lipid nanoparticles

Abstract

The present invention relates to the field of lipid nanoparticles (LNPs), and more specifically to lipid nanoparticles comprising ionized lipids, phospholipids, sterols, PEG lipids, and one or more nucleic acids. The LNPs of the present invention are characterized by having an ionized lipid content exceeding 55 mol% and a cholesterol-to-phospholipid ratio of 1:1 or less, for example, about 1:2 or less. The present invention provides for the use of such LNPs for the extrahepatic delivery of nucleic acid molecules, particularly mRNA; thereby, they are highly suitable for use in the treatment of cancer, autoimmune diseases, hereditary disorders, hematological disorders, aging, fibrosis, neurological disorders, cardiovascular diseases, or infectious diseases.

Description

Technology Field

[0001] The present invention relates to the field of lipid nanoparticles (LNPs); more specifically, to lipid nanoparticles comprising ionizable lipids, phospholipids, sterols, PEG lipids, and one or more nucleic acids. The LNPs of the present invention are characterized by having an ionizable lipid content of more than 55 mol% and a cholesterol-to-phospholipid ratio of 1:1 or less, for example, about 1:2 or less. The present invention provides the use of said LNPs for the extrahepatic delivery of nucleic acid molecules, particularly mRNA, and is accordingly highly suitable for use in treating cancer, autoimmune diseases, genetic disorders, hematologic disorders, aging, fibrosis, neurological disorders, cardiovascular diseases, or infectious diseases. Background Technology

[0002] One of the major challenges in the field of targeted delivery of biologically active substances is often their instability and low cell penetration potential. This is a particularly prominent issue in the delivery of nucleic acid molecules, especially (m)RNA molecules. Therefore, suitable packaging is crucial for proper protection and delivery. Consequently, there is a continuous demand for methods and compositions for packaging biologically active substances, such as nucleic acids.

[0003] In this regard, lipid-based nanoparticle compositions, such as lipoplexes or liposomes, have been used as packaging carriers to transport biologically active substances into cells and / or intracellular compartments. These lipid-based nanoparticle compositions generally comprise mixtures of various lipids, such as cationic lipids, ionized lipids, phospholipids, structural lipids (e.g., sterols or cholesterol), PEG (polyethylene glycol) lipids, ... (as reviewed in the literature [Reichmuth et al., 2016]).

[0004] Lipid-based nanoparticles (LNPs) composed of four lipid mixtures—cationic or ionized lipids, phospholipids, sterols, and PEGylated lipids—were developed to deliver siRNA and mRNA to the liver non-immunogenically after systemic administration. While numerous such lipid compositions are known in the field, in vivo ( in vivo Compositions used for mRNA delivery generally contain an excess of cholesterol relative to phospholipids; for example, Onpattro-type formulations have a molar ratio of ionized lipids / phospholipids / cholesterol / PEG-lipids of 50 / 10 / 38.5 / 1.5, and accordingly, a molar ratio of cholesterol to phospholipids of approximately 4:1. Furthermore, LNPs containing lipids in this molar ratio are known to accumulate primarily in hepatic tissue. If LNPs could accumulate in organs and tissues other than the liver, such as bone marrow and / or the spleen, the clinical utility of such delivery systems could be greatly expanded. Accordingly, the present invention aimed to provide an improved LNP that exhibits increased spleen and bone marrow targeting compared to liver targeting.

[0005] Although research in the field of LNP is currently very active, only a small number of research groups have investigated the effects of reversing the cholesterol-to-phospholipid ratio—that is, providing LNPs with an excess of phospholipids relative to cholesterol. For example, WO2023184038 presents LNPs with cholesterol-to-phospholipid ratios of approximately 1:1 and 1:2 by combining relatively low levels of ionized lipids (up to 45 mol%) and a fixed amount of PEG lipids of 1.5 mol%). However, these researchers focused on the effects of specific molar percentages of various lipids rather than investigating the effect of lipid ratios. They concluded that extrahepatic delivery increases when providing LNPs containing high molar concentrations (30 to 70 mol%) of phospholipids in combination with low ionized lipid content of 5 mol% to 50 mol%. The preferred combination disclosed in the literature includes a phospholipid content of 45 mol% to 55 mol% and an ionized lipid content of less than 40 mol%. Various variants were manufactured, but only one LNP with low ionized lipid content (lcLNP) was tested, which had a molar / molar ratio of ionized lipid / phospholipid / cholesterol / PEG-lipid of 27.4 / 50 / 21.1 / 1.5.

[0006] Additionally, the literature [Kawaguchi et al., 2023 (Journal of Pharmaceutical Sciences* 112; p. 1401-1410)] evaluated the liver targeting of various LNPs and concluded that protein expression in the liver decreased when the molar percentage of cholesterol was reduced from 40 mol% to 20 mol% and 10 mol% following intramuscular or subcutaneous administration of mRNA-LNP. The various conditions used in the test involved a cholesterol-to-phospholipid ratio ranging from approximately 1:4 to 4:1, combining relatively low levels of ionized lipids (up to 52.5 mol%) with a fixed amount of PEG lipids at 1.5 mol%. Similarly, in this study as well, a specific analysis of the effect of the cholesterol-to-phospholipid ratio was not performed.

[0007] WO2022251953 relates to an LNP that exhibits increased expression in target tissues such as the spleen, bone marrow, and / or liver, and shows a preference for the liver compared to spleen and / or bone marrow targeting. Unlike the purpose of the present invention, WO2022251953 does not teach or suggest anything regarding an LNP that prefers spleen and / or bone marrow targeting over liver targeting.

[0008] The inventors have surprisingly discovered that LNPs having relatively high levels of ionized lipids (i.e., at least 55 mol%) and a cholesterol-to-phospholipid ratio of 1:1 or less increase the targeting of LNPs to both the spleen and bone marrow. Further increased bone marrow targeting is achieved when one or more of the following conditions are met: 1) the cholesterol-to-phospholipid ratio is about 1:2 or less, 2) the mol% of ionized lipids is at least 60 mol%, and / or 3) the mol% of PEG-lipids is greater than 1.5 mol% (e.g., 3.0 mol%).

[0009] Summary of the Invention

[0010] In a first embodiment, the present invention provides lipid nanoparticles (LNPs) comprising ionized lipids, phospholipids, sterols, stabilized lipids; and one or more nucleic acid molecules;

[0011] - The above LNP comprises at least 55 mol% of the above ionized lipid;

[0012] - The ratio of the above sterol to phospholipid is 1:1 or less,

[0013] It is characterized by.

[0014] Specifically, the present invention provides lipid nanoparticles (LNPs) comprising ionized lipids, phospholipids, sterols, PEG lipids, and one or more nucleic acid molecules;

[0015] - The above LNP comprises at least 55 mol% of the above ionized lipid;

[0016] - The ratio of the above sterol to phospholipid is 1:1 or less,

[0017] It is characterized by.

[0018] In particular, the present invention provides lipid nanoparticles (LNPs) comprising ionized lipids, phospholipids, cholesterol, PEG lipids; and one or more nucleic acid molecules, and

[0019] - The above LNP comprises at least 55 mol% of the above ionized lipid;

[0020] - The above cholesterol to phospholipid ratio is 1:1 or less,

[0021] It is characterized by.

[0022] In a specific embodiment, the lipid nanoparticles (LNP) defined in this specification comprise a sterol-to-phospholipid ratio of 1:2 or less, preferably a cholesterol-to-phospholipid ratio of 1:2 or less.

[0023] In a further embodiment, the LNP of the present invention comprises at least 0.1 mol% of the stabilized lipid, particularly at least 0.5 mol% of the stabilized lipid, at least 1.0 mol% of the stabilized lipid, 1.5 mol% of the stabilized lipid, particularly at least 2.0 mol% of the stabilized lipid, more particularly at least 2.5 mol% of the stabilized lipid, at least 3.0 mol% of the stabilized lipid, at least 4.0 mol% of the stabilized lipid, at least 5.0 mol% of the stabilized lipid, at least 6.0 mol% of the stabilized lipid, at least 7.0 mol% of the stabilized lipid, at least 8.0 mol% of the stabilized lipid, at least 9.0 mol% of the stabilized lipid, and at least 10.0 mol% of the stabilized lipid.

[0024] In a further embodiment, the LNP of the present invention comprises at least 0.1 mol% of the PEG lipid, particularly at least 0.5 mol% of the PEG lipid, at least 1.0 mol% of the PEG lipid, at least 1.5 mol% of the PEG lipid, particularly at least 2.0 mol% of the PEG lipid, more particularly at least 2.5 mol% of the PEG lipid, at least 3.0 mol% of the PEG lipid, at least 4.0 mol% of the PEG lipid, at least 5.0 mol% of the PEG lipid, at least 6.0 mol% of the PEG lipid, at least 7.0 mol% of the PEG lipid, at least 8.0 mol% of the PEG lipid, at least 9.0 mol% of the PEG lipid, and at least 10.0 mol% of the PEG lipid.

[0025] In another specific embodiment, the molar percentage of ionized lipids in the LNP of the present invention is at least 60 mol%, e.g., at least 65 mol%, at least 70 mol%, at least 75 mol%, at least 80 mol%, or at least 85 mol%.

[0026] In further embodiments, the molar percentage of phospholipids in the LNP of the present invention is greater than 1 mol%, particularly 2 mol% to 30 mol%, more particularly 5 mol% to 28 mol%; more particularly 10 mol% to 27 mol%; and even more particularly 20 mol% to 26 mol%.

[0027] In an additional embodiment, the molar percentage of sterol, preferably cholesterol, in the LNP of the present invention is less than 22 mol%, particularly 10 mol% to 15 mol%, more particularly 12 mol% to 13 mol%.

[0028] In additional embodiments, the PEG lipid in the LNP of the present invention is selected from a list including DMG-PEG and DSG-PEG; in particular, it is DMG-PEG.

[0029] In further embodiments, the present invention provides lipid nanoparticles as defined herein, wherein the phospholipid is selected from a list comprising DOPE, DOPC, DSPC, ESM and mixtures thereof; in particular, DSPC.

[0030] In a further embodiment, the present invention provides lipid nanoparticles as defined herein, wherein the sterol is selected from the group comprising cholesterol, ergosterol, campesterol, oxysterol, antrosterol, cholecalciferol, desmostol, nicasterol, sitosterol, and stigmasterol; preferably, cholesterol.

[0031] In a very specific embodiment, the present invention provides an LNP comprising the following:

[0032] - Approximately 60 mol% of the above ionized lipids;

[0033] - Approximately 25.67 mol% of DSPC;

[0034] - Approximately 12.83 mol% cholesterol; and

[0035] - Approximately 1.5 mol% of DMG-PEG.

[0036] In further specific embodiments, the present invention provides an LNP comprising the following:

[0037] - Approximately 60 mol% of the above ionized lipids;

[0038] - Approximately 24.17 mol% of DSPC;

[0039] - Approximately 12.83 mol% cholesterol; and

[0040] - Approximately 3.0 mol% of DMG-PEG.

