Modified apolipoprotein having a targeting moiety for lipid nanoparticles

Abstract

The present invention relates to a modified apolipoprotein having a targeting moiety. The targeting moiety can be, for example, an antibody or an antigen-binding fragment that enables targeting of a specific cell, tissue, or organ. The modified apolipoprotein can be used as a carrier for a payload, either by itself or when incorporated into lipid nanoparticles. The modified apolipoprotein is utilized for the treatment or prevention of diseases or for targeting a payload to a specific target site.

Description

Technical Field

[0001] The present invention relates to the field of modified apolipoproteins used for the treatment or prevention of disorders. More specifically, the present invention relates to apolipoproteins having targeting moieties, apolipoprotein derivatives, apolipoprotein mimics or apolipoprotein mimic derivatives. The present invention further relates to lipid nanoparticles comprising a modified apolipoprotein and a method for producing such lipid nanoparticles. Finally, the present invention relates to a treatment method using a modified apolipoprotein or a lipid nanoparticle comprising such a modified apolipoprotein.

Background Art

[0002] Introduction The fact that many promising therapeutic agents do not reach or reach insufficiently the target sites they are intended for, or exhibit undesirable off-target effects, is a problem. Furthermore, many therapies can induce an undesirable immune response, resulting in degradation of the therapeutic agent or an undesirable inflammatory reaction. Therefore, there is a continuing need to improve or provide alternative methods for targeting therapeutic agents and to deliver therapeutic agents to their intended targets while avoiding an immune response.

[0003] These problems are addressed, inter alia, by the products and methods as defined in the appended claims.

Summary of the Invention

[0004] The present invention is based on the discovery by the inventors that apolipoproteins, apolipoprotein derivatives, apolipoprotein mimics or apolipoprotein mimic derivatives can be modified to target specific cells, tissues or organs by binding them to a targeting entity (wherein the targeting entity is capable of binding to a molecule on the cell surface of a target cell), and that such modified apolipoproteins can be used as carriers for therapeutic agents when used by themselves, i.e., not as part of lipid nanoparticles, or when used as part of lipid nanoparticles, preferably spherical lipid nanoparticles.

[0005] Furthermore, the inventors have unexpectedly found that by binding a targeting entity to an apolipoprotein, apolipoprotein derivative, apolipoprotein mimic or apolipoprotein mimic derivative, it is possible to easily incorporate the targeting entity into lipid nanoparticles, preferably spherical lipid nanoparticles, comprising an outer layer and a core, and to expose the targeting entity to the environment surrounding the lipid nanoparticles. In this way, the modified apolipoprotein can be targeted to cells, tissues or organs that would not otherwise be reached or would be insufficiently reached, or the modified apolipoprotein can be used to reduce off-target effects.

[0006] In a first aspect, the present invention relates to a modified apolipoprotein comprising an apolipoprotein, apolipoprotein derivative, apolipoprotein mimic or apolipoprotein mimic derivative bound to a targeting entity, wherein the targeting entity is a molecule capable of binding to a molecule on the cell surface of a target cell.

[0007] In a second aspect, the present invention is a lipid nanoparticle comprising an outer layer and a core, wherein the outer layer comprises - phospholipids, - sterols, and - a modified apolipoprotein as disclosed herein Relates to lipid nanoparticles comprising a core, the core comprising at least one core component selected from lipids, cationic lipids, or multivalent molecules.

[0008] In a third aspect, the present invention is a lipid nanoparticle comprising an outer layer and a core, wherein the outer layer comprises - phospholipids, - sterols, and - a modified apolipoprotein as disclosed herein and the core comprises a nucleic acid and a cationic or ionizable cationic lipid.

[0009] In a fourth aspect, the present invention is a method for producing a lipid nanoparticle as disclosed herein, comprising a1) expressing and isolating a modified apolipoprotein as disclosed herein to obtain an isolated modified apolipoprotein, and / or a2) chemically conjugating a targeting moiety to an apolipoprotein or apolipoprotein mimetic to obtain a modified apolipoprotein and isolating the modified apolipoprotein, b) combining the isolated modified apolipoprotein obtained in step a1 and / or step a2 with phospholipids, sterols and optionally a lipid to obtain lipid nanoparticles Relates to a method comprising.

[0010] In a fifth aspect, the present invention is a method for producing lipid nanoparticles, comprising a) rapidly mixing a lipid component in an organic solvent with a nucleic acid in an aqueous buffer to produce lipid nanoparticles, wherein the lipid component comprises a phospholipid, a sterol, a cationic lipid or an ionizable cationic lipid, and the aqueous buffer has a pH of 5.0 or less, and b) rapidly mixing the lipid nanoparticles with one or more modified apolipoproteins as taught herein at a pH of 5.5 - 8.0, preferably 6.0 - 8.0, to produce lipid nanoparticles Relates to a method including the following.

[0011] In a sixth aspect, the present invention relates to a modified apolipoprotein according to the first aspect, or a lipid nanoparticle according to the second or third aspect, for use as a medicament, or a lipid nanoparticle obtained or obtainable by the method according to the fourth or fifth aspect of the present invention.

[0012] In a seventh aspect, the present invention relates to a modified apolipoprotein according to the first aspect, or a lipid nanoparticle according to the second or third aspect, for use in the treatment or prevention of immune-related disorders such as transplant rejection, graft-versus-host disease (GVH), atherosclerosis, infectious diseases, inflammation, autoimmunity, allergies, cancer, genetic disorders, metabolic disorders, neuropathies or tissue trauma, or a lipid nanoparticle obtained or obtainable by the method according to the fourth or fifth aspect of the present invention.

[0013] In an eighth aspect, the present invention relates to the use of a modified apolipoprotein according to the second or third aspect, or a lipid nanoparticle obtained or obtainable by the method according to the fourth or fifth aspect of the present invention, when delivering a compound to a target, preferably the target is a cell, tissue, and / or organ, more preferably the target is a lymphocyte, myeloid cell, tumor cell, endothelial cell, hematopoietic stem cell progenitor cell (HSPC), hematopoietic stem cell (HSC), multipotent progenitor cell (MPP), common myeloid progenitor cell (CMP), or the targeting entity binds to a bacterial, viral, fungal or parasitic protein or antigen. Preferably, the lymphocyte or myeloid cell is selected from monocytes, macrophages, M1-like macrophages, M2-like macrophages, eosinophils, basophils, mast cells, NK cells, B cells, plasma cells, regulatory T cells, hematopoietic stem cells, helper T cells such as Th1, Th2, Th17 or Th22, plasmacytoid dendritic cells, dendritic cells such as conventional DC1 or conventional DC2, or tumor-associated macrophages.

[0014] In a ninth aspect, the present invention relates to an in vitro or ex vivo method for introducing a nucleic acid into a cell, the method comprising contacting the cell with a lipid nanoparticle as disclosed herein, or a lipid nanoparticle obtained by or obtainable by a method as disclosed herein.

[0015] In a tenth aspect, the present invention relates to an in vivo method for introducing a nucleic acid into a cell, the method comprising contacting the cell with a lipid nanoparticle as disclosed herein, or a lipid nanoparticle obtained by or obtainable by a method as disclosed herein.

[0016] In an eleventh aspect, the present invention relates to an in vivo delivery method of a nucleic acid, the method comprising administering to a subject a lipid nanoparticle as disclosed herein, or a lipid nanoparticle obtained by or obtainable by a method as disclosed herein.

[0017] In a twelfth aspect, the present invention relates to a method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a lipid nanoparticle as disclosed herein, or a lipid nanoparticle obtained by or obtainable by a method as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

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Mode for Carrying Out the Invention

[0019] For the purposes of the present invention, the following terms are defined as follows.

[0020] As used herein, the singular terms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes combinations of two or more cells and the like.

[0021] As used herein, the term "and / or" refers to a situation where one or more of the recited instances may occur alone or in combination with at least one of the recited instances and up to all of the recited instances.

[0022] As used herein, the term "antigen" refers to a substance to which the binding portion of an antibody can bind. The specific immunoreactive site within an antigen is known as an "epitope" (or antigenic determinant). The target for an antibody, or an antigen-binding portion thereof, can include an antigen as defined herein.

[0023] As used herein, the term "at least" with respect to a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ··· etc. As used herein, the term "at most" with respect to a particular value means that particular value or less. For example, "at most 5" is understood to be the same as "5 or less", i.e., 5, 4, 3, ···, -10, -11 etc.

[0024] As used herein, the word "comprising" or variations thereof such as "comprises" or "comprising" is understood to include the recited element, integer or step, or group of elements, integers or steps, but not to exclude any other element, integer or step, or group of elements, integers or steps. The verb "comprising" encompasses the verbs "consisting essentially of" and "consisting of".

[0025] As used herein, the term "conventional techniques" refers to situations where the methods for performing conventional techniques used in the methods of the present invention are apparent to those of ordinary skill in the art. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields is well known to those of ordinary skill in the art and is described, for example, in the following references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates, and the series Methods in Enzymology, Academic Press, San Diego.

