtained by dissolving the LNPs in water: % encapsulation = (F t -F i ) / F t x100.

[0183] Particle size and polydispersity index (PDI) were characterized using a Zetasizer Nano ZS™.

[0184] In vivo studies by flow cytometry LNPs with eGFP mRNA concentration of 0.1 mg / mL were injected intravenously (iv) into mice in a volume of 10 μL per mouse formula dose (grams). Spleens and bone marrow were harvested 4 hours, 1 day, and 3 days after LNP injection.

[0185] Spleens and bone marrow were harvested and processed into single cell suspensions. Specifically, mice were anesthetized with 5% isoflurane until loss of reflexes, then exposed to 1% CO2 in air. Spleens and femurs were isolated. Spleens were homogenized and passed through a 70 μm sieve into single cell suspensions. Femurs were centrifuged at 3810 g for 30 s to isolate bone marrow, which was then resuspended in FACS buffer (1x sterile PBS (pH 7.4), 2.5 mM ethylenediaminetetraacetic acid (EDTA), 0.05% (w / v) sodium azide (NaN3), 2% (v / v) bovine serum albumin (BSA)).

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

[0187] Flow cytometry data were analyzed using FlowJo™ version 10 (Becton Dickension™ & Company (BD)). Corresponding immune cell subsets were identified based on the gating schemes shown in Tables 2 and 3 above. eGFP positive (eGFP + Bar graphs of the percentage of eGFP cells versus eGFP mean fluorescence intensity (MFI) were generated using Prism™ version 8 (GraphPad™) software. The gating schemes for spleen and bone marrow are shown in Tables 1 and 2 below.

[0188] Tissue homogenate assay Tissues were removed from mice, placed in 2 mL tubes, and flash frozen in liquid nitrogen. Tissues were then stored at -80°C. Promega™ GLO™ lysis buffer was added to each tube in an appropriate volume to ensure samples remained frozen before adding lysis buffer. Samples were placed in a FastPrep™ homogenizer and the homogenizer was operated at a speed of 6 m / s for 20 seconds, repeated twice for a total of three rounds. Homogenized samples were centrifuged at 12,000 rpm for 10 minutes at room temperature, after which 50 μL of homogenate was added in duplicate to a black plate. The plate was transferred to a plate reader and fluorescence was read at 640 nm excitation / 720 nm emission. Luminescence was measured by adding 50 μL of SteadyGlo™ substrate to the homogenate samples to read the luciferase signal.

[0189] Example 1: Extrahepatic mRNA gene expression in immune cell subsets is significantly increased with the use of high DSPC LNPs The effect of increasing the amount of DSPC from 10 mol% to 50 mol% in 4-component LNPs containing enhanced green fluorescent protein mRNA ("eGFP mRNA") cargo was evaluated in vivo. As shown below, LNPs with increased DSPC levels compared to the Onpattro™ formulation showed significantly increased (>>10%) gene expression in macrophages, monocytes and / or T cells from spleen and / or bone marrow harvested 24 hours and 3 days post-injection compared to the Onpattro™ type formulation.

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

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

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

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

[0194] [Table 2]

[0195] [Table 3]

[0196] The results of mRNA expression detected in splenic sub-cell populations are shown in Figures 1A-F and Figures 2A-C.

[0197] The results of mRNA expression in bone marrow are shown in Figures 3A to 3F.

[0198] As illustrated, the results in Figure 1A-C show that spleens of mice treated with GFP mRNA-containing lcLNP™ with 50 mol% DSPC showed a greater than 20% increase in monocyte and macrophage subpopulations at 4 hours, 24 hours, and 3 days compared to the Onpattro™ formulation containing only 10 mol% DSPC. Similar results were observed at the same time points when the data was plotted using the measured MFI of GFP (Figure 1D-F).