[0041] In a further embodiment, the present invention provides a pharmaceutical composition or vaccine comprising one or more lipid nanoparticles and a pharmaceutically acceptable carrier as defined herein.

[0042] The present invention also provides lipid nanoparticles, pharmaceutical compositions, or vaccines as defined herein for use in human medicine or veterinary medicine, particularly for use in the treatment of cancer, autoimmune diseases, hereditary disorders, hematological disorders, aging, fibrosis, neurological disorders, cardiovascular diseases, or infectious diseases.

[0043] The present invention also relates to the spleen and / or bone marrow in vivo ( in vivo ) Provides a pharmaceutical composition or vaccine comprising one or more lipid nanoparticles as defined herein for use in increasing the gene expression of nucleic acid molecules.

[0044] Finally, the present invention provides nucleic acid molecules to a subject in need within the body ( in vivo A method for delivering ) is provided, the method comprising the step of administering lipid nanoparticles or a pharmaceutical composition such as that defined in this specification to a subject. Brief explanation of the drawing

[0045] Now, referring specifically to the drawings, it is emphasized that the details shown in the drawings are merely illustrative and are presented solely for the purpose of illustrative discussion regarding various embodiments of the present invention. These drawings are presented for the purpose of providing what is considered to be the most useful and easiest explanation of the principles and conceptual aspects of the present invention. Accordingly, there is no intention to show structural details beyond the level necessary for a fundamental understanding of the present invention. This description provided in conjunction with the drawings is intended to enable a person skilled in the art to clearly understand how various forms of the present invention can be practically implemented. Fig. 1. Spleen / liver ratio of mRNA expression when Fluc mRNA LNPs with different molar ratios of ionized lipids, phospholipids, sterols, and PEG-lipids were intravenously injected. The condition with a cholesterol-to-phospholipid ratio of 3.9:1 corresponds to an LNP with a standard lipid molar ratio (INL1 / DSPC / Chol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5) used in the Onpattro formulation. Fig. 2. Luciferase activity in bone marrow when Fluc mRNA LNPs with different molar ratios of ionized lipids, phospholipids, sterols, and PEG-lipids were intravenously injected. The condition with a cholesterol-to-phospholipid ratio of 3.9:1 corresponds to the LNP with the standard lipid molar ratio used in the Onpattro formulation. Fig. 3. Spleen / liver ratio of mRNA expression when Fluc mRNA LNPs with different molar ratios of ionized lipids, phospholipids, sterols, and PEG-lipids were intravenously injected. LNP 5 corresponds to an LNP with a cholesterol-to-phospholipid ratio of 3.9:1 and has the standard lipid molar ratio used in the Onpattro formulation. Fig. 4.Luciferase activity in bone marrow when Fluc mRNA LNPs with different molar ratios of ionized lipids, phospholipids, sterols, and PEG-lipids were intravenously injected. LNP 5 corresponds to an LNP with a cholesterol-to-phospholipid ratio of 3.9:1 and has the standard lipid molar ratio used in the Onpattro formulation. Fig. 5. Luciferase activity in bone marrow when Fluc mRNA LNPs with different molar ratios of ionized lipids, phospholipids, sterols, and PEG-lipids were intravenously injected. LNP 5 corresponds to an LNP with a cholesterol-to-phospholipid ratio of 3.9:1 and has the standard lipid molar ratio used in the Onpattro formulation. Fig. 6 Figure 4 shows the spleen / liver ratio of mRNA expression when Fluc mRNA LNP with the molar ratios of ionized lipids, phospholipids, sterols, and PEG-lipids presented in Table 4 was intravenously injected. A total of five different ionized lipids were used, and the formulation with a cholesterol-to-phospholipid ratio of 3.9:1 has the standard lipid molar ratio used in the Onpattro formulation. Fig. 7 Figure 5 shows the spleen / liver ratio of mRNA expression when Fluc mRNA LNPs with different molar ratios of ionized lipids, phospholipids, sterols, and PEG-lipids presented in Table 5 were intravenously injected. Two different ionized lipids were used in the formulations, and the formulation with a cholesterol-to-phospholipid ratio of 3.9:1 has the standard lipid molar ratio used in the Onpattro formulation. Fig. 8 This example is for an INL 2-containing formulation and shows the amount of Thy1.1-positive cells in a specific hepatocyte type 16 hours after IV injection, measured by flow cytometry. A formulation with a cholesterol-to-phospholipid ratio of 3.9:1 has the standard lipid molar ratio used in the Onpattro formulation. Fig. 9Figure 1 represents the ratio of Thy1.1-positive splenic CD4+ T cells / CD8+ T cells / macrophages to the number of Thy1.1-positive hepatocytes after intravenous injection of Thy1.1 mRNA LNPs with different molar ratios of ionized lipids, phospholipids, sterols, and PEG-lipids. Two different ionized lipids were used in the formulations, and the formulation with a cholesterol-to-phospholipid ratio of 3.9:1 has the standard lipid molar ratio used in the Onpattro formulation. Specific details for implementing the invention

[0046] Detailed description of the invention

[0047] The description and drawings in this specification are merely for illustrating the principles of the invention. Accordingly, it will be understood that a person skilled in the art can devise various configurations that embody the principles of the invention and fall within its scope, even if they are not explicitly described or illustrated in this specification. Furthermore, all embodiments mentioned in this specification are intended primarily for educational purposes to help the reader understand the principles of the invention and the concepts of the inventor(s) who contributed to the advancement of the art, and should not be interpreted as being limited to such specifically mentioned examples or conditions. In addition, all statements in this specification referring to the principles, aspects, and embodiments of the invention, as well as specific embodiments thereof, are intended to encompass their equivalents.

[0048] As used in this specification and the appended claims, the singular forms (“a,” “an,” “the”) include plural objects unless the context clearly indicates otherwise. For example, “RNA polynucleotide (a RNA polynucleotide)” means one RNA polynucleotide or one or more RNA polynucleotides. As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” or “containing,” and “contains,” have an inclusive or open-ended meaning and do not exclude additional, unmentioned members, elements, or method steps. When referring to numerical ranges with both ends, the mentioned ends are included as well as all numbers and fractions contained within each range.

[0049] In this specification, the terms “about” or “approximately,” used to refer to measurable values ​​such as parameters, quantities, temporal durations, etc., mean that they include a variation of + / - 10% or less, e.g., + / - 5% or less, from the said specific value within a range appropriate for carrying out the disclosed invention. The value itself referred to by the modifiers “about” or “approximately” should also be understood as specifically and preferably disclosed. Thus, where a ratio of 1:2 is specified, it also includes a slight variation of said ratio, which includes a variation of 10% meaning between 1:1.8 and 1:2.2 or a variation of 5% meaning between 1:1.9 and 1:2.1. Likewise, a 1:1 ratio means that it includes a 10% variation between 1:0.9 and 1:1.1, or a 5% variation between 1:0.95 and 1:1.05.

[0050] The term “one or more,” such as one or more members of a group of members, is clear in itself, but to give additional examples, this term encompasses, above all, any one of the members, or any two or more of the members, for example, any three or more (≥), four or more (≥), five or more (≥), six or more (≥), or seven or more (≥), and up to all of the members.

[0051] Throughout this specification, references to "one embodiment" or "an embodiment" mean that specific features, structures, or characteristics described in relation to such embodiments are included in at least one embodiment of the present invention. Accordingly, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment, but may. Furthermore, specific features, structures, or characteristics disclosed in this specification may be combined in one or more embodiments in a suitable manner that would be obvious to a person skilled in the art. Additionally, while some embodiments described in this specification may include only some of the features included in other embodiments, combinations of features from different embodiments are also included within the scope of the present invention and will be understood by a person skilled in the art as constituting separate embodiments. For example, any of the disclosed embodiments and / or claims may be used in any combination.

[0052] All documents cited in this specification are incorporated herein by reference in their entirety.

[0053] Unless otherwise specified, all terms used to disclose the present invention, including technical and scientific terms, have the meaning generally understood by a person skilled in the art to which the present invention pertains. For further guidance, definitions of terms may be included to help better understand the teachings of the present invention.

[0054] As described in detail above, the present invention provides lipid nanoparticles (LNPs) comprising ionized lipids, phospholipids, sterols, stabilized lipids; and one or more nucleic acid molecules, and

[0055] - The above LNP comprises at least 55 mol% of the above ionized lipid;

[0056] - The ratio of the above sterol to phospholipid is 1:1 or less,

[0057] It is characterized by.

[0058] As described in detail above, the present invention provides lipid nanoparticles (LNPs) comprising ionizable lipids, phospholipids, sterols, PEG lipids; and one or more nucleic acid molecules, and

[0059] - The above LNP comprises at least 55 mol% of the above ionized lipid;

[0060] - The ratio of the above sterol to phospholipid is 1:1 or less,

[0061] It is characterized by.

[0062] In particular, the present invention provides lipid nanoparticles (LNPs) comprising ionizable lipids, phospholipids, cholesterol, PEG lipids; and one or more nucleic acid molecules, and

[0063] - The above LNP comprises at least 55 mol% of the above ionized lipid;

[0064] - The above cholesterol to phospholipid ratio is 1:1 or less,

[0065] It is characterized by.

[0066] Specifically, in the case of these LNPs, it was found that significantly increased cargo delivery to extrahepatic tissues, such as the spleen and / or bone marrow, is achieved, as demonstrated in the examples section.

[0067] The lipid nanoparticles described herein particularly comprise a nucleic acid. In the context of the present invention, "nucleic acid" is deoxyribonucleic acid (DNA) or, preferably, ribonucleic acid (RNA), more preferably mRNA. Nucleic acids according to the present invention include genomic DNA, cDNA, mRNA, and molecules produced recombinantly and chemically synthesized. Nucleic acids according to the present invention may be single-stranded or double-stranded and may be in the form of a linear or circular molecule closed by covalent bonds. Nucleic acids may be used for introduction into cells, i.e., transfection, in the form of RNA, prepared, for example, through in vitro transcription from a DNA template. Additionally, RNA may be modified through stabilization sequences, capping, and / or polyadenylation prior to application.

[0068] In the context of the present invention, the term “RNA” refers to a molecule comprising ribonucleotide residues and preferably composed wholly or substantially of ribonucleotide residues. “Ribonucleotide” refers to a nucleotide having a hydroxyl group at the 2’ position of a β-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinant RNA, as well as modified RNA distinguished from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such modification may include adding a non-nucleotide material, e.g., to the end(s) of the RNA or internally, e.g., to one or more nucleotides of the RNA. Additionally, nucleotides within the RNA molecule may include non-standard nucleotides, such as non-naturally generated nucleotides or chemically synthesized nucleotides or deoxynucleotides. Such modified RNA may be referred to as an analog. Nucleic acids may be included in a vector. As used herein, the term "vector" includes any vector known to a person skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs) or yeast artificials, or analogs of naturally generated RNA.The term RNA also includes, but is not limited to, small activating RNA (saRNA) and trans-amplifying RNA (taRNA), and also includes other types of RNA such as circular RNA (circRNA), siRNA (small interfering RNA), gRNA (guide RNA), base editor RNA, and self-amplifying RNA.