[0026] As used herein, the term "identity" refers to a measure of identity of nucleotide or amino acid sequences. Generally, sequences are aligned so as to obtain the highest degree of match. "Identity" itself has a meaning recognized in the art and can be calculated using published techniques. See, for example, (Computational Molecular Biology, Lesk, A.M., ED., Oxford University Press, New York, 1988, Biocomputing: Informatics And Genome Projects, Smith, D.W., ED., Academic Press, New York, 1993, Computer Analysis Of Sequence Data, Part I, Griffin, A.M., And Griffin, H.G., EDS., Humana Press, New Jersey, 1994, Sequence Analysis In Molecular Biology, Von Heinje, G., Academic Press, 1987, and Sequence Analysis Primer; Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). There are several methods for measuring identity between two nucleotide or amino acid sequences, and at the same time, the term "identity" is well-known to those skilled in the art (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48: 1073). Methods commonly used to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide To Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., Siam J. Applied Math (1988) 48: 1073).Methods for determining identity and similarity are codified in computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1): 387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215: 403).

[0027] By way of example, a polynucleotide having a nucleotide sequence that has at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of a particular sequence is intended to have a nucleotide sequence that is identical to the reference sequence, provided that the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference amino acid sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted and / or replaced with another nucleotide, and / or up to 5% of the number of nucleotides of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or at any location between those terminal positions, and may be scattered individually between nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0028] Similarly, a polypeptide having an amino acid sequence that has at least, for example, 95% "identity" to the reference amino acid sequence of SEQ ID NO: X is intended to have an amino acid sequence of the polypeptide that is identical to the reference sequence, provided that the amino acid sequence may contain up to 5 amino acid changes per 100 amino acids of the respective reference amino acids of SEQ ID NO: X. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted, or replaced with another amino acid, or up to 5% of the number of amino acids of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence, or anywhere between those terminal positions, and may occur individually among the residues in the reference sequence, or may be scattered in one or more contiguous groups within the reference sequence.

[0029] As used herein, the term "in vitro" refers to experiments or measurements performed using components of an organism isolated from their natural conditions.

[0030] As used herein, the term "ex vivo" refers to experiments or measurements performed in or on tissue derived from an organism in an external environment with minimal alteration of natural conditions.

[0031] As used herein, the terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" are intended to encompass DNA molecules and RNA molecules. The nucleic acid (molecule) can be single-stranded or double-stranded, but is preferably double-stranded DNA or single-stranded or double-stranded RNA, more preferably single-stranded or double-stranded RNA.

[0032] The terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" are well understood in the art. For further guidance, the terms generally refer to any length polymer (preferably a linear polymer) essentially composed of nucleoside units. Nucleoside units generally include a heterocyclic base and a sugar group. As heterocyclic bases, inter alia, purine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) widely present in naturally occurring nucleic acids, as well as pyrimidine bases, other naturally occurring bases (e.g., xanthine, inosine, hypoxanthine), and chemically or biochemically modified (e.g., methylated) bases, unnatural bases or derivatized bases can be mentioned. Exemplary modified nucleic acid bases include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, but are not limited thereto. In particular, 5-methylcytosine substitution has been shown to enhance nucleic acid duplex stability. Sugar groups include, inter alia, pentose (pentofuranose) groups such as ribose and / or 2-deoxyribose, which are common to naturally occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, and modified or substituted sugar groups (2'-O-alkylated, e.g., 2'-O-methylated or 2'-O-ethylated sugars, e.g., ribose; 2'-O-alkyloxyalkylated, e.g., 2'-O-methoxyethylated sugars, e.g., ribose; or 2'-O,4'-C-alkylidene linked, e.g., 2'-O,4'-C-methylene linked or 2'-O,4'-C-ethylene linked sugars, e.g., ribose; 2'-fluoro-arabinose, etc., but are not limited thereto).The nucleoside units are linked to each other by any one of a number of known internucleoside linkages, including, inter alia, phosphodiester linkages common to naturally occurring nucleic acids, and further modified phosphate or phosphonate-based linkages such as phosphorothioates, alkyl phosphorothioates such as methyl phosphorothioate, phosphorodithioates, alkyl phosphonates such as methyl phosphonate, alkyl phosphonothioates, phosphotriesters such as alkyl phosphotriester, phosphoramidates, phosphoropiperazidates, phosphoromorpholidates, cross-linked phosphoramidates, cross-linked methylene phosphonates, cross-linked phosphorothioates; and still further, siloxanes, carbonates, sulfamates, carboalkoxys, acetamidates, carbamates such as 3'-N-carbamate, morpholino, borano, thioethers, 3'-thioacetals, and sulfones. Preferably, the internucleoside linkage can be a phosphate-based linkage including a modified phosphate-based linkage, for example, more preferably, a phosphodiester, phosphorothioate or phosphorodithioate linkage, or a combination thereof. Also, the term "nucleic acid" includes, but is not limited to, any other nucleic acid base-containing polymer such as nucleic acid mimetics, including peptide nucleic acid (PNA), peptide nucleic acid having a phosphate group (PHONA), locked nucleic acid (LNA), morpholino phosphorodiamidate backbone nucleic acid (PMO), cyclohexene nucleic acid (CeNA), tricyclo-DNA (tcDNA), and nucleic acids having a backbone segment with an alkyl linker or an amino linker (see, for example, Kurreck 2003 (Eur J Biochem 270: 1628-1644)). "Alkyl", when used in this context, includes, in particular, lower hydrocarbon moieties, for example, straight-chain or branched, saturated or unsaturated hydrocarbons having from C1 to C4, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl.

[0033] Nucleic acids as contemplated herein may include naturally occurring nucleosides, modified nucleosides, or mixtures thereof. Modified nucleosides may include modified heterocyclic bases, modified sugar moieties, modified internucleoside linkages, or combinations thereof. The term "nucleic acid" more preferably includes DNA, RNA, and DNA / RNA hybrid molecules, specifically hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesized) DNA, RNA, or DNA / RNA hybrids. Nucleic acids may be naturally occurring, e.g., may exist in nature or be isolated from nature, may be recombinant, i.e., produced by recombinant DNA technology, and / or may be partially or wholly chemically or biochemically synthesized. "Nucleic acid" can be double-stranded, partially double-stranded, or single-stranded. In the case of single-stranded, the nucleic acid can be a sense strand or an antisense strand. Further, the nucleic acid can be circular or linear.

[0034] As used herein, the term "sequence" when referring to a nucleotide, or "nucleic acid sequence", "nucleotide sequence", or "polynucleotide sequence" refers to the order of nucleic acids and / or polynucleotides, or nucleotides therein. Within the context of the present invention, a first nucleic acid sequence may be contained within or overlap with a further nucleic acid sequence.

[0035] As used herein, the terms "subject" or "individual" or "animal" or "patient" or "mammal" are used interchangeably and refer to any subject for which a diagnosis, prognosis, or therapy is desired, particularly a mammalian subject. Examples of mammalian subjects include humans, laboratory animals, livestock, and zoo animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, bears, sport animals, or pet animals. As defined herein, a subject may be alive or dead. Samples can be taken from a subject postmortem, i.e., after death, and / or samples can be taken from a living subject.

[0036] As used herein, the terms "treatment", "treating", "alleviating", "reducing", or "ameliorating" are used interchangeably and refer to an approach for obtaining beneficial or desirable results, including but not limited to therapeutic benefit. Therapeutic benefit means eradication or amelioration of, or reduction (or delay) in the progression of, a primary disease being treated. Also, therapeutic benefit is achieved by eradication or amelioration of, or reduction (or delay) in the progression of, one or more of the physiological symptoms associated with the primary disease, such that an improvement or deceleration or reduction in decline is observed in the patient, even though the patient may still be suffering from the primary disease.

[0037] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which a nucleic acid molecule capable of being transported is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. The term "vector" may also refer to a viral particle containing a nucleic acid of interest (i.e., a viral vector).

[0038] Part of the present invention contains materials subject to copyright protection (including but not limited to figures, device photographs, or any other aspect of this submission for which copyright protection is available or may be available). Since the patent document or the patented invention is found in the patent file or record of the Patent Office, the copyright owner does not object to the copying of the patent document or the patented invention by anyone, but reserves all copyrights in other circumstances.

[0039] Various terms relating to the methods, compositions, uses, and other aspects of the present invention are used throughout the specification and claims. Such terms, unless otherwise noted, should be given their ordinary meaning in the technical field to which the invention pertains. Other specifically defined terms should be construed in a manner consistent with the definitions provided herein. Preferred materials and methods are described herein, but any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present invention.

[0040] Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0041] The present invention is based on the inventors' discovery that apolipoproteins can be modified to target specific cells, tissues, organs, or pathogens. The inventors have found that a modified apolipoprotein that binds to a targeting entity enables the targeting of lipid nanoparticles containing such modified apolipoprotein. Since lipid nanoparticles (or modified apolipoproteins) can carry a payload such as a pharmaceutical, this enables the direct targeting of the pharmaceutical to a desired target site. Furthermore, the modified apolipoprotein of the present invention also enables the preparation of lipid nanoparticles, where the targeting entity is consequently exposed to the environment (i.e., the aqueous environment) surrounding the apolipoprotein lipid nanoparticles.