[0199] Figures 2A-2C show representative scatter plots of eGFP expression levels in monocyte / macrophage cell populations in the spleen at single cell resolution at 24 hours post-injection for the data shown in Figures 1B and 1E. To better visualize, mRNA-lcLNP™ with 50 mol% DSPC contained significantly more eGFP than mRNA-Onpattro™. + The cells contained eGFP + There were two-fold more cells in the mRNA-lcLNP™ than in the mRNA-Onpattro™ (eGFP positive cells were 18.5% in the Onpattro™ and 39.3% in the lcLNP™).

[0200] Similarly, Figure 3A-C shows that in the bone marrow at 24 hours and 3 days after injection, monocyte and macrophage subpopulations of mice treated with mRNA-lcLNPs containing 50 mol% DSPC were significantly higher than green fluorescent protein positive cells (GFP + The data show that green fluorescent protein positive cells (GFP) were increased by over 20% compared to the Onpattro™ formulation. Similar results were observed after 24 hours and 3 days when the data was plotted using the measured MFI of GFP (Figure 3D-F). Furthermore, T cell subpopulations in the bone marrow of mice treated with mRNA-lcLNP containing 50 mol% DSPC showed a significant increase in green fluorescent protein positive cells (GFP) compared to the Onpattro™ formulation at time points greater than 24 hours. + cells) increased by more than 20% (Figures 3B, 3C, 3E, and 3F).

[0201] Figures 4A-4C show representative scatter plots of eGFP expression levels in T cell populations in bone marrow at single cell resolution at 24 hours post-injection for the data shown in Figures 3B and 3E. As better visualized, eGFP expression levels in mRNA-lcLNP™ compared to mRNA-Onpattro™ in T cell populations after 24 hours were significantly higher. + There was a five-fold increase in cells (eGFP-positive cell counts were 2.98% in Onpattro™ and 19.5% in lcLNP™ containing 50 mol% DSPC).

[0202] Example 2: Use of high DSPC LNPs incorporating various ionizable lipids significantly increases mRNA gene expression in immune cell subsets Example 1 shows that increasing DSPC content in a four-component mRNA-LNP system containing the ionizable sulfur lipid MF019 results in a significant increase in extrahepatic mRNA expression. To examine the effect of increasing DSPC in a four-component LNP system containing a different ionizable lipid, mRNA gene expression in spleen and bone marrow was examined in the same formulation as in Example 1, but incorporating the ionizable lipid MC3 (DLin-MC3-DMA) instead.

[0203] As described below, similar trends in bone marrow and spleen mRNA expression were observed as DSPC content increased, but the ionizable lipid in the formulation was MC3.

[0204] The following two four-component eGFP mRNA formulations containing MC3 ionizable lipid, DSPC, cholesterol, and PEG-lipid were compared. [Table 4]

[0205] As shown, the bar graph in Figure 5A shows that at 24 hours, T cell, monocyte and macrophage subpopulations, as well as neutrophils, in the bone marrow of mice treated with GFP mRNA-containing lcLNP™ with 50 mol% DSPC showed a greater than 20% increase in GFP-positive cells over the Onpatro™ formulation with only 10 mol% DSPC. The same general trend was observed when the data was plotted using the measured MFI of GFP (Figure 5B).

[0206] Figures 6A-6C show representative scatter plots showing eGFP expression levels of T cell populations at single cell resolution in bone marrow for the data shown in Figures 5A and 5B. To allow better visualization, mRNA-lcLNP™ with 50 mol% DSPC (Figure 6C) had significantly more eGFP positive cells than mRNA-Onpattro™ LNPs (Figure 6B). eGFP positive cells were at least 4-fold greater for mRNA-lcLNP™ than for mRNA-Onpattro™ (Figure 6B shows that only 4.75% of cells were positive for eGFP, compared to 18.3% in Figure 6C).

[0207] FIG. 7 is a bar graph showing that at 24 hours, monocyte and macrophage (mono / macs) subpopulations in the spleens of mice treated with GFP mRNA-containing lcLNP™ with 50 mol% DSPC showed approximately a 2-fold increase in GFP-positive cells than the Onpatro™ formulation with only 10 mol% DSPC.