[0069] According to the present invention, the term “RNA” includes, preferably relates to, “mRNA” meaning “messenger RNA,” and is also related to a “transcript” that can be generated using DNA as a template and codes for a peptide or protein. mRNA generally comprises a 5’ untranslated region (5’-UTR), a protein or peptide coding region, and a 3’ untranslated region (3’-UTR). mRNA has a limited half-life in cells and in vitro. Preferably, mRNA is generated through in vitro transcription using a DNA template. In one embodiment of the present invention, the RNA is obtained through in vitro transcription or chemical synthesis. In vitro transcription methodologies are known to those skilled in the art. For example, various in vitro transcription kits are available commercially.

[0070] In a specific embodiment of the present invention, the mRNA molecule is an mRNA molecule encoding an immune-regulatory protein. Depending on the specific application, such a protein may be an immune-stimulating protein, an immune-tolerizing protein, or an immune-suppressive protein. In this embodiment, the present invention is particularly suitable for use in the treatment of cancer, autoimmune diseases, hereditary disorders, hematological disorders, aging, fibrosis, neurological disorders, cardiovascular diseases, or infectious diseases.

[0071] In the context of the present invention, the term "mRNA molecules encoding immune-modulating proteins" refers to mRNA molecules encoding proteins that help maintain a balanced and appropriately responding immune response by regulating or altering the activity of the immune system. Immune-modulating proteins can exert various effects on the immune system, including suppressing excessive responses or promoting more robust defenses when necessary. These proteins play a crucial role in preventing excessive inflammation or immune responses that could harm body tissues by ensuring that the immune system responds appropriately to various challenges.

[0072] In the context of the present invention, the term "mRNA molecules encoding immune-stimulating proteins" refers to mRNA molecules encoding proteins capable of enhancing or activating the immune system, which is the body's defense mechanism against pathogens such as bacteria, viruses, and other harmful entities. These proteins can stimulate various components of the immune system, including antigen-presenting cells (e.g., dendritic cells) and other immune cells, to induce an enhanced and more effective immune response. Immunostimulating proteins may be naturally occurring or may be engineered for therapeutic purposes in the development of vaccines or immunotherapies. They play a crucial role in supporting the body's ability to recognize and eliminate external invaders and contribute to the overall health and function of the immune system.

[0073] In the context of the present invention, the term "mRNA molecules encoding immune-tolerizing proteins" refers to mRNA molecules encoding proteins that induce or promote immune tolerance within the body. Immune tolerance is a core aspect of immune system function, as it involves the ability to effectively respond to external invaders, such as pathogens, while simultaneously recognizing and tolerating the body's own cells and tissues. Proteins possessing immune-tolerant properties help regulate immune responses to prevent the phenomenon known as autoimmunity, in which the immune system attacks healthy self cells. These proteins can regulate immune cells and signaling pathways to establish a state of tolerance, thereby preventing the development of inappropriate or excessive immune responses to self-antigens. Proteins that induce immune tolerance are receiving particular attention in the context of autoimmune diseases, where the immune system mistakenly targets and damages the body's own tissues.

[0074] In the context of the present invention, the term "mRNA molecules encoding immune-suppressive proteins" refers to mRNA molecules encoding proteins that suppress or inhibit the activity of the immune system. These proteins play a crucial role in regulating immune responses to prevent excessive reactions that may cause tissue damage or autoimmune disorders. Immunosuppressive proteins are commonly used in medical settings to intentionally weaken immune activity, such as in the context of organ transplantation or the treatment of autoimmune diseases. These proteins may act by suppressing the function of immune cells, blocking signaling pathways involved in immune activation, or promoting a state of immune tolerance.

[0075] In another specific embodiment, the mRNA molecule is an mRNA molecule encoding an antigen and / or a disease-specific protein. Alternatively, the mRNA molecule may code for a protein suitable for use in gene therapy, such as protein replacement therapy, enzyme replacement therapy, RNAi-based therapy, CRISPR-Cas-based therapy, addition of transcription factors, generation of specific chemokines, immunomodulators, and / or antisense oligonucleotide therapy.

[0076] According to the present invention, the term “antigen” comprises any molecule, preferably a peptide or protein, comprising at least one epitope that induces an immune response and / or is a target of an immune response; thus, the term “antigen” also implies encompassing a minimal epitope of antigen origin. As defined herein, “minimal epitope” means the smallest structure capable of inducing an immune response. Preferably, in the context of the present invention, an antigen is a molecule that induces an immune response, preferably specific to the corresponding antigen or a cell expressing the corresponding antigen, after optionally undergoing processing. In particular, the “antigen” relates to a molecule that, after optionally undergoing processing, is presented by an MHC molecule and reacts specifically with T lymphocytes (T cells).

[0077] In a specific embodiment, the antigen is a target-specific antigen, which may be a tumor antigen, an auto-antigen, or a bacterial, viral, or fungal antigen. The target-specific antigen may be derived from any one of the following: (a) total mRNA isolated from target cell(s), one or more specific target mRNA molecules, (a) protein lysates of target cell(s), (a) specific proteins derived from target cell(s), or synthetic target-specific peptides or proteins and synthetic mRNA or DNA encoding the target-specific antigen or a peptide derived therefrom.

[0078] To avoid any misunderstanding, the LNP of the present invention may comprise a single mRNA molecule, or may comprise a plurality of mRNA molecules, for example, one or more mRNA molecules encoding an immuno-regulatory protein and / or a combination of one or more mRNA molecules encoding an antigen and / or disease-specific protein or a protein for use in gene therapy.

[0079] In a very specific embodiment, the mRNA molecule encoding an immune-regulatory protein may be combined with one or more mRNA molecules encoding an antigen and / or disease-specific protein or a protein for use in gene therapy.

[0080] As used herein, the term “nanoparticle” refers to any particle having a diameter suitable for systemic administration, particularly intravenous administration of nucleic acids, generally having a diameter of less than 1000 nanometers (nm), preferably less than 500 nm, more preferably, for example, 40 to 200 nm; preferably 40 to 160 nm, preferably 40 to 100 nm, having a diameter of less than 200 nm.

[0081] In very specific embodiments, the nanoparticles of the present invention may be in the form of lipid nanoparticles or lipid nanoparticle compositions comprising a combination of lipids as defined herein. Lipid nanoparticles (LNPs) are generally known as nanoscale particles composed of a combination of different lipids. While such LNPs may include a number of different types of lipids, the LNPs of the present invention are generally composed of a combination of ionized lipids, phospholipids, sterols, and PEG lipids.

[0082] In the context of the present invention, the term lipid nanoparticle (LNP), also referred to as solid lipid nanoparticle (SLNP), means a nanoparticle containing lipids. These are commonly used as pharmaceutical drug delivery systems or pharmaceutical formulations. LNPs as drug delivery carriers were first approved in 2018 and are currently being used in several RNA-based vaccine candidates. Lipid nanoparticles are generally spherical with an average diameter of 10 to 1,000 nanometers and possess a lipid core matrix capable of solubilizing lipophilic molecules. As used herein, the term lipid is used in a broader sense and includes triglycerides, diglycerides, monoglycerides, fatty acids, steroids (e.g., cholesterol), and waxes. Biological membrane lipids, such as phospholipids, sphingomyelins, bile acids, and sterols, are commonly used as stabilizers for LNPs.

[0083] Accordingly, in the context of the present invention, the nanoparticles disclosed herein further comprise one or more additional lipids, e.g., helper lipids (e.g., phospholipids), sterols and / or stabilizing lipids (e.g., PEGylated lipids and variants thereof), regardless of whether they act as stabilizers.

[0084] In the context of the present invention, the term lipid means a chemically defined substance that is insoluble in water but soluble in alcohols, ethers, and chloroform, among others. Ionizable or cationic lipids are generally lipids composed of three parts: an amine head group, a linker moiety, and a hydrophobic tail. In the context of a compound or lipid, the term "ionizable" or "otherwise cationic" means that there is any uncharged group within the compound or lipid that can accept an ion (typically an H+ ion) and become positively charged. Alternatively, any uncharged group within the compound or lipid can accept an electron and become negatively charged.

[0085] In the context of the present invention, any type of ionizable lipid may be appropriately used. In very specific embodiments, suitable ionizable lipids are those defined in WO2022136641, PCT / EP2024 / 069656, or WO2024084056. In very specific embodiments, the ionizable lipid is S-Ac7-DOg:

[0086]

[0087] Accordingly, in specific embodiments, the present invention,

[0088] - Ionized lipids that are S-Ac7-Dog;

[0089] - Phospholipids;

[0090] - Sterols;

[0091] - PEG lipids; and

[0092] - Provides lipid nanoparticles comprising one or more nucleic acid molecules; particularly nucleic acid molecules; and

[0093] - The above LNP comprises at least 55 mol% of the above ionized lipid; and

[0094] - The above cholesterol to phospholipid ratio is 1:1 or less,

[0095] It is characterized by.

[0096] In the context of the present invention, the term "phospholipid" refers to an auxiliary lipid molecule consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" composed of a phosphate group. Since these two components are often connected to each other by a glycerol molecule, the phospholipid of the present invention is preferably a glycerol-phospholipid. Additionally, the phosphate group is often modified into a simple organic molecule such as choline (i.e., forming phosphocholine) or ethanolamine (i.e., forming phosphoethanolamine).

[0097] Suitable auxiliary lipids or phospholipids in the context of the present invention may be selected from a list including the following: 1,2-dioleoyl- sn -Glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl- sn-Glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OchemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dipitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), egg yolk sphingomyelin (ESM), and mixtures thereof. The phospholipids include, but are not limited to, sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-1'-(3'-oleoyl-2'-hydroxy)-glycerol (BMP), 1,2-distearoyl-sn-glycero-3-phosphate; Or it may be anionic phospholipids such as 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), or salts thereof.

[0098] In a more specific embodiment, the auxiliary lipid or phospholipid is selected from a list including the following: 1,2-dioleoyl- sn -Glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl- sn -Glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), egg yolk sphingomyelin (ESM) and mixtures thereof; in particular DSPC.

[0099] In specific embodiments, the present invention,

[0100] - Ionizable lipids;

[0101] - DSPC phospholipid;

[0102] - Sterols;

[0103] - PEG lipids; and

[0104] - Provides lipid nanoparticles comprising one or more nucleic acid molecules; in particular, nucleic acid molecules, and

[0105] - The above LNP comprises at least 55 mol% of the above ionized lipid; and

[0106] - The above cholesterol to phospholipid ratio is 1:1 or less,

[0107] It is characterized by.

[0108] Accordingly, in specific embodiments, the present invention,

[0109] - Ionized lipid S-Ac7-Dog

[0110] - phospholipid DSPC;

[0111] - Sterols;

[0112] - PEG lipids; and

[0113] - Provides lipid nanoparticles comprising one or more nucleic acid molecules; in particular, nucleic acid molecules, and

[0114] - The above LNP comprises at least 55 mol% of the above ionized lipid; and

[0115] - The above cholesterol to phospholipid ratio is 1:1 or less,

[0116] It is characterized by.