[0042] Accordingly, in a first aspect, the present invention relates to a modified apolipoprotein comprising an apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, or apolipoprotein mimetic derivative bound to a targeting entity, wherein the targeting entity is a molecule capable of binding to a molecule on the cell surface of a target cell.

[0043] The modified apolipoprotein can be used by itself, i.e., not as part of lipid nanoparticles. In this way, the modified apolipoprotein can function as a carrier for delivering a payload (e.g., a therapeutic agent) to a target site. The payload can be bound to the modified apolipoprotein, for example, by a covalent bond. The targeting entity can be used to target the modified apolipoprotein to a specific site such as a cell, tissue, organ, or pathogen.

[0044] As used herein, the term "bound" when referring to a modified apolipoprotein can refer to a stable or semi-stable association such as, but not limited to, a peptide bond, a sulfur bond, a protein-protein interaction, or a protein-ligand interaction, such as a covalent bond, a hydrogen bond, or a van der Waals force, or a combination thereof.

[0045] Targeting entity As used herein, a targeting entity refers to a molecule that, when bound to a protein such as an apolipoprotein, enables the protein to bind to a different target (or in other words, a target different from what it would bind to if the targeting entity were not fused to the protein) and / or to bind to its intended target with a higher affinity than it would bind without the targeting entity being fused to the protein.

[0046] In certain embodiments, the targeting entity may be capable of binding to different targets, such as a particular subset of target cells, including binding to a particular subset of the set of cells that are normally bound by the modified apolipoprotein. For example, apoA1 is known to bind to receptors on bone marrow cells, and thus, the targeting entity can be used to bind to a particular subtype of bone marrow cells. For example, as described elsewhere herein, SIRP alpha as a targeting entity may be capable of targeting immunosuppressive macrophages. Non-limiting examples of targeting entities are antibodies or antigen-binding fragments thereof or antibody fragments, routing peptides or routing proteins, and preferably, the routing peptides or routing proteins are ligands of receptors present on the target.

[0047] Thus, in one embodiment, the targeting entity is - an antibody or an antigen-binding fragment of an antibody, - a protein ligand, a protein-binding domain, or a protein-binding fragment thereof, - a peptide, - a peptidomimetic, or - a glycopolymer selected from.

[0048] As used herein, the term "target" refers to an object to which the modified apolipoprotein or targeting entity preferentially binds. The target can refer to a receptor or cell surface molecule, such as a protein or proteoglycan, lipid, phospholipid, sugar, glycopolymer, cell, cell type, tissue or tissue type or organ.

[0049] Apolipoproteins are recognized to bind to specific ligands. For example, various apolipoproteins found in various lipoproteins (e.g., HDL, LDL, VLDL, etc.) are thought to be involved in targeting and binding and thus differences in lipoprotein function. Without wishing to be bound by theory, some apolipoproteins have amphiphilic properties and are involved in binding to lipids in an aqueous environment together with phospholipids and / or sterols, while different parts of the molecule are thought to be involved in interacting with other molecules, e.g., binding to protein receptors. Furthermore, apolipoproteins are assumed to be able to circulate as proteins, i.e., not as lipoproteins. Thus, the ability to modify the binding affinity of apolipoproteins (by adding targeting entities) provides an interesting opportunity to fine-tune the targeting or binding of apolipoproteins. Several uses are envisioned for such modified apolipoproteins.

[0050] First, the targeting entity can simply be used to reroute apolipoproteins or lipoproteins, for example, to effect a change in lipid homeostasis. For example, it can be assumed that the LDL or HDL values in the blood of a subject can be altered by using apolipoproteins combined with the targeting entity. This can potentially be utilized in the treatment of lipid disorders such as high blood cholesterol levels.

[0051] Second, the targeting entity can be used to reroute a lipoprotein (lipid nanoparticle) having a payload to a predetermined target. Lipoproteins or lipid nanoparticles provide an interesting way to carry payloads such as pharmaceutical compounds. Thereby, delivery of lipophilic compounds through the blood is enabled when the compound can be dissolved in the lipid core of the lipoprotein / lipid nanoparticle. A further advantage is that the lipoprotein consists of a naturally occurring compound and is thus recognized as native by the immune system, avoiding the induction of an immune response by the pharmaceutical compound.

[0052] Third, the targeting entity may be further combined with the payload and can thus enable targeting of the payload. As described above, apolipoproteins can essentially function as carriers for the payload. It is an advantage that apolipoproteins significantly reduce the removal of the payload and enable the use of payloads (such as cytokines) that are easily removed from the blood in a therapeutic setting. One possible problem is that the payload / apolipoprotein combination does not reach the intended target of the payload (i.e., the site, cell, tissue, or organ where it is intended to exert its effect). This is solved by further including the targeting entity.

[0053] Data generated by the inventors suggests that modified apolipoproteins can be successfully rerouted to different targets by using the targeting entity. Thus, in one embodiment, the targeting entity is selected from an antibody or an antigen-binding fragment thereof, a rerouting peptide or a rerouting protein, preferably, where the rerouting peptide or rerouting protein is a ligand of a receptor present on the target. In one embodiment, the targeting entity may be an antibody or an antigen-binding fragment thereof. In principle, any type of antigen-binding molecule is assumed to be usable as the targeting entity for the modified apolipoprotein according to the invention.

[0054] In certain embodiments, the targeting entity binds to an antigen that is highly expressed on, or is present exclusively on, myeloid cells. Suitable targets are known to those of skill in the art, and non-limiting examples are co-stimulatory molecules such as CD11b, CD11c, CD14 or CD80, CD83, CD86, CD40 or HLA-DR. Thus, in one embodiment, the targeting entity is a myeloid targeting peptide or protein selected from an antibody or an antigen-binding fragment thereof that binds to CD11b, CD11c, CD14, CD80, CD83, CD86, CD40 or HLA-DR.

[0055] In other certain embodiments, the targeting entity may be a protein ligand that binds to a receptor or factor expressed on myeloid cells, non-limiting examples of which are CD40L (CD154) and the Fc domain, although those of skill in the art will recognize other suitable ligands or co-factors. Thus, in one embodiment, the targeting entity is a myeloid targeting peptide or myeloid targeting protein, where the myeloid targeting protein or myeloid targeting peptide is selected from CD40L (CD154) and the Fc domain.

[0056] In one embodiment, the targeting entity is an antibody or an antigen-binding fragment thereof, where the antibody or antigen-binding fragment thereof is a fragment antigen-binding region (Fab), Fab2, single-chain variable fragment (scFv), scFv-Fc, dAb-Fc, free light chain antibody, half antibody, bispecific Fab2, Fab3, trispecific Fab3, diabody, bispecific diabody, tribody, trispecific tribody, minibody, IgG, immunoglobulin new antigen receptor (IgNAR), monovalent IgG, VhH, nanobody, or variable domain of a new antigen receptor (VNAR), or an antigen-binding fragment thereof, preferably selected from camelid or shark VhH or derivatives thereof or antigen-binding fragments thereof.

[0057] The antibody or antigen-binding fragment may also be a designed antigen-binding protein such as, but not limited to, an affibody, FN3 domain, DARPin, or de novo designed protein receptor. Antibodies or antigen-binding fragments thereof having a lower molecular weight are recognized as being preferred due to their reduced size. Thus, in a preferred embodiment, the antibody or antigen-binding fragment thereof is a Fab, scFv, single-domain antibody, V h H or VNAR.

[0058] In one embodiment, the targeting entity may be a rerouting peptide. Non-limiting examples of rerouting peptides are receptor-binding peptides and / or ligand-mimicking peptides. Thus, in one embodiment, the rerouting peptide is selected from programmed cell death protein 1 (PD1) or signal regulatory protein alpha (SIRPa). SIRP alpha as a targeting entity may enable targeting immunosuppressive macrophages. However, it is understood that any peptide having binding specificity for a cell surface receptor may be used as the targeting entity.

[0059] In one embodiment, the targeting entity may be a protein such as a receptor ligand, receptor, or interacting protein. Alternatively, the targeting entity may be a portion of a protein, such as a protein or ligand-binding domain. Thus, in one embodiment, the rerouting protein is selected from, for example, CD40L or GP120. CD40L can be used to target cells expressing the CD40 receptor. GP120 can be used to directly bind to the CD4 T cell coreceptor. However, it is understood that any protein having binding specificity for a cell surface receptor may be used as the targeting entity.

[0060] In one embodiment, the targeting entity may be a sugar polymer. The sugar polymer can be used to direct an apolipoprotein modified with the sugar polymer to a specific site or receptor. For example, the apolipoprotein can be modified with mannose to direct the modified apolipoprotein to the mannose receptor, which is generally expressed on macrophages, dendritic cells, and endothelial cells. Those skilled in the art recognize other sugar polymers that can be used for similar purposes and which cells can be targeted using them. As used herein, the term sugar polymer refers to any polysaccharide and thus any molecule containing at least two saccharides.

[0061] As used herein, the term lipoprotein refers to particles of at least one apolipoprotein and lipid molecules, generally nanoparticles, dispersed or dissolved in an aqueous environment.