[0208] Figures 8A-8C show representative scatter plots showing eGFP expression levels of monocyte / macrophage cell populations at single cell resolution in the spleen for the data shown in Figure 7. mRNA-lcLNP™ with 50 mol% DSPC (Figure 8C) had significantly more eGFP positive cells than mRNA-Onpattro™ (Figure 8B). eGFP positive cells were at least 2-fold greater for mRNA-lcLNP™ than for mRNA-Onpattro™. (In Figure 8B, 5.78% of cells were positive, while in Figure 8C, 12.4% were positive for eGFP).

[0209] Example 3: mRNA gene expression in immune cell subsets is significantly increased with DSPC-rich LNPs but not with sphingomyelin The effect of increasing the content of another neutral lipid, egg sphingomyelin (ESM), on in vivo mRNA expression was evaluated using the same formulations as in Examples 1 and 2, but substituting ESM for DSPC. Surprisingly, increased mRNA expression in extrahepatic tissues in high DSPC LNPs was not observed in LNPs with elevated levels of ESM.

[0210] The formulations investigated are shown in Table 5 below. [Table 5]

[0211] As illustrated, the bar graph in Figure 9A shows that 24 hours after injection, cell subpopulations in the bone marrow (erythrocytes, B cells, T cells, monocytes / macrophages, and neutrophils) of mice treated with GFP mRNA-containing lcLN™ with 50 mol% DSPC showed a >20% increase in GFP-positive cells in most cell types measured for the same formulations with egg sphingomyelin (ESM) as the neutral lipid. The same general trend was observed when the data was plotted using the measured MFI of GFP (Figure 9B).

[0212] Figures 10A and 10B show representative scatter plots depicting eGFP expression levels of T cell populations at single cell resolution in bone marrow for the data shown in Figures 9A and 9B. To better visualize, mRNA-lcLNP™ with 50 mol% DSPC (Figure 10A) had approximately 4-fold more eGFP positive cells (eGFP + 1.0%) than the egg sphingomyelin (ESM) formulation (Figure 10B). + was 18.3 for lcLNP™ and 4.26 only for ESMLNP).

[0213] FIG. 11A is a bar graph showing that various subpopulations in the spleens of mice treated with GFP mRNA-containing lcLNP™ with 50 mol% DSPC at 24 hours showed an increase in GFP-positive cells compared to the otherwise identical formulation containing ESM.

[0214] The increase in EGF mRNA positive cells in monocytes and macrophages was approximately six-fold greater in DSPC LNPs compared to ESM LNPs (Figure 11A). When the same data was plotted to show mean fluorescence intensity (MFI; Figure 11B), the same general trend was observed.

[0215] Figures 12A and 12B show representative scatter plots showing eGFP expression levels of monocyte / macrophage cell populations at single cell resolution in the spleen for the data shown in Figure 11. mRNA-lcLNP™ with 50 mol% DSPC (Figure 12A) had significantly more eGFP-positive cells than the same formulation where DSPC was replaced with egg sphingomyelin (Figure 12B). eGFP-positive cells were at least 20-fold greater for mRNA-lcLNP™ with DSPC than for ESM LNP.

[0216] Example 4: mRNA gene expression is significantly increased with high DSPC LNPs in various tissues and organs, reducing hepatotoxicity This example demonstrates that LNPs with high levels of DSPC show increased mRNA expression in the spleen, lung, kidney, heart, bone marrow, abdomen, back and ear 24 hours after injection compared to Onpattro™ type formulations.

[0217] The LNPs in Table 6 below were examined. [Table 6]

[0218] The N / P for each formulation was 9, and the control was phosphate buffered saline (PBS). Luciferase and LNP mRNA coding for were administered to CD-1 mice at 1 mg / kg and 5 mg / kg, and tissue homogenates were quantified for luciferase 24 hours after administration. The ionizable cationic lipid MF019 is described in WO2022 / 155728A1. The ionizable cationic lipid NTx-C16 has the following structure: [ka]

[0219] Figures 13A and 13B show that the fold change for lcLNP™ samples with greater than 50 mol% DSPC versus Onpatro™-type formulations with 10 mol% DSPC was greater than 1 for each extrahepatic organ investigated at both doses of 1 mg / kg and 5 mg / kg, respectively.