[0117] In the context of the present invention, the term 'sterol,' also known as steroid alcohol, is a subgroup of steroids that may be produced naturally in plants, animals, and fungi, or by some bacteria. In the context of the present invention, any suitable sterol selected from a list including, e.g., cholesterol, ergosterol, campesterol, oxysterol, antrosterol, cholecalciferol, desmostrol, nicasterol, sitosterol, and stigmasterol; preferably, cholesterol may be used.

[0118] In another specific embodiment, the present invention,

[0119] - Ionized lipids;

[0120] - phospholipid DSPC;

[0121] - Cholesterol, a sterol;

[0122] - PEG lipids; and

[0123] - Provides lipid nanoparticles comprising one or more nucleic acid molecules; in particular, mRNA molecules, and

[0124] - The above LNP comprises at least 55 mol% of the above ionized lipid;

[0125] - The above cholesterol to phospholipid ratio is 1:1 or less,

[0126] It is characterized by.

[0127] Accordingly, in specific embodiments, the present invention,

[0128] - Ionized lipid S-Ac7-Dog

[0129] - phospholipid DSPC;

[0130] - Cholesterol, a sterol;

[0131] - PEG lipids; and

[0132] - Provides lipid nanoparticles comprising one or more nucleic acid molecules; in particular, mRNA molecules, and

[0133] - The above LNP comprises at least 55 mol% of the above ionized lipid; and

[0134] - The above cholesterol to phospholipid ratio is 1:1 or less,

[0135] It is characterized by.

[0136] In the context of the present invention, the term "stabilizing lipid" means a component that contributes to maintaining the structural integrity and stability of lipid nanoparticles. In the context of the present invention, stabilizing lipids may be, for example, PEGylated lipids and their variants, sphingolipids, and glycerolipids.

[0137] In the context of the present invention, the term “PEG lipid” or otherwise “PEGylated lipid” means any suitable lipid modified with a PEG (polyethylene glycol) group. The PEG lipid of the present invention may be a C14-PEG lipid, a C16-PEG lipid, or a C18-PEG lipid.

[0138] C14-PEG lipids comprise a fatty acid tail containing 14 C-atoms, including a polyethylene glycol (PEG) moiety that determines the molecular weight of the lipid. In certain embodiments, the C14-PEG lipid is a diC14-PEG lipid, i.e., a lipid having two C14 lipid tails. In additional embodiments, the diC14-PEG lipid is based on dimyristoyl, i.e., two C14 tails, and is selected from a list including, for example: (dimyristoyl-based)-PEG2000 lipids, e.g., DMG-PEG2000 lipids (1,2-dimyristoyl- rac -Glycero-3-methoxypolyethylene glycol-2000) or 2-dimyristoyl- sn -Glycero-3-phosphoethanolamine glycol-2000(DMPE-PEG2000).

[0139]

[0140]

[0141] C16-PEG lipids include a polyethylene glycol (PEG) moiety that determines the molecular weight of the lipid, and a fatty acid tail comprising 16 carbon atoms. In certain embodiments, the C16-PEG lipid is a diC16-PEG lipid, i.e., a lipid having two C16 lipid tails. In certain embodiments, the diC16-PEG lipid is, for example, N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}(C16 PEG2000 ceramide).

[0142]

[0143] C18-PEG lipids comprise a fatty acid tail containing 18 carbon atoms, including a polyethylene glycol (PEG) moiety that determines the molecular weight of the lipid. In certain embodiments, the C18-PEG lipid is a diC18-PEG lipid, i.e., a lipid having two C18 lipid tails. In certain embodiments, the diC18-PEG lipid is selected from a list including the following: (distearoyl-based)-PEG lipids, e.g., DSG-PEG2000 lipid (2-distearoyl- rac -glycero-3-methoxypolyethylene glycol-2000) or DSPE-PEG2000 lipid (1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; or (dioleoyl-based)-PEG2000 lipids, e.g., DOG-PEG2000 lipids (1,2-dioleoyl- rac-glycerol) or DOPE-PEG2000 lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000])

[0144]

[0145]

[0146]

[0147]

[0148] In a very specific embodiment, the PEG-lipid is DMG-PEG.

[0149] Accordingly, in specific embodiments, the present invention,

[0150] - Ionized lipids;

[0151] - phospholipid DSPC;

[0152] - Cholesterol, a sterol;

[0153] - PEG lipid DMG-PEG; and

[0154] - Provides lipid nanoparticles comprising one or more nucleic acid molecules; in particular, mRNA molecules, and

[0155] - The above LNP comprises at least 55 mol% of the above ionized lipid;

[0156] - The above cholesterol to phospholipid ratio is 1:1 or less,

[0157] It is characterized by.

[0158] Accordingly, in certain embodiments, the present invention,

[0159] - Ionized lipid S-Ac7-Dog

[0160] - phospholipid DSPC;

[0161] - Cholesterol, a sterol;

[0162] - PEG lipid DMG-PEG; and

[0163] - Provides lipid nanoparticles comprising one or more nucleic acid molecules; in particular, mRNA molecules, and

[0164] - The above LNP comprises at least 55 mol% of the above ionized lipid;

[0165] - The above cholesterol to phospholipid ratio is 1:1 or less,

[0166] It is characterized by.

[0167] The inventors have also confirmed that extrahepatic delivery of the LNP of the present invention may be further enhanced when one or more of the following conditions are met: 1) the ratio of cholesterol to phospholipids is 1:2 or less, 2) the molar percentage of ionized lipids is at least 60 mol% and / or 3) the molar percentage of PEG-lipids is greater than 1.5 (e.g., 3.0 mol%). In particular, these conditions lead to increased bone marrow delivery, as demonstrated in the Examples section.

[0168] Accordingly, in a specific embodiment, one or more of the following conditions apply:

[0169] - The lipid nanoparticles (LNPs) defined herein contain cholesterol to phospholipid in a ratio of 1:1 or less, particularly 1:2 or less.

[0170] - The LNP of the present invention comprises at least 1.5 mol% of PEG lipid, in particular at least 2.0 mol% of PEG lipid, more particularly at least 2.5 mol% of said PEG lipid, e.g., about 3.0 mol% of said PEG lipid, and / or

[0171] - The molar percentage of ionized lipids in the LNP of the present invention is at least 55 mol%; in particular at least 60 mol%,

[0172] - The molar percentage of phospholipids in the LNP of the present invention is greater than 1 mol%, particularly 2 mol% to 30 mol%, more particularly 5 mol% to 28 mol%; more particularly 10 mol% to 27 mol%; and even more particularly 20 mol% to 26 mol%.

[0173] - The molar percentage of cholesterol in the LNP of the present invention is less than 22 mol%, particularly 10 mol% to 15 mol%, more particularly 12 mol% to 13 mol%.

[0174] Accordingly, the present invention relates to a cholesterol-to-phospholipid ratio of 1:1 or less, for example, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1: 3.9, 1 : 4, 1 : 4.1, 1 : 4.2, 1 : 4.3, 1 : 4.4, 1 : 4.5, 1 : 4.6, 1 : 4.7, 1 : 4.8, 1 : 4.9, 1 : 5, 1 : 5.1, 1 : 5.2, 1 : 5.3, 1 : 5.4, 1 : 5.5, 1 : 5.6, 1 : 5.7, 1 : 5.8, 1 : 5.9, 1 : 6, 1 : 6.1, 1 : 6.2, 1 : 6.3, 1 : 6.4, 1 : 6.5, 1 : 6.6, 1 : 6.7, 1 : 6.8, 1 : 6.9, 1 : 7, Provides LNPs less than or equal to 1 : 7.1, 1 : 7.2, 1 : 7.3, 1 : 7.4, 1 : 7.5, 1 : 7.6, 1 : 7.7, 1 : 7.8, 1 : 7.9, 1 : 8, 1 : 8.1, 1 : 8.2, 1 : 8.3, 1 : 8.4, 1 : 8.5, 1 : 8.6, 1 : 8.7, 1 : 8.8, 1 : 8.9, 1 : 9, 1 : 9.1, 1 : 9.2, 1 : 9.3, 1 : 9.4, 1 : 9.5, 1 : 9.6, 1 : 9.7, 1 : 9.8, 1 : 9.9, and 1 : 10.

[0175] In a further embodiment, the LNP of the present invention comprises at least 0.1 mol% of the stabilized lipid, particularly at least 0.5 mol% of the stabilized lipid, at least 1.0 mol% of the stabilized lipid, 1.5 mol% of the stabilized lipid, particularly at least 2.0 mol% of the stabilized lipid, more particularly at least 2.5 mol% of the stabilized lipid, at least 3.0 mol% of the stabilized lipid, at least 4.0 mol% of the stabilized lipid, at least 5.0 mol% of the stabilized lipid, at least 6.0 mol% of the stabilized lipid, at least 7.0 mol% of the stabilized lipid, at least 8.0 mol% of the stabilized lipid, at least 9.0 mol% of the stabilized lipid, and at least 10.0 mol% of the stabilized lipid.

[0176] The present invention comprises at least 0.1 mol% of PEG lipid, at least 0.2 mol% of PEG lipid, at least 0.3 mol% of PEG lipid, at least 0.4 mol% of PEG lipid, at least 0.5 mol% of PEG lipid, at least 0.6 mol% of PEG lipid, at least 0.7 mol% of PEG lipid, at least 0.8 mol% of PEG lipid, at least 0.9 mol% of PEG lipid, at least 1.0 mol% of PEG lipid, at least 1.1 mol% of PEG lipid, at least 1.2 mol% of PEG lipid, at least 1.3 mol% of PEG lipid, at least 1.4 mol% of PEG lipid, at least 1.5 mol% of PEG lipid, for example, about 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2.0 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%. Mole%, 2.5 mole%, 2.6 mole%, 2.7 mole%, 2.8 mole%, 2.9 mole%, 3.0 mole%, 3.1 mole%, 3.2 mole%, 3.3 mole%, 3.4 mole%, 3.5 mole%, 3.6 mole%, 3.7 mole%, 3.8 mole%, 3.9 mole%, 4.0 mole%, 4.1 mole%, 4.2 mole%, 4.3 mole%, 4.4 mole%, 4.5 mole%, 4.6 mole%, 4.7 mole%, 4.8 mole%, 4.9 mole%, 5.0 mole%, 5.1 mole%, 5.2 mole%, 5.3 mole%, 5.4 mole%, 5.5 mole%, 5.6 mole%, 5.7 mole%, 5.8 mole%, 5.9 Mole%, 6.0 Mole%, 6.1 Mole%, 6.2 Mole%, 6.3 Mole%, 6.4 Mole%, 6.5 Mole%, 6.6 Mole%, 6.7 Mole%, 6.8 Mole%, 6.9 Mole%, 7.0 Mole%, 7.1 Mole%, 7.2 Mole%, 7.3 Mole%, 7.4 Mole%, 7.5 Mole%, 7.6 Mole%, 7.7 Mole%, 7.8 Mole%, 7.9 Mole%, 8.0 Mole%, 8.1 Mole%, 8.2 Mole%, 8.3 Mole%, 8.4 Mole%, 8.5 Mole%, 8.6 Mole%, 8.7 Mole%, 8.8 Mole%, 8.9 Mole%, 9.0 Mole%, 9.1 Mole%, 9.2 Mole%, 9.3 Mole%, 9.4 Mole%, 9.5 mole%, 9.6 mole%, 9.7 mole%, 9.8 mole%, 9.9 mole%, 10.We additionally provide LNP containing 0 mole% or more.