[0062] As used herein, the term rerouting refers to targeting a modified apolipoprotein to a target different from the one to which it normally binds, or reducing the binding of the modified apolipoprotein to its normal target, or preventing off-target binding. For example, apolipoprotein a1 (apoA1) is known to bind to receptors on myeloid cells, and thus a targeting entity can be used to bind to different cells and reduce binding to myeloid cells (e.g., myeloid cells can be monocytes, dendritic cells, tissue macrophages or granulocytes).

[0063] In one embodiment, the targeting entity binds to a protein on the surface of lymphocytes, myeloid cells, tumor cells, endothelial cells, hematopoietic stem cell progenitor cells (HSPCs), hematopoietic stem cells (HSCs), multipotent progenitor cells (MPPs), myeloid common progenitor cells (CMPs), or the targeting entity binds to a bacterial, viral, fungal or parasitic protein or antigen, Preferably, the targeting entity binds to a protein on the surface of lymphocytes, bone marrow cells, or tumor cells, or the targeting entity binds to a bacterial, viral, fungal, or parasitic protein or antigen, Preferably, the lymphocyte or bone marrow cell is selected from monocytes, macrophages, M1-like macrophages, M2-like macrophages, eosinophils, basophils, mast cells, NK cells, B cells, plasma cells, regulatory T cells, hematopoietic stem cells, granulocytes, helper T cells such as Th1, Th2, Th17, or Th22, plasmacytoid dendritic cells, dendritic cells such as conventional DC1 or conventional DC2, or tumor-associated macrophages, More preferably, the lymphocyte or bone marrow cell is selected from monocytes, macrophages, M1-like macrophages, M2-like macrophages, eosinophils, basophils, mast cells, NK cells, B cells, plasma cells, regulatory T cells, hematopoietic stem cells, helper T cells such as Th1, Th2, Th17, or Th22, plasmacytoid dendritic cells, dendritic cells such as conventional DC1 or conventional DC2, or tumor-associated macrophages.

[0064] In certain embodiments, the bone marrow cell may be a blood cell derived from a progenitor cell for granulocytes, monocytes, erythrocytes, or platelets.

[0065] In one embodiment, the targeting entity is CD1a, CD1b, CD1c, CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD15u, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79α, CD79β, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CD125, CD126, CD127, CDw128, CD129, CD130, CDw131, CD132, CD133, CD134, CD135, CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CD146, CD147,CD148, CD150, CD151, CD152, CD153, CD154, CD155, CD156a, CD156b, CD157, CD158, CD158a, CD158b, CD159a, CD160, CD161, CD162, CD162R, CD163, CD164, CD165, CD166, CD167a, CD168, CD169, CD170, CD171, CD172a, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179a, CD179b, CD180, CD183, CD184, CD195, CDw197, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD207 (CLEC4K), CD208, CD209 (CLEC4L), CDw210, CD212, CD213a1, CD213a2, CDw217, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD236, CD236R, CD238, CD239, CD240CE, CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR8, CCR9, CLEC1A, CLEC1B, CLEC2A, CLEC2B, CLEC3A, CLEC3B, CLEC4A, CLEC4C (CD303), CLEC4D, CLEC4J CLEC4E, CLEC4F, CLEC4G, ASGR1 (CLEC4H1), ASGR2 (CLEC4H2), FCER2 (), CLEC4M, CLEC5A, CLEC6A, CLEC7A, OLR1 (CLEC8A), CLEC9A, CLEC10A, CLEC11A, CLEC12A, CLEC12B, CD302 (CLEC13A), LY75 (CLEC13B), PLA2R1 (CLEC13C), MRC1 (CLEC13D), MRC2 (CLEC13E), CLEC14A, CLEC16A, CLEC17A, KLRA1, KLRB1 (CLEC5B), KLRC1, KLRC2, KLRC3Selected from KLRC4, KLRD1, KLRF1 (CLEC5C), KLRG1 (CLEC15A), KLRG2 (CLEC15B), KLRK1, AGC1, ATRNL1, BCAN, CD248, CHODL, CL-K1-Ia, CL-K1-Ib, CL-K1-Ic, CLECSF5, COLEC10, COLEC11, COLEC12, CSPG3, FCER2, FREM1, HBXBP, LAYN, LOC348174, LOC728276, MAFA, MBL2, MGC34761, MICL, MRC1L1, PAP, PKD1, PKD1L2, PRG2, PRG3, REG1A, REG1B, REG3A, REG3G, REG4, SELE, SELL, SELP, SFTPA1, SFTPA2, SFTPA2B, SFTPD, SRCL, THBD, VCAN, alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelial tumor antigen (ETA), tyrosinase, and melanoma-associated antigen (MAGE). Preferably, it binds to a protein selected from CD14, CD11b, CD357 (GITR), CD193, CD123, CD117, CD56, CD19, CD38, CD25, CD133, CXCR3, CCR3, CD196, CCR10, CD64, CD206, CLEC4C (CD303), CLEC9A, CD1c, CD163, alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelial tumor antigen (ETA), tyrosinase, and melanoma-associated antigen (MAGE). Binds to a protein selected from:

[0066] These targets can be used to target specific cell types or subsets of cells. Those skilled in the art will recognize which markers may be appropriate for which cell types (s), for example, the following proteins can be used to target specific cells:

[0067] Cells of the innate immune system: - Monocytes: CD14 - Macrophages: CD11b - Natural lymphocytes: CD357 (GITR) - Eosinophils: CD193 - Good base ball: CD123 - Mast cell: CD117

[0068] Cells of the adaptive immune system: - NK cell: CD56 - B cell: CD19 - Plasma cell: CD38 - Regulatory T cell: CD25

[0069] Subtypes of immune cells: - Bone marrow progenitor cells: - Hematopoietic stem cells: CD133 - Helper T cell subsets: Th1: CXCR3, Th2: CCR3, Th17: CD196, Th22: CCR10 - Macrophage polarization state (M1-like or M2-like): M1: CD64, M2: CD206 (mannose receptor) (e.g., targeted by apolipoprotein conjugated to a nanobody specific for this receptor, or apolipoprotein conjugated to mannose) - Subtypes of dendritic cells: Plasmacytoid dendritic cells: CD303, Conventional DC1: CLEC9A, Conventional DC2: CD1c - Tumor-associated macrophages: CD163 (or targeted by a nanobody against a tumor antigen).

[0070] In certain embodiments, the targeting entity can bind to non-myeloid cells, such as non-myeloid immune cells, preferably non-myeloid leukocytes, more preferably lymphocytes, more preferably T cells, and even more preferably CD8+ T cells.

[0071] In certain embodiments, the targeting entity can bind to cells that are not myeloid cells but can differentiate into myeloid cells such as hematopoietic stem cell progenitors (HSPCs) like hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), or common myeloid progenitors (CMPs).

[0072] In certain embodiments, the targeting entity can bind to non-myeloid cells, such as non-myeloid immune cells or endothelial cells. Endothelial cells can be targeted using a targeting entity capable of binding to a surface marker of endothelial cells. For example, endothelial cells can be targeted using a targeting entity capable of binding to a factor VIII-related antigen such as factor VIII, a targeting entity capable of binding to CD31 / PECAM-1 such as CD31, a targeting entity capable of binding to angiotensin-converting enzyme (ACE / CD143) such as angiotensin, a targeting entity capable of binding to CD34 such as L-selectin, or a targeting entity capable of binding to endoglin (CD105).

[0073] In certain embodiments, the non-myeloid cells are lymphocytes such as T cells, B cells, or natural killer (NK) cells. Preferably, the lymphocytes are T cells, more preferably CD8+ T cells.

[0074] In certain embodiments, the targeting entity is an antibody or an antigen-binding fragment thereof that binds to, for example, specifically binds to CD8.

[0075] In certain embodiments, when the target cell is a T cell, the targeting entity can be an antibody or an antigen-binding fragment thereof that binds to, preferably specifically binds to CD8. For example, the targeting entity can be VHH CD8 containing an amino acid sequence as described in Woodham A.W. et al., Nanobody-antigen conjugates elicit HPV-specific antitumor immune responses, Cancer Immunology Research, 2018, Vol. 6, issue 7 and as shown in Supplemental Table 1 of the above reference.

[0076] For example, apolipoprotein nanoparticles targeting CD8 as described herein can also be used to generate chimeric antigen receptor (CAR) T cells in vivo. In particular, apolipoprotein nanoparticles targeting CD8 can be used to deliver plasmid DNA (pDNA) encoding a command to reprogram T cells, linear or circular RNA (e.g., linear or circular mRNA), and / or gene editing components in vivo. For example, a targeted nuclease can be used to introduce nucleic acids into cells. For example, as the targeted nuclease, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZNFs), clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR / Cas9, CRISPR / Cpfl, and combinations thereof, such as single guide RNA such as single guide RNA using CRISPR / Cas or other gene editing components, can be used, but are not limited thereto. In certain examples, pDNA or RNA encoding a chimeric antigen receptor (CAR) is delivered to T cells, enabling them to recognize and kill tumor cells bearing cognate ligands. In another specific example, pDNA or RNA encoding a chimeric antigen receptor (CAR) is delivered to T cells, enabling them to recognize and eliminate pathological cells such as activated fibroblasts in fibrotic disorders.