[0220] FIG. 13C shows the serum activity (U / L) of enzymes that correlate with hepatotoxicity for the lcLNP™ samples and Onpatro™ from Table 6 above. In particular, the figure shows the levels of liver serum enzymes alanine transaminase (ALT) (left) and aspartate transaminase (AST) (right) 24 hours after injection of CD-1 mice with a dose of 5 mg / kg LNP-mRNA encoding luciferase. Serum ALT and AST activities (U / L) were elevated after injection of Onpatro™ (nMC3), whereas their activities were relatively low after injection of lcLNP™ samples with 50 mol% DSPC. Since ALT and AST are markers of hepatotoxicity, LNPs with elevated phosphatidylcholine show reduced hepatotoxicity compared to Onpatro™.

[0221] Example 5: mRNA gene expression of LNPs with elevated levels of various phosphatidylcholine lipids The following in vivo data demonstrates that LNPs with elevated levels of various phosphatidylcholine lipids show increased mRNA expression in spleen, heart, lung, kidney, muscle tissue, bone marrow (BM) and ear compared to Onpattro™-type formulations.

[0222] The following mRNA-LNPs were prepared with high levels of DSPC, DOPC, POPC, DPPC, and combinations of DSPC and DOPC at various molar ratios as shown in Table 7 below. [Table 7]

[0223] The luciferase-encoding mRNA and N / P of each formulation are shown below. Tissue homogenate luciferase levels were measured in CD-1 mice 24 hours after injection as previously shown. The ionizable cationic lipid nMC3 is described as described in co-owned and co-pending WO2022 / 246571, and the C-109 ionizable lipid is described in U.S. Provisional Application No. 63 / 410,261, filed September 27, 2022 (Compound 7 with a ketal head group), which is incorporated herein by reference.

[0224] The particle size and polydispersity index (PDI), as well as the % entrapment of mRNA for each formulation (A-J in Table 7 above) are shown in FIG. 14A.

[0225] FIG. 14B shows that luminescence intensity / mg liver was elevated for the Onpattro™-type formulation (LNP A), while the remaining LNPs with various levels of DSPC, DOPC, POPC, DPPC at 50 mol%, or a combination of DSPC and DOPC (combined content 50 mol%) were close to baseline levels.

[0226] Figures 14C-I show the luminescence intensity / mg organ or tissue for spleen, heart, lung, kidney, muscle, bone marrow (BM) and ear at 24 hours after injection. LNPs B and C with 50 mol% DSPC showed the highest luminescence intensity / mg organ or tissue compared to Onpatro™ in extrahepatic organs / tissues, but LNP F with 50 mol% DPPC also had acceptable luminescence intensity in many of the organs / tissues tested (spleen, heart, kidney, bone marrow (BM) and ear). Dioleoylphosphatidylcholine (DOPC and POPC) LNPs (LNPs D and E) showed low luminescence intensity in the liver of each organ / tissue, but LNPs containing a mixture of dioleoylphosphatidylcholine (DOPC) and DSPC (LNPs G-J) showed high luminescence intensity in many of the organs / tissues examined in this study (see Figures 14C-I). These results indicate that LNPs with elevated levels of phosphatidylcholine lipids with saturated chains and phase transition temperatures above physiological temperatures (DSPC and DPPC) improve delivery of mRNA beyond the liver compared to LNPs with unsaturated chains (DOPC or POPC) and phase transition temperatures below physiological temperatures. However, combinations of phosphatidylcholine lipids with high phase transition temperatures (e.g., DSPC or DPPC) and low phase transition temperatures (e.g., DOPC or POPC) may provide improved extrahepatic delivery as well (e.g., LNPs G, H, and I shown in Figures 14C, 14D).