[0177] The present invention also provides an LNP comprising ionized lipids in a molar percentage of at least 55 mol%, e.g., 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, 60 mol%, 61 mol%, 62 mol%, 63 mol%, 64 mol%, 65 mol%, 66 mol%, 67 mol%, 68 mol%, 69 mol%, 70 mol%, 71 mol%, 72 mol%, 73 mol%, 74 mol%, 75 mol%, 76 mol%, 77 mol%, 78 mol%, 79 mol%, 80 mol%, 81 mol%, 82 mol%, 83 mol%, 84 mol%, 85 mol% or more.

[0178] The present invention further comprises an LNP containing phospholipids in a molar percentage greater than 1 mol%, e.g., 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol% or more. Provides

[0179] The present invention further provides an LNP containing cholesterol in a molar percentage of less than 22 mol%, e.g., 22 mol%, 21 mol%, 20 mol%, 19 mol%, 18 mol%, 17 mol%, 16 mol%, 15 mol%, 14 mol%, 13 mol%, 12 mol%, 11 mol%, 10 mol%, 9 mol%, 8 mol%, 7 mol%, 6 mol%, 5 mol%, 3 mol%, 2 mol%, 1 mol%, 0.4 mol% or less.

[0180] Therefore, the present invention also,

[0181] - At least 55 mol% of ionized lipids, in particular at least 60 mol%

[0182] - More than 1 mol% of phospholipids, particularly 2 mol% to 40 mol%, more particularly 10 mol% to 30 mol%, more particularly 20 mol% to 26 mol%.

[0183] - Less than 22 mol% sterol, preferably cholesterol; particularly 0.4 mol% to 20 mol%, more particularly 2 mol% to 18 mol%, more particularly 10 mol% to 15 mol%, more particularly 12 mol% to 13 mol%;

[0184] - at least 1.5 mol% of PEG lipid, particularly at least 2.0 mol% of PEG lipid, more particularly at least 2.5 mol% of the PEG lipid, e.g., at least 3.0 mol% of the PEG lipid, at least 4.0 mol% of the PEG lipid, at least 5.0 mol% of the PEG lipid, at least 6.0 mol% of the PEG lipid, at least 7.0 mol% of the PEG lipid, at least 8.0 mol% of the PEG lipid, at least 9.0 mol% of the PEG lipid, at least 10.0 mol% of the PEG lipid; and

[0185] - Provides lipid nanoparticles containing one or more nucleic acid molecules, particularly mRNA molecules, and

[0186] The above cholesterol to phospholipid ratio is characterized by being 1:1 or less, particularly 1:2 or less.

[0187] In specific embodiments, the present invention,

[0188] - At least 55 mol% of S-Ac7-dOg; in particular at least 60 mol%

[0189] - DSPC of more than 1 mol%; particularly 10 mol% to 30 mol%, more particularly 20 mol% to 26 mol%;

[0190] - Less than 22 mol% cholesterol, particularly 10 mol% to 15 mol%, more particularly 12 mol% to 13 mol%;

[0191] - at least 1.5 mol% of DMG-PEG lipids, particularly at least 2.0 mol% of DMG-PEG lipids, more particularly at least 2.5 mol%, e.g., about 3.0 mol% of DMG-PEG lipids, and

[0192] - Provides lipid nanoparticles comprising one or more nucleic acid molecules; in particular, mRNA molecules, and

[0193] The above cholesterol to phospholipid ratio is characterized as being 1:1 or less; in particular, 1:2 or less.

[0194] In another specific embodiment, the present invention,

[0195] - At least 55 mol% of S-Ac7-dOg; in particular at least 60 mol%

[0196] - DSPC of more than 1 mol%; particularly 10 mol% to 30 mol%, more particularly 20 mol% to 26 mol%;

[0197] - Less than 22 mol% cholesterol, particularly 10 mol% to 15 mol%, more particularly 12 mol% to 13 mol%;

[0198] - at least 1.5 mol% of DSG-PEG lipids, particularly at least 2.0 mol% of DSG-PEG lipids, more particularly at least 2.5 mol%, e.g., about 3.0 mol% of DSG-PEG lipids, and

[0199] - Provides lipid nanoparticles comprising one or more nucleic acid molecules; in particular, mRNA molecules, and

[0200] The above cholesterol to phospholipid ratio is characterized as being 1:1 or less; in particular, 1:2 or less.

[0201] In very specific embodiments, the present invention,

[0202] - Approximately 60 mol% of the above ionized lipids;

[0203] - Approximately 25.67 mol% phospholipids;

[0204] - Approximately 12.83 mol% cholesterol; and

[0205] - About 1.5% PEG lipids,

[0206] Provides an LNP containing

[0207] In another very specific embodiment, the present invention,

[0208] - Approximately 60 mol% of S-Ac7-Dog;

[0209] - Approximately 25.67 mol% of DSPC;

[0210] - Approximately 12.83 mol% cholesterol; and

[0211] - Approximately 1.5 mol% of DMG-PEG lipids,

[0212] Provides an LNP containing

[0213] This LNP is specifically characterized by exhibiting very high spleen-centric cargo delivery and high bone marrow cargo delivery.

[0214] In another very specific embodiment, the present invention,

[0215] - Approximately 60 mol% of the above ionized lipids;

[0216] - Approximately 19.25 mol% phospholipids;

[0217] - Approximately 19.25 mol% cholesterol; and

[0218] - About 1.5% PEG lipids,

[0219] Provides an LNP containing

[0220] Still in additional specific embodiments, the present invention,

[0221] - Approximately 60 mol% of S-Ac7-dOg;

[0222] - Approximately 19.25 mol% of DSPC;

[0223] - Approximately 19.25 mol% cholesterol; and

[0224] - Approximately 1.5 mol% of DMG-PEG,

[0225] Provides an LNP containing

[0226] In very specific embodiments, the present invention,

[0227] - Approximately 60 mol% of the above ionized lipids;

[0228] - Approximately 24.17 mol% phospholipids;

[0229] - Approximately 12.83 mol% cholesterol; and

[0230] - Approximately 3.0 of PEG lipids,

[0231] Provides an LNP containing

[0232] Still in additional specific embodiments, the present invention,

[0233] - Approximately 60 mol% of S-Ac7-dOg;

[0234] - Approximately 24.17 mol% of DSPC;

[0235] - Approximately 12.83 mol% cholesterol; and

[0236] - Approximately 3.0 mol% of DMG-PEG,

[0237] Provides an LNP containing

[0238] This LNP is specifically characterized by exhibiting high splenic-centered cargo delivery and very high bone marrow cargo delivery.

[0239] Particularly noteworthy LNPs may have the following ionized lipid / phospholipid / cholesterol / PEG lipid ratios:

[0240] - 60 / 25.67 / 12.83 / 1.5

[0241] - 60 / 19.25 / 19.25 / 1.5

[0242] - 60 / 24.17 / 12.83 / 3.0

[0243] Accordingly, the present invention provides an LNP having the following ionized lipid / DSPC / cholesterol / PEG lipid ratio:

[0244] - 60 / 25.67 / 12.83 / 1.5, where PEG lipids = DMG-PEG

[0245] - 60 / 25.67 / 12.83 / 1.5, where PEG lipids = DSG-PEG

[0246] - 60 / 19.25 / 19.25 / 1.5, where PEG lipids = DMG-PEG

[0247] - 60 / 19.25 / 19.25 / 1.5, where PEG lipids = DSG-PEG

[0248] - 60 / 24.17 / 12.83 / 3.0, where PEG lipids = DMG-PEG

[0249] - 60 / 24.17 / 12.83 / 3.0, where PEG lipids = DSG-PEG

[0250] In addition, the present invention provides an LNP having the following S-Ac7-dOg / DSPC / cholesterol / PEG lipid ratio:

[0251] - 60 / 25.67 / 12.83 / 1.5, where PEG lipids = DMG-PEG

[0252] - 60 / 25.67 / 12.83 / 1.5, where PEG lipids = DSG-PEG

[0253] - 60 / 19.25 / 19.25 / 1.5, where PEG lipids = DMG-PEG

[0254] - 60 / 19.25 / 19.25 / 1.5, where PEG lipids = DSG-PEG

[0255] - 60 / 24.17 / 12.83 / 3.0, where PEG lipids = DMG-PEG

[0256] - 60 / 24.17 / 12.83 / 3.0, where PEG lipids = DSG-PEG

[0257] When mol% is used in the context of the present invention, it means the mol% of a specified component based on empty nanoparticles that do not contain nucleic acids. This means that the mol% of said component is calculated based on the total amount of ionized lipids, phospholipids, sterols, and PEG lipids present in the LNP.

[0258] The inventors have discovered that the LNP of the present invention is particularly suitable for extrahepatic delivery of nucleic acids, regardless of whether it is immunogenic or non-immunogenic. Accordingly, the present invention provides an LNP comprising one or more nucleic acid molecules, such as DNA or RNA, more specifically mRNA.

[0259] In the context of the present invention, "immunogenic delivery of nucleic acid molecules" means delivering nucleic acid molecules to a cell, thereby causing the nucleic acid molecules to come into contact with the cell and be internalized and / or expressed within the cell, thereby inducing an immune response.

[0260] Given the predominant extrahepatic tissue targeting of LNPs after IV administration, the LNPs of the present invention are particularly suitable for non-immunogenic approaches, including but not limited to gene editing, protein replacement, gene editing of HSCs, addition of transcription factors, introduction of chemokines, introduction of immunomodulators, and in situ CAR formation of immune cells.

[0261] Protein replacement therapy involves administering specific proteins or nucleic acids encoding them to patients who have a deficiency or dysfunction of those proteins due to genetic disorders or other pathological conditions. The purpose of this therapy is to restore normal function by supplementing the deficient or defective proteins. This approach is commonly used to treat diseases where specific proteins are absent or do not function properly.

[0262] Through gene editing, scientists can precisely modify the DNA of organisms, for example, in hematopoietic stem cells (HSCs) or T-cells. This technology enables the addition, removal, or alteration of genetic material at specific locations within the genome. The most widely known and used gene editing technology is CRISPR-Cas9, but there are also other technologies such as Transcription Activator-Like Effector Nuclease (TALEN) and Zinc Finger Nuclease (ZFN). Because HSCs possess self-renewing capabilities and the ability to differentiate into various blood cell lineages, using HSCs in this approach is particularly suitable for treating genetic blood disorders and certain immunodeficiencys.

[0263] In situ CAR formation of immune cells through nucleic acid administration involves directly modifying immune cells within the patient's body to express a chimeric antigen receptor (CAR). This method aims to overcome several limitations associated with conventional CAR-T cell therapy, which generally involves collecting T cells from a patient, genetically modifying them in a laboratory to express a CAR, and then re-injecting them back into the patient.