[0077] Apolipoprotein nanoparticles that target CD8 as described herein can also be used to modify CD8+ T cell function. For example, CD8+ T cell function can be modified by altering receptor interactions between cytokines using the above-described CD8-targeting apolipoprotein nanoparticles to deliver plasmid DNA (pDNA) encoding instructions for cytokine production in T cells, linear or circular RNA (e.g., linear or circular mRNA), and / or the above-described gene editing components. In certain examples, pDNA or mRNA encoding cytokines such as IFN-y, IL-2, IL-10, IL-12, IL-15 can be delivered to T cells to enable them to secrete the cytokines. This can reprogram the immunosuppressive tumor microenvironment into an antitumor phenotype. In another specific example, pDNA or mRNA encoding cytokine receptors such as IFN-yR, IL-2R, IL-12R, or IL-15R may be delivered to T cells. CD8+ T cell function may also be modified through the regulation of immune checkpoints. For example, CD8+ T cell function can be modified by downregulating immune checkpoints using the above-described CD8-targeting apolipoprotein nanoparticles to deliver antisense oligonucleotides, small interfering RNA (siRNA), mRNA, and / or the above-described gene editing components that encode instructions for downregulating immune checkpoints in T cells. In certain examples, siRNA targeting PD1, CTLA4, SHP-2, LAG3, or TIM-3 or the above-described gene editing components may be delivered to T cells for downregulation of checkpoint molecules that inhibit cytotoxic T cell function in cancer.

[0078] In yet another example, apolipoprotein nanoparticles targeting CD8 as described herein can be used for the re-polarization of pathogenic CD8+ T cells in autoimmunity towards a tolerogenic phenotype. In particular, apolipoprotein nanoparticles targeting CD8 can be used to deliver an antisense oligonucleotide, small interfering RNA (siRNA), mRNA, or a gene editing component as described above that encodes a command to re-polarize pathogenic T cells. In a specific example, mRNA encoding FOXP3 can be delivered to T cells and change their phenotype to regulatory T cells (Tregs). In another specific example, CD8+IL17+ T cells can be re-polarized via delivery of siRNA targeting IL-17 or the RORyt transcription factor.

[0079] In one embodiment, the modified apolipoprotein comprises aapoA1 together with VHH8CD8.

[0080] In one embodiment, the VHHCD8-apoA1 modified apolipoprotein comprises, consists of, or is encoded by a nucleic acid having a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 7 or SEQ ID NO: 1, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 8 or SEQ ID NO: 2.

[0081] In certain embodiments, when the target cell is a T and / or B cell, the targeting entity can be PD1, CD40L, or GP120.

[0082] Apolipoprotein Apolipoproteins are proteins that bind to lipids such as triglycerides and cholesterol to form lipoproteins. Apolipoproteins transport lipids (and fat-soluble vitamins) in blood, cerebrospinal fluid, and lymph. The lipid components of lipoproteins are insoluble in water. However, due to their surfactant-like (amphiphilic) properties, apolipoproteins and other amphiphilic molecules (such as phospholipids) can surround the lipids and create water-soluble lipoprotein particles themselves, and thus can be transported through water-based circulation (i.e., blood, lymph). In addition to stabilizing the structure of lipoproteins and solubilizing lipid components, apolipoproteins interact with lipoprotein receptors and lipid transport proteins, thereby participating in the uptake and clearance of lipoproteins.

[0083] In lipid transport, apolipoproteins function as structural components of lipoprotein particles, ligands for cell surface receptors, and lipid transport proteins, and cofactors for enzymes. Different lipoprotein particles contain different classes of apolipoproteins that affect their functions. For example, apolipoprotein A1 (apoA1) is a major structural protein component of high-density lipoproteins (HDL), but it is present in smaller amounts in other lipoproteins, and HDL contains other apolipoproteins. The present invention is not assumed to be limited to specific types of apolipoproteins. Thus, in one embodiment, the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, or apolipoprotein mimetic derivative is selected from apoA1, apoA-1 Milano, apoA2, apoA4, apoA5, apoB, apoB48, apoB100, apoC-I, apoC-II, apoC-III, apoC-IV, apoD, apoE, apoF, apoH, apoL, apoL1, apoL2, apoL3, apoL4, apoL5, apoL6, apoLD1, apoO, apoOL, and apoM, or combinations thereof, or mimetics or derivatives thereof. Preferably, it is selected from apoA1, apoA2, apoA4, apoA5, apoB100, apoC-I, apoC-II, apoC-III, apoC-IV, apoD, apoE, apoF, apoH, apoL and apoM, or mimetics or derivatives thereof, More preferably, it is selected from apoA1, apoA2, apoA4, apoA5, apoB100, apoC-I, apoC-II, apoC-III, apoC-IV and apoE or mimetics or derivatives thereof, Even more preferably, it is selected from apoA1, apoA4, apoA5, apoB100, apoC-III and apoE or mimetics or derivatives thereof, Most preferably, it is selected from apoA1, apoB100 and apoE or mimetics or derivatives thereof.

[0084] In certain embodiments, the apolipoprotein component of the modified apolipoprotein is apoA1 or an apoA1 mutant.

[0085] In certain embodiments, the apolipoprotein component may also be an apolipoprotein fragment. Preferably, the apolipoprotein fragment retains the biological activity of the full-length apolipoprotein, such as the ability of the apolipoprotein to be incorporated into lipid nanoparticles or target to the bone marrow compartment. In certain embodiments, the apolipoprotein fragment comprises at least the ATP-binding cassette subfamily A member 1 (ABCA1), ATP-binding cassette subfamily G member 1 (ABCG1) and / or scavenger receptor class B type 1 (SR-BI) binding regions of the full-length apolipoprotein, thereby enabling binding to bone marrow cells. In certain embodiments, the apolipoprotein fragment comprises at least an alpha helix of the full-length apolipoprotein. These helices are hydrophilic on one side (interacting with the aqueous environment) and hydrophobic on the other side (interacting with the lipids in the particle).

[0086] In certain embodiments, the fragment may be an N- and / or C-terminal truncated form of the full-length peptide, polypeptide or protein from which it is derived.

[0087] In certain embodiments, the fragment may comprise at least about 30%, such as at least about 50% or at least about 70%, preferably at least about 80%, such as at least about 85%, more preferably at least about 90%, even more preferably at least about 95% or even about 99% of the amino acid sequence length of the full-length peptide, polypeptide or protein from which it is derived. For example, as long as it does not exceed the length of the full-length peptide, polypeptide, or protein, the fragment may comprise 5 or more contiguous amino acids, or 10 or more contiguous amino acids, or 20 or more contiguous amino acids, or 30 or more contiguous amino acids, such as 40 or more contiguous amino acids, such as 50 or more contiguous amino acids, such as 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, or 200 or more contiguous amino acids of the corresponding full-length peptide, polypeptide or protein.

[0088] In certain embodiments, an apolipoprotein component, such as an apolipoprotein fragment, comprises the bone marrow-binding portion of the full-length apolipoprotein.

[0089] In certain embodiments, the apolipoprotein component is an apolipoprotein mutant comprising a mutation that enables chemical conjugation of the apolipoprotein to a targeting entity, where the targeting entity is a molecule capable of binding to a molecule on the cell surface of a target cell. In certain embodiments, the apolipoprotein component may also be an apolipoprotein mutant comprising a substitution from serine to cysteine, such as a mutant defined by SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12 as described elsewhere herein.

[0090] Peptide sequences related to various proteins described in this specification, or nucleic acid sequences related to genes encoding various proteins described in this specification, are readily available to those skilled in the art from, for example, the UCSC Genome Browser (http: / / genome.ucsc.edu / ), the Ensembl Genome Browser (https: / / www.ensembl.org), and NCBI (https: / / www.ncbi.nlm.nih.gov / protein). Consensus sequences for various proteins or genes can be readily derived from these sources, but it is understood that certain variations may exist due to, but not limited to, genetic variations and multiple splice variants of the genes. Therefore, when referring to a specific protein, this should be interpreted as encompassing sequence variations due to genetic variations and splice variants. Therefore, as used herein, when referring to a specific protein, this refers to a corresponding consensus protein sequence as obtained from the Ensembl Genome Browser, or a consensus nucleic acid (gene) sequence as obtained from the Ensembl Genome Browser, or a protein sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding consensus protein sequence as obtained from the Ensembl Genome Browser, or a gene sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding consensus gene sequence as obtained from the Ensembl Genome Browser, or a nucleic acid sequence that encodes a protein that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding consensus protein sequence as obtained from the Ensembl Genome Browser.

[0091] An apolipoprotein mimetic is a synthetic peptide or protein that mimics the function or structure of an apolipoprotein. Several apolipoprotein mimetics are known. For example, Wolska et al. (Cells. 2021 Mar; 10(3): 597., which is incorporated by reference in its entirety) reviewed various apoA1, apoE, and apoC-II mimetics described in the literature. For example, most aapoA1 mimetic peptides are designed based on their ability to mostly excrete cholesterol from cells. Since this process has not been shown to depend on specific protein-protein interactions, most aapoA1 mimetic peptides are simply amphipathic helices and, in fact, many of them have no primary amino acid homology to apoA1. Exemplary aapoA1 mimetics are aapoA1 mimetic 18A, aapoA1 mimetic 2F, and aapoA1 mimetic 37pA.