[0227] Example 6: Various LNPs with high phosphatidylcholine content (30 mol% or higher) and different ionizable lipids show enhanced extrahepatic mRNA expression in vivo The in vivo data below demonstrates that LNPs formulated with elevated levels of phosphatidylcholine lipids (e.g., 30-50 mol% DSPC) and a variety of different ionizable cationic lipids demonstrate enhanced expression of mRNA in the bone marrow (BM), spleen, skin, heart, lung, small intestine (SI), cecum, and colon.

[0228] A panel of mRNA LNPs, shown in Table 8, was prepared with varying PC content, different ionizable cationic lipids and N / P. The encapsulated mRNA encodes luciferase. Tissue homogenate luciferase levels were measured 24 hours after injection in CD-1 mice as previously shown. [Table 8-1] [Table 8-2]

[0229] The ionizable cationic lipids used in the panel of LNPs are shown in Table 9 below. [Table 9]

[0230] In vivo data for tissue homogenates of liver, spleen, bone marrow (BM), abdominal skin, heart, lung, small intestine (SI), cecum and colon where DSPC was elevated (>30 mol%) in the LNP panel of Table 8 are shown in Figures 15A-15H.

[0231] Example 7: LNPs with high phosphatidylcholine content have unique morphology Cryo-TEM images of lipid nanoparticles composed of MF019 / DSPC / Chol / PEG-DMG (27.4 / 50 / 21.1 / 1.5 mol:mol) encapsulating mRNA encoding luciferase were obtained and are shown in FIG.

[0232] Images of lipid nanoparticles encapsulating mRNA with high levels of phosphatidylcholine lipids have a morphology with electron-dense regions contained within the bilayer. The core is then surrounded by a structure consistent with a lipid bilayer, as shown in Figure 16. The morphology unique to LNPs with increased phosphatidylcholine lipids may result in improved in vivo delivery properties of LNPs to extrahepatic (non-liver) target sites, as observed in previous examples.

[0233] These examples are intended to illustrate the preparation of specific lipid nanoparticle mRNA preparations and their properties and are not intended to limit the scope of the invention.

[0234] As used herein, the articles "a" or "an" are intended to include both the singular and the plural, unless otherwise specified.

Claims

1. Lipid nanoparticles for extrahepatic delivery of mRNA, wherein the lipid nanoparticles are: (i) mRNA and; (ii) Phosphatidylcholine lipids in an amount of 30 mol% to 70 mol%; (iii) an ionizable cationic lipid in an amount of 5 mol% to 50 mol%; (iv) Sterols selected from cholesterol or its derivatives; (v) A hydrophilic polymer lipid conjugate present with a lipid content of 0.5 mol% to 5 mol%; Each lipid content is a relative value to the total lipid content of the lipid nanoparticles, wherein the lipid nanoparticles are lipid nanoparticles.

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

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

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

5. The lipid nanoparticles according to claim 1, wherein the phosphatidylcholine is a mixture of two phosphatidylcholine lipids.

6. The lipid nanoparticles according to claim 5, wherein the mixture comprises distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylcholine (DOPC).

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

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

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

10. The lipid nanoparticle according to claim 1 or 2, wherein the ionizable lipid is an aminolipid.

11. The lipid nanoparticles according to claim 1 or 2, wherein the ionizable cationic lipid is present in an amount of less than 40 mol%.

12. The lipid nanoparticle according to claim 1 or 2, wherein the hydrophilic polymer lipid conjugate is a polyethylene glycol lipid conjugate.

13. The lipid nanoparticles according to claim 1 or 2, wherein the sterol is present in an amount of 15 mol% to 45 mol% based on the total lipids present in the lipid nanoparticles.

14. The lipid nanoparticles according to claim 1 or 2, wherein the sterol is present in an amount of 18 mol% to 40 mol% based on the total lipids present in the lipid nanoparticles.

15. The lipid nanoparticles according to claim 1, wherein, when measured in extrahepatic tissue or organ, the gene expression of the mRNA increases by at least 10% in vivo, and optionally, the extrahepatic tissue or organ is the spleen, bone marrow, lung, kidney, heart, abdominal skin, back skin and / or ear.

Images