[0264] The amount of nucleic acid in the above LNP is generally expressed as a molar ratio, that is, the ratio of cationic lipids (ionized lipids) to RNA phosphate. In the context of the present invention, the molar ratio of the above LNP is between about 3 / 1; in particular between about 4 / 1 and 16 / 1. Alternatively, the amount of nucleic acid in the above LNP may be expressed as an N / P ratio, that is, the ratio of nitrogen atoms in ionized lipids to phosphate groups in nucleic acid. In the context of the present invention, the N / P ratio of the above LNP is between about 3 / 1; in particular between about 4 / 1 and 16 / 1.

[0265] In a further embodiment, the present invention provides a pharmaceutical composition comprising one or more LNPs as defined herein. Such a pharmaceutical composition is particularly suitable as a vaccine. Accordingly, the present invention also provides a vaccine comprising one or more LNPs according to the present invention.

[0266] In the context of the present invention, the term “vaccine” as used herein refers to any preparation for providing adaptive immunity (antibody and / or T cell response) against a disease. To this end, a vaccine in this specification comprises at least one nucleic acid molecule encoding an antigen that induces an adaptive immune response. Such antigen may exist in the form of a microorganism in a weakened or dead form, a protein or peptide, or a nucleic acid encoding the antigen. In the context of the present invention, an antigen means a protein or peptide that is recognized as a foreign substance by the host’s immune system and thereby stimulates the production of antibodies against such antigen for the purpose of fighting against it. A vaccine may be for prophylactic purposes (e.g., to prevent or mitigate the effects of future infection by any natural or wild-type pathogen) or for therapeutic purposes (e.g., to actively treat or alleviate the symptoms of an ongoing disease). The administration of a vaccine is referred to as vaccination.

[0267] The vaccine of the present invention may be used to induce an immune response, in particular an immune response against a disease-associated antigen or cells expressing a disease-associated antigen, e.g., an immune response against cancer. Accordingly, the vaccine may be used for the prophylactic and / or therapeutic treatment of a disease involving a disease-associated antigen or cells expressing a disease-associated antigen, e.g., cancer. Preferably, the immune response is a T cell response. In one embodiment, the disease-associated antigen is a tumor antigen. The antigen encoded by the RNA contained in the nanoparticles described herein is preferably a disease-associated antigen, or induces an immune response against a disease-associated antigen or cells expressing a disease-associated antigen.

[0268] The LNP and vaccine of the present invention are specifically intended for intravenous administration, that is, direct infusion of liquid substances into a vein. The intravenous route is the fastest method for delivering liquids and drugs throughout the body, i.e., the entire body. Accordingly, the present invention provides not only a vaccine for intravenous administration but also uses for the intravenous administration of the disclosed vaccine and LNP. Accordingly, the vaccine and LNP of the present invention can be administered intravenously. Furthermore, the present invention provides uses for the vaccine and LNP according to the present invention, wherein the vaccine is administered intravenously.

[0269] In particular, it has been found that the immunogenicity of the LNP of the present invention increases with multiple immunizations. Accordingly, in certain embodiments, the LNP defined herein is intended for use in vaccination, wherein the LNP is administered at least twice, preferably at least three times, at specific intervals.

[0270] The present invention also provides an LNP, a pharmaceutical composition, and a vaccine according to the present invention for use in human medicine or veterinary medicine. It is also intended that the LNP, the pharmaceutical composition, and the vaccine according to the present invention be used for human medicine or veterinary medicine. Finally, the present invention provides a method for preventing and treating human and veterinary disorders by administering the LNP, the pharmaceutical composition, and the vaccine according to the present invention to subjects in need thereof.

[0271] These pharmaceutical compositions are particularly suitable for various fields such as prophylactic vaccines, therapeutic vaccines, protein replacement therapy, gene editing, gene silencing, and small molecule delivery.

[0272] The present invention also provides uses for the LNP, pharmaceutical composition, or vaccine according to the present invention for the immunogenic delivery of one or more nucleic acid molecules. Accordingly, the LNP, pharmaceutical composition, and vaccine of the present invention are highly useful for the treatment of various human and veterinary disorders. Thus, the present invention provides the LNP, pharmaceutical composition, and vaccine of the present invention for use in the treatment of cancer, autoimmune diseases, infectious diseases, hematological disorders, aging, fibrosis, neurological disorders, or cardiovascular diseases.

[0273] In a specific embodiment, the nanoparticles and pharmaceutical compositions defined herein may be used to induce an immune response in a subject by administering to the subject a pharmaceutical composition in which the active ingredient is an immunostimulating oligonucleotide.

[0274] For example, the present invention provides a vaccine comprising one or more nanoparticles according to the present invention. To this end, the vaccine as defined herein comprises at least one active ingredient, e.g., a nucleic acid molecule, e.g., an mRNA molecule encoding an antigen that induces an adaptive immune response. Such antigen may exist in the form of a microorganism in a weakened or dead form, a protein or peptide, or a nucleic acid encoding an antigen, e.g., a disease-associated antigen, e.g., a tumor antigen. The vaccine may be for prophylactic purposes (e.g., to prevent future infection by any natural or "wild-type" pathogen or to mitigate its effects) or for therapeutic purposes (e.g., to actively treat or alleviate symptoms of an ongoing disease).

[0275] In another aspect, the nanoparticles and pharmaceutical compositions defined herein may be used to treat a disease or disorder characterized by low expression of a polypeptide in a subject by providing the pharmaceutical composition of the present invention to a subject, for example, in the context of protein replacement therapy and / or in the treatment of a hereditary disorder, wherein the active ingredient is a plasmid encoding said polypeptide or a functional variant or fragment thereof.

[0276] Still in additional embodiments, the nanoparticles and compositions defined herein may be used as a transfection agent comprising the composition or nanoparticles described herein, said composition or nanoparticles comprising nucleic acid. This agent can efficiently deliver nucleic acid into the cell upon contact with the cell. Still in yet another embodiment relates to a method of obtaining or forming the composition or nanoparticles described herein and delivering nucleic acid into the cell by bringing said composition or lipid particles into contact with the cell.

[0277] The lipid nanoparticles of the present invention can be prepared according to the protocol specified in the Examples section. More generally, the LNPs can be prepared using a method comprising the following:

[0278] - A step of preparing a first alcoholic composition comprising the above ionized lipid, the above phospholipid, the above sterol, the above PEG lipid, and a suitable alcoholic solvent;

[0279] - A step of preparing a second aqueous composition comprising one or more nucleic acids and an aqueous solvent;

[0280] - A step of mixing the first composition and the second composition in a microfluidic mixing device.

[0281] More specifically, lipid components are mixed at an appropriate concentration within an alcoholic vehicle such as ethanol. To this, an aqueous composition containing nucleic acids is added and then loaded into a microfluidic mixing device.

[0282] The purpose of microfluidic mixing is to achieve thorough and rapid mixing of multiple samples (i.e., lipid phases and nucleic acid phases) within a microscale device. Such sample mixing is generally achieved by enhancing the diffusion effect between different species flows. To this end, various microfluidic mixing devices may be used, for example, as reviewed in the literature [Lee et al., 2011]. A particularly suitable microfluidic mixing device according to the present invention is the NanoAssemblr from Precision Nanosystems. Alternatively, LNP can be prepared using T-mixing utilizing a T-joint connecting two Harvard Apparatus syringe pumps.

[0283] Other techniques suitable for preparing the LNP of the present invention include dispersing the components in a suitable dispersion medium, e.g., an aqueous solvent and an alcoholic solvent, and applying one or more of the following methods: ethanol dilution, simple hydration, sonication, heating, vortexing, ether injection, French press, cholic acid method, Ca 2+ Fusion method, freeze-thaw method, reverse phase evaporation method, T-junction mixing, microfluidic hydrodynamic focusing, staggered herringbone mixing, etc.

[0284] Examples

[0285] Example 1: In Vivo Experiment

[0286] Bioluminescence imaging after IV administration of Fluc mRNA-containing LNP

[0287] Materials and Methods

[0288] animal

[0289] Mice were housed in IVCs under conditions free of specific pathogens. All animal experiments were performed with the approval of the Ethics Committee, and animal care followed established guidelines. Six-week-old (20 to 23 g) female Balb / C mice were obtained from Charles River International Laboratories, Inc. (France) and housed (up to 5 mice per cage) with free access to water and standard laboratory animal food (chow).

[0290] intravenous injection

[0291] For intravenous injection, mice were placed in a warming chamber to dilate the veins for up to 10 minutes, or the cage was placed under a red lamp for 10 to 20 minutes. Once adequately warmed, the mice were restrained individually. The tails were wiped with gauze soaked in 70% ethanol. The needle of a BD microfine syringe (20 to 25 G) containing LNP solution was carefully inserted into one of the lateral tail veins, and the plunger was slowly pressed to inject a dose of up to 200 μL (based on 20 to 25 g mice). The needle was removed from the vein, and the injection site was gently compressed with dry gauze to stop local bleeding. The animals were then observed in the cage for at least 10 minutes. Each injection corresponded to 10 μg of Fluc mRNA, and each treatment group included 5 mice.

[0292] Luminous imaging

[0293] Imaging was performed 24 hours after IV injection of the LNP formulation. 100 μL of D-luciferin (30 mg / mL) was injected intraperitoneally (ip) into each mouse. The mice were then placed in an anesthesia induction chamber supplied with oxygen (0.4 to 0.8 L / min) and isoflurane (5%) until they reached an anesthetic state. The flow of isoflurane was then reduced (3%) to maintain anesthesia until they were ready for imaging. Mice were placed in stages inside the IVIS Lumina II (PerkinElmer), with up to three mice per group, using the same anesthetic flow (isoflurane 3 to 4%). Imaging was performed using field of view D with parameters set to luminescence, automatic exposure including background overlay, and medium level binning (4). Imaging of the animals was performed within 15 minutes of luciferin injection (at the time of the signal peak). After capturing in vivo images, mice were sacrificed by cervical dislocation and dissected, and imaging was performed on the liver, spleen, and femur, respectively, under similar imaging conditions.

[0294] mRNA synthesis

[0295] Firefly luciferase (Fluc) mRNA was prepared from a linearized peTheRNA vector (see WO2015071295) using eTheRNA-optimized in vitro transcription (IVT) reaction conditions and purified via cellulose. N1ψ-modified mRNA was produced by completely substituting uridine with N1-methylpseudouridine (N1ψ).

[0296] Example 1a: Cholesterol-to-phospholipid ratio affects the biodistribution of LNP

[0297] LNP manufacturing

[0298] Lipid-based nanoparticles were prepared by microfluidic mixing of mRNA solution and lipid solution in sodium acetate buffer (100 mM, pH 4) in a 2:1 volume ratio using a NanoAssemblr Benchtop (Precision Nanosystems) at a rate of 9 mL / min or 16 mL / min. The lipid solution contained a mixture of ionized lipid (S-Ac7-DOg), DSPC (Avanti), cholesterol (Sigma), and DMG-PEG2000 (Avanti).