[0092] For example, apoE has several putative anti-atherogenic functions and many different types of apoE-based peptides have been reported. One of the main goals in the design of these peptides is to promote the hepatic clearance of apoB-containing lipoproteins. Since apoE can bind to its receptor only when bound to lipids, these peptides usually have not only a receptor-binding motif derived from the N-terminal domain of apoE, but also a lipid-binding region based on the C-terminal domain of apoE or some other sequences.

[0093] For example, apoC-II mimetics are described based on either a shortened first helix (18A) linked to the LPL activation domain of apoC-II or a native apoC-II helix with amino acid substitutions in both the first and second helices to enhance the dual-helix binding to lipoproteins.

[0094] Accordingly, as used herein, an apolipoprotein mimetic refers to a synthetic protein or peptide that shares structural and / or functional features with each apolipoprotein. For example, the shared structural features may be the presence of a primary, secondary or tertiary peptide structure such as a peptide sequence, an alpha helix or a beta sheet or the three-dimensional structure of the peptide, or the functional features may be similarity in binding to a particular target such as a receptor. Preferably, the apolipoprotein is capable of binding to lipids in a manner similar to the corresponding apolipoprotein, and more preferably is capable of forming lipid particles.

[0095] In certain embodiments, the apolipoprotein mimetic may be capable of binding to bone marrow cells to the same or a similar extent as each apolipoprotein. For example, an apolipoprotein mimetic of apoA1 is preferably capable of binding to bone marrow cells to the same or a similar extent as apoA1.

[0096] In one embodiment, the modified apolipoprotein is a combination of apoA1 and a targeting moiety. In one embodiment, the modified apolipoprotein is a combination of an apoA1 mimetic and a targeting moiety. In one embodiment, the modified apolipoprotein is a combination of apoE and a targeting moiety.

[0097] In one embodiment, the modified apolipoprotein is a combination of an apolipoprotein component (e.g., an apoA1 mutant, preferably a human apoA1 mutant) and a targeting moiety. In one embodiment, the modified apolipoprotein is a combination of an apoA1 mimetic and a targeting moiety. In one embodiment, the modified apolipoprotein is a combination of apoE and a targeting moiety.

[0098] As an example, the human apoA1 protein sequence is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) accession number NP_001304947.1 (isoform 1 preproprotein), and Uniprot (www.uniprot.org) accession number P02647.1.

[0099] In certain embodiments, the apolipoprotein component is apoA1 (e.g., such that the first 18 amino acids thereof form a signal peptide, derived from the human precursor of apoA1 as defined by SEQ ID NO: 9) or an apolipoprotein component derived from apoA1 (e.g., as defined by SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12). In certain embodiments, the apolipoprotein component is human apoA1 as defined by SEQ ID NO: 13.

[0100] For example, a reactive handle may be used to chemically conjugate an apolipoprotein component to a targeting body. Thus, an apolipoprotein component derived from apoA1 containing cysteine instead of serine at position 147 (such as as defined by SEQ ID NO: 11) or position 279 (such as as defined by SEQ ID NO: 12) may be useful for preparing a modified apolipoprotein. In one embodiment, the apolipoprotein component has an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, or consists of, consists essentially of, or contains amino acids encoded by a nucleic acid having a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.

[0101] Note that sequences such as those defined by SEQ ID NO: 14, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12 contain at the N-terminus the amino acid sequence GLVPRGSIDD (SEQ ID NO: 16), which is a thrombin cleavage site. For example, a sequence such as that defined by SEQ ID NO: 15 contains a 6His tag, followed by the amino acid sequence GLVPRGSIDD (SEQ ID NO: 16) at the N-terminus. Here, the thrombin cleavage site can be used to remove the N-terminal His tag from the peptide.

[0102] In one embodiment, the apolipoprotein component comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 10, wherein SEQ ID NO: 10 includes a cysteine at position 7 of SEQ ID NO: 10.

[0103] In one embodiment, the apolipoprotein component comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 11, wherein SEQ ID NO: 10 includes a cysteine at position 150 of SEQ ID NO: 11.

[0104] In one embodiment, the apolipoprotein component comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 12, wherein SEQ ID NO: 10 includes a cysteine at position 239 of SEQ ID NO: 12.

[0105] As used herein, when referring to a modified apolipoprotein, the term fusion protein is to be construed as an apolipoprotein to which a targeting moiety is to be covalently attached. The covalent attachment can result from in-frame coding of a peptide or protein sequence by a nucleotide sequence encoding the fusion protein. Alternatively, the covalent attachment can result from a covalent bond between the targeting moiety and the apolipoprotein through a sulfur bond such as, for example, a thioether bond formed at a cysteine residue of the apolipoprotein. Further, the payload, if it is a peptide or protein, may optionally be a fusion protein with a modified apolipoprotein. The targeting moiety and / or the payload and / or the apolipoprotein (or a mimetic or derivative thereof) may include site-specific incorporation of unnatural amino acids such as para-azidophenylalanine, which is understood to be usable in subsequent (strain-promoted) "click" (conjugation) reactions with alkyne-modified reagents.

[0106] The modified apolipoprotein may include a linker, such as a flexible linker, between the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative and the targeting moiety. The linker may be a glycine-serine linker such as (GGS) n - linker (wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably a (GGS)4 (SEQ ID NO: 23)-linker.

[0107] In certain embodiments, the modified apolipoprotein may include one or more tags, for example, at the N-terminus and / or C-terminus of the modified apolipoprotein. One or more tags such as a 6His tag or a strep tag may enable purification of the modified apolipoprotein.

[0108] The targeting entity can be covalently attached to any part of an apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative. A linker, such as a flexible linker, can be used to enable such a covalent bond.

[0109] In certain embodiments, the targeting entity is located at the N- or C-terminus of the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative in the modified apolipoprotein.

[0110] As used herein, an immune response refers to a reaction that occurs within an organism by the immune system. The immune response can be a innate immune response or an adaptive immune response or the complement immune system. The immune responses as referred to herein include, but are not limited to, secretion of pro-inflammatory molecules; secretion of anti-inflammatory molecules; phagocytosis; production, presentation or secretion of antibodies; antigen presentation; activation, proliferation, suppression or differentiation of immune cells; binding of immune cells to a target, or initiation of an immune-related cell signaling cascade.

[0111] Payload In one embodiment, the modified apolipoprotein further comprises a payload. The payload can be attached to the modified apolipoprotein, for example, by a covalent bond. As used herein, a payload refers to a molecule to be delivered to a target site. Non-limiting examples are nucleic acids or nucleic acid analogs, therapeutic agents, biologics, cytokines, chemokines, hormones, growth factors, or combinations thereof, but one of ordinary skill in the art may recognize additional types of payloads. The payload is an immunomodulatory molecule and can interfere with, alter, stimulate or suppress the innate immune response or the adaptive immune response or the complement immune system. The payload can be a protein, a peptide or an organic compound. The payload can be isolated from a natural source or obtained therefrom, or can be cloned or synthesized.

[0112] Thus, in one embodiment, the payload is selected from nucleic acids or nucleic acid analogs, therapeutic agents, biologics, cytokines, chemokines, hormones, growth factors, or combinations thereof.

[0113] In one embodiment, the payload may be a cytokine. Cytokines are known to those skilled in the art to be small proteins on the order of about 5-20 kDa and are important in cell signaling. For example, cytokines can refer to four-alpha-helix bundle family cytokines such as the IL-2 subfamily, interferon (IFN) subfamily or IL-10 subfamily; the IL-1 family; cysteine knot cytokines such as the TGF beta family; the IL-17 family. Thus, cytokines are preferably selected from IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3 / IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33, BAFF, 4-1BBL, TNFSF8, CD40LG, CD70, CD95L / CD178, EDA-A1, TNFSF14, LTA / TNFB, LTB, TNF alpha, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF15, TNFSF4, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1, IFNE, IFNG, IFNZ, IFNA8, IFNA5 / IFNaG, IFNω / IFNW1, CLCF1, CNTF, IL11, IL31, IL6, leptin, LIF, OSM, IL10, IL19, IL20, IL22, IL24, IL28B, IL28A, IL29, TGF-beta1 / TGFB1, TGF-beta2 / TGFB2, TGF-beta3 / TGFB3. In a preferred embodiment, the cytokine is selected from the IL-2 subfamily, interferon subfamily, IL-10 subfamily, IL-1 family, TGF beta family, or IL-17 family, or combinations thereof, and more preferably, where the cytokine is selected from IL-1β, IL-2, IL-4, IL-38, or combinations thereof.

[0114] In one embodiment, the modified apolipoprotein is a fusion protein of the modified apolipoprotein and IL-1B. IL-1B is also known as IL1B, IL-1β, IL1F2 or interleukin 1 beta, and is a cytokine protein encoded by the IL1B gene in humans. In one embodiment, the modified apolipoprotein is a fusion protein of the modified apolipoprotein and IL-2. IL-2 is also known as IL2 TCGF, lymphokine or interleukin 2, and is an interleukin that regulates the activity of white blood cells involved in immunity. In one embodiment, the modified apolipoprotein is a fusion protein of the modified apolipoprotein and IL-4. IL-4 is also known as BSF-1, IL4 or interleukin 4, and is a cytokine that induces the differentiation of naive helper T cells. In one embodiment, the modified apolipoprotein is a fusion protein of the modified apolipoprotein and IL-38. IL-38 is also known as IL38, IL1F10, interleukin 38, interleukin 1 family member 10 or IL1-theta, and is a protein encoded by the IL1F10 gene in humans.