[0299] Focusing on the cholesterol-to-phospholipid ratio, LNPs were prepared with varying molar ratios of ionized lipids / auxiliary lipids / cholesterol / DMG-PEG2000. All LNPs were encapsulated with Fluc mRNA as reporter mRNA, and the mRNA / ionized lipid molar ratio was set to 1 / 10.

[0300] A list of relevant physicochemical properties for various LNP compositions based on INL 1 (ionized lipid = S-Ac7-DOg), evaluated for cholesterol to phospholipid ratios, is presented in Table 1 below.

[0301] [Table 1]

[0302]

[0303] Figure 1 shows the spleen-to-liver ratio for mRNA expression when Fluc mRNA LNP was intravenously injected at the molar ratios presented in Table 1. This spleen-to-liver ratio is the in vitro mean luminance of the spleen obtained via IVIS at 24 hours after injection ( ex vivo average radiance) (normalized as a function of photons / second / cm² / steradian) is used in vitro ( ex vivoIt was calculated as a ratio derived by dividing by the average luminance. Here, LNPs with a cholesterol-to-phospholipid ratio of 1:1 or less showed a shift in biodistribution skewed toward the spleen compared to LNPs with a higher cholesterol-to-auxiliary lipid ratio. The condition with a cholesterol-to-phospholipid ratio of 3.9:1 corresponds to an LNP having the standard lipid molar ratio (DLin-MC3-DMA / DSPC / Chol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5) used in the Onpattro formulation.

[0304] Figure 2 shows luciferase activity in the bone marrow of an excised femur at 24 hours after injection. The femur was crushed to expose the bone marrow to oxygen, enabling visualization using IVIS. Luciferase activity is in vitro ( ex vivo It is expressed as average luminance (normalized as a function of photons / second / cm² / steradian). Here, the case where the cholesterol to auxiliary lipid ratio is 1:2 showed a higher signal in the bone marrow compared to other LNPs containing an LNP with a cholesterol to phospholipid ratio of 3.9:1, which is the standard lipid molar ratio used in Onpattro formulations.

[0305] Example 1b: The PEG-lipid type and amount of LNP having a low cholesterol-to-phospholipid ratio affect the biodistribution of LNP.

[0306] LNP manufacturing

[0307] Lipid-based nanoparticles were prepared by microfluidic mixing of mRNA solution dissolved in sodium acetate buffer (100 mM, pH 4) and lipid solution in a 2:1 volume ratio using a NanoAssemblr Benchtop (Precision Nanosystems) at a rate of 9 mL / min or 16 mL / min. The lipid solution contained a mixture of ionized lipids (S-Ac7-Dog), DSPC (Avanti), cholesterol (Sigma), and PEG lipids selected from DMG-PEG2000 or DSG-PEG2000 (Avanti), as shown in Tables 2 and 3.

[0308] LNPs were prepared with varying molar ratios of ionized lipids / auxiliary lipids / cholesterol / PEG lipids, with the cholesterol-to-phospholipid ratio set low at 1:2. These LNPs were compared to a standard formulation with a cholesterol-to-phospholipid ratio of 3.9:1. All LNPs were encapsulated with Fluc mRNA as a reporter mRNA, and the mRNA-to-ionized lipid molar ratio was set to 1 / 10.

[0309] Table 2 presents a list of physicochemical properties for various LNP compositions based on INL 1 (S-Ac7-Dog) with different amounts of DMG-PEG2000 and a cholesterol-to-phospholipid ratio of approximately 1:2.

[0310] [Table 2]

[0311]

[0312] Figure 3 shows the relative spleen-to-liver ratio of mRNA expression when Fluc mRNA LNP was intravenously injected at the molar ratios presented in Table 2. This spleen-to-liver ratio is based on in vitro (IVIS) of the spleen obtained via IVIS at 24 hours post-injection. ex vivo ) Average luminance (normalized as a function of photons / second / cm² / steradians) in vitro ( ex vivoIt was calculated as a ratio derived by dividing by the average luminance. According to the data presented below, the combination of 60% molar percentage of ionized lipids and a cholesterol-to-phospholipid ratio of 1:2 was found to be advantageous compared to the condition of 35% molar percentage of ionized lipids and a cholesterol-to-phospholipid ratio of 1:2, as well as compared to containing an LNP with a cholesterol-to-phospholipid ratio of 3.9:1, which is the standard lipid molar ratio used in Onpattro formulations.

[0313] Figure 4 shows luciferase activity in the bone marrow of an excised femur at 24 hours after injection. The femur was crushed to expose the bone marrow to oxygen, enabling visualization using IVIS. Luciferase activity is in vitro ( ex vivo It is expressed as average luminance (normalized as a function of photons / second / cm² / steradian). LNP 4, containing 3 mol% DMG-PEG-2000 molar percentage in combination with 60% ionized lipids and a cholesterol-to-phospholipid ratio of 1:2, showed significantly higher signals in bone marrow compared to LNPs with lower mol% ionized lipids and / or lower mol% DMG-PEG-2000.

[0314] Table 3 presents a list of physicochemical properties for various LNP compositions based on INL 1 (S-Ac7-Dog) with different amounts of DSG-PEG2000 and a cholesterol-to-phospholipid ratio of approximately 1:2.

[0315] [Table 3]

[0316]

[0317] Figure 5 shows luciferase activity in the bone marrow of an excised femur at 24 hours after injection. The femur was crushed to expose the bone marrow to oxygen, enabling visualization using IVIS. Luciferase activity is in vitro ( ex vivo) is expressed as average luminance (normalized as a function of photons / second / cm² / steradian). LNP 8 and LNP 9, containing 60% ionized lipid molar percentage in combination with DSG-PEG2000 and a cholesterol-to-phospholipid ratio of 1:2, showed significantly higher signals in bone marrow compared to LNPs containing lower mol% ionized lipids and LNP5, which represents an LNP with a cholesterol-to-phospholipid ratio of 3.9:1 and a standard lipid molar ratio used in Onpattro formulations.

[0318] Example 1c: The ratio of cholesterol to phospholipids determines the biodistribution of LNP independently of ionized lipids.

[0319] LNP manufacturing

[0320] Lipid-based nanoparticles were prepared by mixing mRNA solution and lipid solution in sodium acetate buffer (100 mM, pH 4) at a volume ratio of 2:1 (INL 1) or 3:1 (INL 2 / INL 3 / INL 4 / INL 5) using a T-mixing device at a rate of 9 mL / min, 12 mL / min, or 20 mL / min. The lipid solution contained a mixture of ionized lipids (INL 1 and INL 2 are both covered by patent application WO2022136641, INL 3 and INL 4 are both covered by patent application PCT / EP2024 / 069656, and INL 5 is covered by patent application WO2024084056), DSPC (Avanti), cholesterol (Sigma), and DMG-PEG2000 (Avanti).

[0321] Focusing on the cholesterol-to-phospholipid ratio, LNPs were prepared with different molar ratios of ionized lipids / auxiliary lipids / cholesterol / DMG-PEG2000. Fluc mRNA was encapsulated as reporter mRNA in all LNPs, and the mRNA / ionized lipid molar ratio was set to 1 / 10 for INL 1 and 1 / 6 for INL 2, INL 3, INL 4, and INL 5.

[0322] A list of relevant physicochemical properties for various LNP compositions is presented in Table 4 below.

[0323] [Table 4]

[0324]

[0325] Figure 6 shows the spleen-to-liver ratio of mRNA expression when Fluc mRNA LNP was intravenously injected at the molar ratios presented in Table 4. This spleen-to-liver ratio is based on in vitro (IVIS) of the spleen obtained via IVIS at 24 hours post-injection. ex vivo ) Average luminance (normalized as a function of photons / second / cm² / steradians) in vitro ( ex vivo It was calculated as a ratio derived by dividing by the average luminance. Here, LNPs with a cholesterol-to-phospholipid ratio of 1:2 exhibited a bias in biodistribution skewed toward the spleen compared to LNPs with a higher cholesterol-to-phospholipid ratio. As can be seen in Figure 6, similar effects on the spleen-to-liver ratio were observed regardless of the ionized lipids used in the LNP formulation. The condition with a cholesterol-to-phospholipid ratio of 3.9:1 corresponds to LNPs with standard lipid molar ratios used in the Onpattro formulation (INL / DSPC / Chol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5).

[0326] Example 1d: The combination of cholesterol to phospholipid ratio and ionized lipid percentage determines the biodistribution of LNP independently of ionized lipids.

[0327] LNP manufacturing

[0328] Lipid-based nanoparticles were prepared by microfluidically mixing an mRNA solution and a lipid solution in sodium acetate buffer (100 mM, pH 4) in a 3:1 volume ratio using a T-mixing device at a rate of 12 mL / min or 20 mL / min. The lipid solution contained a mixture of ionized lipids (INL 2 is covered by patent application WO2022136641 and INL 5 is covered by patent WO2024084056), DSPC (Avanti), cholesterol (Sigma), and DMG-PEG2000 (Avanti).

[0329] Focusing on the cholesterol-to-phospholipid ratio, LNPs were prepared with varying molar ratios of ionized lipids / auxiliary lipids / cholesterol / DMG-PEG2000. All LNPs were encapsulated with Fluc mRNA as reporter mRNA, and the mRNA / ionized lipid molar ratio was set to 1:6 for all formulations.

[0330] A list of relevant physicochemical properties of various LNP compositions based on INL 2 and INL 5 is presented in Table 5 below.

[0331] [Table 5]

[0332]

[0333] Figure 7 shows the spleen-to-liver ratio of mRNA expression when Fluc mRNA LNP was intravenously injected at the molar ratios presented in Table 5. This spleen-to-liver ratio is based on in vitro (IVIS) of the spleen obtained via IVIS at 24 hours post-injection. ex vivo ) Average luminance (normalized as a function of photons / second / cm² / steradians) in vitro ( ex vivoIt was calculated as a ratio derived by dividing by the average luminance. Here, LNP with a cholesterol-to-phospholipid ratio of 1:2 exhibited a bias in biodistribution skewed toward the spleen compared to LNP with a higher cholesterol-to-assisted lipid ratio. Furthermore, this data re-demonstrated that the combination of 60% molar percentage of ionized lipids and a cholesterol-to-phospholipid ratio of 1:2 was favorable compared to conditions with a similar cholesterol-to-phospholipid ratio and 35% ionized lipid percentage. These results were already evident in cases using previous alternative ionized lipids, suggesting that the effect is independent of the structure of the ionized lipids.

[0334] Flow cytometry-based cell affinity analysis after IV administration of Thy1.1 mRNA-containing LNP

[0335] Materials and Methods

[0336] animal

[0337] Mice were housed in IVCs under conditions free of specific pathogens. All animal experiments were performed with the approval of the ethics committee, and animal care was carried out according to established guidelines. Six-week-old female Balb / C mice (20 to 23 g) were supplied by Charles River International Laboratories, Inc. (France) and housed (up to 5 mice per cage), and were given free access to water and standard laboratory animal feed.