[0115] In one embodiment, the payload may be a chemokine. The chemokine is preferably selected from CCL1 / TCA3, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2 / MCP-1, CCL20, CCL21, CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 / LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 / Ppbp, CXCL9, IL8 / CXCL8, XCL1, XCL2, FAM19A1, FAM19A2, FAM19A3, FAM19A4 and FAM19A5. In an alternative embodiment, the chemokine is selected from CC chemokines, CXC chemokines, C chemokines, CX3C chemokines or combinations thereof.

[0116] In one embodiment, the payload is a hormone. Hormones are known to those skilled in the art as signaling molecules in multicellular organisms that are transported to distal organs to regulate physiological functions and behavior. In one embodiment, the hormone is selected from adrenaline (also known as epinephrine), melatonin, noradrenaline (also known as norepinephrine), triiodothyronine, thyroxine, dopamine, prostaglandins, leukotrienes, prostacyclin, thromboxane, amylin (also known as pancreatic amyloid polypeptide), anti-Müllerian hormone (also known as Müllerian-inhibiting factor / hormone), adiponectin, adrenocorticotropic hormone (also known as corticotropin), angiotensinogen, angiotensin, antidiuretic hormone (also known as vasopressin, arginine vasopressin), atrial natriuretic peptide (also known as atriopeptin), brain natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, cortistatin, enkephalin, endothelin, erythropoietin, follicle-stimulating hormone, galanin, gastric inhibitory polypeptide, gastrin, ghrelin, glucagon, glucagon-like peptide-1, gonadotropin-releasing hormone, growth hormone-releasing hormone, hepcidin, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, insulin-like growth factor (also known as somatomedin), leptin, lipotropin, luteinizing hormone, melanocyte-stimulating hormone, motilin, orexin, osteocalcin, oxytocin (also known as pitocin), pancreatic polypeptide, parathyroid hormone, pituitary adenylate cyclase-activating peptide, prolactin (also known as luteotropic hormone), prolactin-releasing hormone, relaxin, renin, secretin, somatostatin (also known as growth hormone-inhibiting hormone or growth hormone-release-inhibiting hormone or somatotropin-release-inhibiting factor or somatotropin-release-inhibiting hormone), thrombopoietin, thyroid-stimulating hormone (also known as thyrotropin), thyrotropin-releasing hormone, vasoactive intestinal peptide, guanylin or uroguanylin.

[0117] In one embodiment, the payload is a growth factor. Growth factors are known to those skilled in the art as naturally occurring substances capable of stimulating cell growth, wound healing, and in some cases cell differentiation. In one embodiment, the growth factor is adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor, bone morphogenetic protein (BMP), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3, erythropoietin (EPO), fibroblast growth factor (FGF), fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 3 (FGF3), fibroblast growth factor 4 (FGF4), fibroblast growth factor 5 (FGF5), fibroblast growth factor 6 (FGF6), fibroblast growth factor 7 (FGF7), fibroblast growth factor 8 (FGF8), fibroblast growth factor 9 (FGF9), fibroblast growth factor 10 (FGF10), fibroblast growth factor 11 (FGF11), fibroblast growth factor 12 (FGF12), fibroblast growth factor 13 (FGF13), fibroblast growth factor 14 (FGF14), fibroblast growth factor 15 (FGF15), fibroblast growth factor 16 (FGF16), fibroblast growth factor 17 (FGF17), fibroblast growth factor 18 (FGF18), fibroblast growth factor 19 (FGF19), fibroblast growth factor 20 (FGF20), fibroblast growth factor 21 (FGF21), fibroblast growth factor 22 (FGF22), fibroblast growth factor 23 (FGF23), fetal bovine somatotropin (FBS), glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, artemin, growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, keratinocyte growth factor (KGF), migration-stimulating factor (MSF),Macrophage-stimulating protein (MSP), also known as hepatocyte growth factor-like protein (HGFLP), myostatin (GDF-8), neuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3), neuregulin 4 (NRG4), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalarase (RNLS) - anti-apoptotic survival factor, T cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor, transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11 and WNT16. In a preferred embodiment, the growth factor is selected from VEGF, EGF, CNTF, LIF, Ephrin, FGF, GDNF, HDF, HDGF, IGF, KGF, MSF, NRG, BDNF, NGF, neurotrophin, PGF, PDGF, RNLS, TCGF, TGF, TNF, WNT or combinations thereof.,

[0118] In one embodiment, the payload is a hematopoietic growth factor. In one embodiment, the hematopoietic growth factor is selected from IL-3, CSF-1 (M-CSF), GM-CSF, G-CSF, members of the IL-12 family of interleukins or erythropoietin or combinations thereof.

[0119] Lipid nanoparticles The modified apolipoproteins as described herein are envisioned to be used as proteins or as lipid nanoparticles. As described above, apolipoproteins can circulate on their own (i.e., not incorporated into lipoproteins or lipid nanoparticles). This use may be suitable for delivering a payload, such as a cytokine, to a target site. Apolipoproteins can circulate as proteins, but are also known to be able to form lipoproteins in situ. However, it may also be suitable to include the modified apolipoprotein in lipid nanoparticles. Thus, in one aspect, the invention relates to lipid nanoparticles comprising one or more modified apolipoproteins as described herein. Without being bound by any hypothesis, it is believed that apolipoproteins, apolipoprotein derivatives, apolipoprotein mimics or apolipoprotein mimic derivatives can function as scaffolds that assist in the formation of nanoparticles together with phospholipids and sterols.

[0120] Apolipoproteins, apolipoprotein derivatives, apolipoprotein mimics or apolipoprotein mimic derivatives form part of the lipid nanoparticle structure. In certain embodiments, at least a portion of the modified apolipoprotein is exposed to the environment surrounding the lipid nanoparticle (i.e., the aqueous environment). Typically, a portion of the apolipoprotein, apolipoprotein derivative, apolipoprotein mimic or apolipoprotein mimic derivative is exposed to the environment surrounding the lipid nanoparticle (see, for example, Figure 2). Further, fusion of a targeting entity to an apolipoprotein, apolipoprotein derivative, apolipoprotein mimic or apolipoprotein mimic derivative typically allows the targeting entity to be fully exposed to the environment surrounding the apolipoprotein lipid nanoparticle (see, for example, Figure 2). In other words, in certain embodiments, the targeting entity is not embedded within the lipid nanoparticle. As a result, the targeting entity can move freely and exert its natural function(s), such as its cell targeting function.

[0121] In certain embodiments, the lipid nanoparticles are not phospholipid bilayers.

[0122] In certain embodiments, the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine, and phosphatidylglycerol or combinations thereof. In further certain embodiments, the phospholipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (PHPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dilauroylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dilauroylphosphatidylserine (DLPS), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or combinations thereof.

[0123] In certain embodiments, the sterol is selected from cholesterol, desmosterol, stigmasterol, β-sitosterol, ergosterol, hopanoids, hydroxysteroids, phytosterols, steroids, hydrogenated cholesterol, campesterol, zymosterol, or combinations thereof.

[0124] The modified apolipoprotein may further be conjugated to a payload. The apolipoprotein may also be an apolipoprotein mimetic or derivative. Generally, two types of nanoparticles can be distinguished: discoidal nanoparticles and spherical nanoparticles. Both contain an outer layer of phospholipids, a sterol, and a modified apolipoprotein as described herein. The two differ in that discoidal nanoparticles lack a core, resulting in the formation of a discoidal shape, whereas spherical nanoparticles further contain a substantially hydrophobic core. The core may usually contain lipids, but not necessarily. Preferably, the lipid nanoparticles are spherical. Spherical nanoparticles allow for the conjugation of (more) more fusion proteins, depending on size, for example, than discoidal nanoparticles. Furthermore, spherical nanoparticles facilitate the encapsulation of larger payloads, such as mRNA or gene editing components, or a large amount of payload (e.g., in the case of small molecule drugs or small lipophilic payloads), compared to discoidal nanoparticles.

[0125] In certain embodiments, the lipid nanoparticles contain a payload, the lipid nanoparticles contain a core surrounded by a surface layer, where the core contains the payload and the surface layer contains an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic, or an apolipoprotein mimetic derivative, a phospholipid, a targeting entity, and a sterol.

[0126] In one embodiment, the invention relates to lipid nanoparticles comprising an outer layer and optionally a core, wherein the outer layer comprises - a phospholipid, - a sterol, and - a modified apolipoprotein according to any one of the preceding claims and wherein the core, when present, comprises at least one core component selected from lipids, cationic lipids, or multivalent molecules. When referring to the outer layer of the lipid nanoparticles, the outer layer is essentially a phospholipid monolayer further containing additional components, at least cholesterol and the modified apolipoprotein.