[0338] intravenous injection

[0339] For intravenous injection, mice were placed in a warming chamber to dilate their tail veins for up to 10 minutes. Once the veins were sufficiently dilated, the mice were immobilized individually. The tails were wiped with gauze soaked in 70% ethanol. The needle of a BD micro-syringe (20-25G) filled with LNP suspension was carefully inserted into one of the tail veins, and the plunger was slowly pressed to inject a dose of up to 200 μL. The needle was removed from the vein, and the injection site was gently pressed with dry gauze to stop local bleeding. The mice were then observed for LNP-induced reactogenicity in their cages for at least 10 minutes. Each injection corresponded to 5 μg or 20 μg of Thy1.1 mRNA, and each treatment group included 5 mice.

[0340] Flow cytometry

[0341] All mice were euthanized 16 hours after IV injection of the LNP formulation. Both the liver and spleen were excised and collected in MACS Tissue Storage Solution (Miltenyi Biotec). Following the manufacturer's instructions, the organs were mechanically and enzymatically separated using gentleMACS Tissue Dissociator and the Liver or Spleen Dissociation Kit (Miltenyi). Subsequently, red blood cells were removed using RBC Lysis Buffer (Thermo Fisher Scientific), the remaining cells were resuspended in FACS buffer (PBS containing 1% BSA and 2.5 mM EDTA), and the liver single-cell suspension was applied to a 40 μm strainer. After counting cells using ViaStain AOPI Staining Solution and Cellaca MX 2FL (Revvity), for each staining condition, 2 million viable cells were pre-stained using the LIVE / DEAD Fixable Near IR (876) Viability Kit (Thermo Fisher Scientific) and Purified Rat Anti-Mouse CD16 / CD32 (BD Biosciences), and then stained with one of the following antibody cocktails: Antibody Cocktail 1 (hepatic myeloid immune cell staining) containing CD90.1-BV786, TIM4-PE, CD11b-BV605 (BD Biosciences), Ly6G-PE-Cy5, Ly6C-AF700, CD31-PE-Cy7, CD146-APC (BioLegend), CD45-BUV496, and F4 / 80-eFluor450 (Thermo Fisher Scientific); CD11b-BV605, CD8-FITC (BD Biosciences), CD4-BV421, CD19-BV785 (BioLegend), CD90.1-APC, CD45-BUV496, CD3-PerCP-eFluor710, and NK1.Antibody cocktail 2 containing 1-PE-eFluor610 (Thermo Fisher Scientific) (hepatic lymphoid immune cell staining); antibody cocktail 3 containing CD3-BUV661, CD8-FITC (BD Biosciences), CD4-BV421, CD19-BV785 (BioLegend), CD90.1-APC, CD45-BUV496, and NK1.1-PE-eFluor610 (Thermo Fisher Scientific) (splenic lymphoid immune cell staining); Antibody cocktail 4 (staining of splenic myeloid immune cells) containing CD11b-BV605, CD11c-BV650 (BD Biosciences), Ly6C-AF700, CD3-biotin, CD19-biotin, NKp46-biotin (BioLegend), CD90.1-APC, CD45-BUV496, F4 / 80-eFluor450, and Ly6G-PE (Thermo Fisher Scientific). Cells stained with antibody cocktail 4 were subsequently stained with streptavidin-FITC (BD Biosciences). Flow cytometry analysis was performed using a CytoFLEX LX flow cytometer (Beckman Coulter) and FlowJo software (FlowJo LLC, BD Biosciences).

[0342] mRNA synthesis

[0343] Thymic cell antigen 1.1 (Thy1.1) mRNA was prepared from a linearized peTheRNA vector (see WO2015071295) using eTheRNA-optimized in vitro transcription (IVT) reaction conditions and purified through cellulose. N1ψ-modified mRNA was produced by completely substituting the uridine with N1-methylpseudouridine (N1ψ).

[0344] Example 1e: Effect of cholesterol to phospholipid ratio on cell orientation.

[0345] LNP manufacturing

[0346] Lipid-based nanoparticles were prepared by T-conjugation mixing of mRNA solution and lipid solution in sodium acetate buffer (100 mM, pH 4) in a 2:1 volume ratio (INL 1) or a 3:1 volume ratio (INL 2) at a rate of 9 mL / min (INL 1) or 12 mL / min (INL 2). The lipid solution contained a mixture of ionized lipids (both INL 1 and INL 2 are covered by patent application WO2022136641), DSPC (Avanti), cholesterol (Sigma), and DMG-PEG2000 (Avanti).

[0347] Focusing on the cholesterol-to-phospholipid ratio, LNPs were prepared with different molar ratios of ionized lipids / auxiliary lipids / cholesterol / DMG-PEG2000. Thy1.1 mRNA was encapsulated in all LNPs as reporter mRNA, and the mRNA / ionized lipid molar ratio was set to 1 / 10 for INL 1 and 1 / 6 for INL 2.

[0348] A list of relevant physicochemical properties for various LNP compositions is presented in Table 6 below.

[0349] [Table 6]

[0350]

[0351] Figure 8 illustrates the INL 2-containing formulation, showing the amount of Thy1.1-positive cells in specific hepatocyte types confirmed by flow cytometry 16 hours after IV injection. As can be seen in the graph, when the cholesterol-to-phospholipid ratio was changed from 3.9:1 to 1:2 while increasing the molar percentage of ionized lipids to 60 mol%, a distinct drop in expression was observed in most cell types, including hepatocytes, liver sinusoidal endothelial cells, neutrophils, monocytes, B cells, CD4+ and CD8+ T cells, NKT cells, and NK cells. These data demonstrate that the molar composition of the formulation enables de-targeting of the liver.

[0352] Additionally, Figure 9 shows the ratio of Thy1.1-positive splenic CD4+ T cells / CD8+ T cells / macrophages to the number of Thy1.1-positive hepatocytes. This once again demonstrates the bias in biodistribution skewed toward the spleen by lowering the cholesterol-to-phospholipid ratio, further showing that this is independent of the chemical structure of ionized lipids in the formulation.

Claims

Claim 1 A lipid nanoparticle (LNP) comprising an ionized lipid, a phospholipid, a sterol, a stabilized lipid; and one or more nucleic acid molecules, wherein the LNP comprises at least 55 mol% of the ionized lipid; and the ratio of the sterol to the phospholipid is 1:1 or less. Claim 2 Lipid nanoparticles (LNP) according to claim 1, wherein the ratio of sterol to phospholipid is 1:2 or less. Claim 3 A lipid nanoparticle according to either claim 1 or 2, wherein the LNP comprises at least 0.1 mol% of the stabilized lipid, particularly at least 0.5 mol% of the stabilized lipid, particularly at least 1.0 mol% of the stabilized lipid, particularly at least 1.5 mol% of the stabilized lipid, particularly at least 2.0 mol% of the stabilized lipid, more particularly at least 2.5 mol% of the stabilized lipid, at least 3.0 mol% of the stabilized lipid, at least 4.0 mol% of the stabilized lipid, at least 5.0 mol% of the stabilized lipid, at least 6.0 mol% of the stabilized lipid, at least 7.0 mol% of the stabilized lipid, at least 8.0 mol% of the stabilized lipid, at least 9.0 mol% of the stabilized lipid, and at least 10.0 mol% of the stabilized lipid. Claim 4 A lipid nanoparticle according to any one of claims 1 to 3, wherein the stabilized lipid is a PEG lipid or a variant thereof. Claim 5 A lipid nanoparticle according to claim 4, wherein the LNP comprises at least 0.1 mol% of the PEG lipid, particularly at least 0.5 mol% of the PEG lipid, particularly at least 1.0 mol% of the PEG lipid, particularly at least 1.5 mol% of the PEG lipid, particularly at least 2.0 mol% of the PEG lipid, more particularly at least 2.5 mol% of the PEG lipid, at least 3.0 mol% of the PEG lipid, at least 4.0 mol% of the PEG lipid, at least 5.0 mol% of the PEG lipid, at least 6.0 mol% of the PEG lipid, at least 7.0 mol% of the PEG lipid, at least 8.0 mol% of the PEG lipid, at least 9.0 mol% of the PEG lipid, and at least 10.0 mol% of the PEG lipid. Claim 6 Lipid nanoparticles according to any one of claims 1 to 5, wherein the molar percentage of the phospholipid is greater than 1 mol%, particularly 2 mol% to 40 mol%, more particularly 2 mol% to 30 mol%, more particularly 5 mol% to 28 mol%; more particularly 10 mol% to 27 mol%; more even more particularly 20 mol% to 26 mol%. Claim 7 Lipid nanoparticles according to any one of claims 1 to 6, wherein the molar percentage of the sterol is less than 22 mol%, particularly 0.4 mol% to 20 mol%, more particularly 2 mol% to 18 mol%; more particularly 10 mol% to 15 mol%, further more particularly 12 mol% to 13 mol%. Claim 8 A lipid nanoparticle according to any one of claims 4 to 7, wherein the PEG lipid is selected from a list including DMG-PEG and DSG-PEG. Claim 9 A lipid nanoparticle according to any one of claims 1 to 8, wherein the phospholipid is selected from a list comprising DOPE, DOPC, DSPC, ESM and mixtures thereof; particularly DSPC. Claim 10 A lipid nanoparticle according to any one of claims 1 to 9, wherein the sterol is selected from a list including cholesterol, ergosterol, campesterol, oxysterol, antrosterol, cholecalciferol, desmostol, nicasterol, sitosterol, and stigmasterol; preferably cholesterol. Claim 11 In any one of claims 1 to 10, the lipid nanoparticles wherein the LNP comprises: - about 60 mol% of the ionized lipid; - about 25.67 mol% of DSPC; - about 12.83 mol% of cholesterol; and - about 1.5 mol% of DMG-PEG. Claim 12 In any one of claims 1 to 11, the lipid nanoparticles wherein the LNP comprises: - about 60 mol% of the ionized lipid; - about 24.17 mol% of DSPC; - about 12.83 mol% of cholesterol; and - about 3.0 mol% of DMG-PEG. Claim 13 A pharmaceutical composition or vaccine comprising one or more lipid nanoparticles and a pharmaceutically acceptable carrier as defined in any one of claims 1 to 12. Claim 14 Lipid nanoparticles defined in any one of claims 1 to 12 or a pharmaceutical composition or vaccine defined in claim 13 for use in human medicine or veterinary medicine. Claim 15 Lipid nanoparticles defined in any one of claims 1 to 12 or a pharmaceutical composition or vaccine defined in claim 13 for use in the treatment of cancer, autoimmune disease, hereditary disorder, hematological disorder, aging, fibrosis, neurological disorder, cardiovascular disease or infectious disease. Claim 16 Lipid nanoparticles defined in any one of claims 1 to 12 or a pharmaceutical composition or vaccine defined in claim 13 for use in increasing gene expression of nucleic acid molecules in the spleen and / or bone marrow. Claim 17 mRNA to the target in need in vivo ( in vivo A method for delivering, comprising the step of administering to a subject a lipid nanoparticle defined in any one of claims 1 to 12 or a pharmaceutical composition or vaccine defined in claim 13.

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