[0127] As used herein, lipid nanoparticles refer to an assembly of phospholipids and sterols, and one or more apolipoproteins soluble in an aqueous solution. The particles may contain lipids, in which case the lipids are encapsulated by phospholipids and sterols. The nanoparticles may further contain additional components such as additional proteins or payloads. Thus, in one embodiment, the lipid nanoparticles as defined herein further contain lipids.

[0128] In certain embodiments, the lipid nanoparticles include native (e.g., unfused) apolipoproteins, apolipoprotein derivatives, apolipoprotein mimics or apolipoprotein mimic derivatives in addition to apolipoproteins, apolipoprotein derivatives, apolipoprotein mimics or apolipoprotein mimic derivatives that form part of a modified apolipoprotein as described herein. In further embodiments, the lipid nanoparticles as defined herein further contain a payload as defined above herein. The payload may be, for example, a pharmaceutical compound. The lipid nanoparticle core is particularly suitable for lipophilic payloads but can also be used for amphiphilic molecules. The pharmaceutical compound may be an organic compound, a peptide, a protein, a nucleic acid or nucleic acid analog, a biologic or a lipid. Thus, in one embodiment, the lipid nanoparticles further contain a payload, preferably where the payload is selected from a nucleic acid or nucleic acid analog, a therapeutic agent, a biologic or a combination thereof. Alternatively, the payload may be bound to the outer layer of the lipid nanoparticles, for example, to a (modified) apolipoprotein, or the payload may be included in the phospholipid layer.

[0129] For example, the payload may be a nucleic acid or a nucleic acid analog. Examples can be, but are not limited to, mRNA, siRNA, sgRNA, miRNA, piRNA, snRNA, snoRNA, srRNA or tsRNA. Nucleic acid analogs can be peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA), threose nucleic acids (TNA) and hexitol nucleic acids (HNA), or mixtures or combinations thereof. When the nucleic acid payload is contained in the lipophilic core of the nanoparticle, it is understood that the charged backbone of the nucleic acid needs to be neutralized, for example, by a cationic lipid.

[0130] In certain embodiments, the lipid nanoparticle comprises a nucleic acid and a cationic or ionizable cationic lipid.

[0131] In certain embodiments, the nucleic acid and the cationic or ionizable cationic lipid are contained by the core, and the modified apolipoprotein and phospholipid are contained by the outer layer.

[0132] Accordingly, the present invention also encompasses lipid nanoparticles comprising an outer layer and a core, wherein the outer layer comprises - a phospholipid, - a sterol, and - a modified apolipoprotein as taught herein and the core comprises a nucleic acid and a cationic or ionizable cationic lipid.

[0133] This type of nucleic acid-containing apolipoprotein particles is described in WO 2022 / 268913 pamphlet, the entire content of which is incorporated herein by reference. The nanoparticles are designed to complex nucleic acids, which are inherently hydrophilic, using helper molecules to draw the nucleic acids into the hydrophobic nanoparticle core. For this purpose, cationic hydrophobic molecules are used. The cationic groups can complex with the anionic phosphate groups in the sugar-phosphate backbone via ionic interactions. The hydrophobic portion of the helper molecule forms a shell around the hydrophilic nucleic acid molecule. The cationic helper molecule can be permanently charged or ionizable. Cationic helper molecules include a wide variety of commercially available or in-house synthesized molecules, but the cationic helper molecule needs to follow two general criteria: 1) a positively charged group to enable complex formation with the negatively charged sugar-phosphate backbone. 2) a hydrophobic portion to form a hydrophobic shell and enable incorporation into the nanoparticle core. The content of the cationic material in the nanoparticle formulation can range from a cationic to anionic ratio of 1:1 to 25:1. This ratio, often referred to as the N / P (nitrogen / phosphate) ratio, is based on the number of positive charges (often nitrogen-based) in the (ionizable) cationic lipid versus the number of negative charges (usually phosphate) in the nucleic acid payload. Thus, the N / P ratio is the ratio between the cumulative molar amount (N) of cationic and / or ionizable groups in the cationic or ionizable lipid component(s) and the cumulative molar amount (P) of phosphate groups in the nucleic acid component(s). In certain embodiments, the N / P ratio of the nanoparticles as taught herein is 1 to 25, 1 to 20, 1 to 15, 1 to 12, 1 to 9, 1 to 6, or 1 to 3. For example, the N / P ratio of the nanoparticles as taught herein can be 3, 6, 9, or 12.

[0134] Although not bound by theory, the inventors believe that the nucleic acid-containing nanoparticles described herein have an outer layer comprising mainly modified apolipoproteins, phospholipids and optional sterols, and a core comprising a cationic or ionizable cationic lipid and a nucleic acid. More specifically, the core of the nanoparticle comprises an aggregate of nucleic acids that interact with a (ionizable) cationic lipid, where this core of the nanoparticle is surrounded by a lipid shell comprising, consisting essentially of, or consisting of modified apolipoproteins, phospholipids and optional sterols. According to this understanding, the nucleic acid is located within (i.e., inside) the nanoparticle and is not located on the outer surface of the nanoparticle and / or is not exposed around the nanoparticle. In certain embodiments, the payload (i.e., nucleic acid) of the nanoparticles of the invention is not bound by ionic interactions at the outer (surface) of the particle. Binding of the nucleic acid to the outer surface of the particle is undesirable as it leaves the nucleic acid highly exposed to the surroundings, perhaps making the particle more toxic, as well as leading to rapid (in vivo) degradation of the nucleic acid payload. According to this understanding, the modified apolipoprotein is located on the outer surface of the nanoparticle and / or is exposed around the nanoparticle.

[0135] The core of the nanoparticle may be solid and may not have or possess a significant aqueous void or reservoir within the core. In certain embodiments, the core of the nanoparticle is non-aqueous.

[0136] In certain specific embodiments, the core of the nanoparticle is not surrounded by a lipid bilayer such as that present in a vesicle-like or liposome particle having a lipid bilayer surrounding, for example, an aqueous core.

[0137] In certain embodiments, the nanoparticles do not contain a synthetic (non-natural) hydrophilic polymer or a (lipid) conjugate of such a polymer, such as most notably polyethylene-glycol (PEG). As a result, such nanoparticles do not induce an undesirable immune response, particularly upon repeated administration.

[0138] In certain embodiments, the nanoparticle core further comprises a filler, preferably a filler selected from triacylglycerols and cholesterol acyl esters, or combinations thereof, for example, where the triacylglycerol is tricaprylin and / or the cholesterol acyl ester is cholesteryl caprylate and / or cholesteryl oleate. Cholesteryl acetate may also be used as a filler material. Still other filler materials that may be applied are diglycerides or triglycerides or other esters derived from C1-C18 carboxylic acids, preferably C6-C18 fatty acids, where these carboxylic acids and fatty acids may be saturated or unsaturated. Preferably, the filler is a triglyceride derived from C6-C18 fatty acids. In addition to the nucleic acid and the cationic helper molecule, additional hydrophobic filler molecules may be included in the core of the nanoparticle formulation. Its main use is to alter the physicochemical properties of the nanoparticles and / or improve stability.

[0139] In certain embodiments, the nucleic acid is RNA, DNA or a nucleic acid analog.

[0140] In certain embodiments, the RNA is microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), transfer RNA (tRNA), tRNA-derived small RNA (tsRNA), small regulatory RNA (srRNA), messenger RNA (mRNA), modified mRNA, ribosomal RNA (rRNA), long non-coding RNA (lncRNA), or guide RNA (gRNA), or combinations and / or modifications thereof.

[0141] In certain embodiments, the DNA is single-stranded or double-stranded DNA.

[0142] In certain embodiments, the nucleic acid is an antisense oligonucleotide, and the antisense oligonucleotide is a single-stranded DNA or RNA consisting of nucleotides or nucleoside analogs containing a modified phosphodiester backbone or 2'-ribose.

[0143] In certain embodiments, the nucleotide or nucleoside analog is selected from locked nucleic acid (LNA), bridged nucleic acid (BNA), morpholino or peptide nucleic acid (PNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), hexitol nucleic acid (HNA), or mixtures or combinations thereof.

[0144] In certain embodiments, the nucleic acid is conjugated and the nucleic acid conjugate is incorporated into the nanoparticles of the present invention. The nucleic acid conjugate includes, for example, a lipid conjugate with a phospholipid, or a sterol such as cholesterol, or a hydrophobic alkyl chain. The nucleic acid conjugate also includes a conjugate with an oligomer or polymer. Preferably, these oligomers or polymers have hydrophobic properties.

[0145] In certain embodiments, the nucleic acid is incorporated into the nanoparticle by itself or "as is", i.e., the nucleic acid is not conjugated.

[0146] As used herein, the term ionizable cationic lipid has a neutral charge at physiological pH (e.g., at pH 7-7.5, preferably at pH 7.3-7.5, such as at pH 7.4), and is protonated or positively charged at lower pH (e.g., at pH 1-5, preferably at pH 1-4, such as at pH 4). Ionizable cationic lipids are understood to be particularly useful because they can be protonated at low pH and thus promote binding to hydrophilic nucleic acids. Subsequently, by raising the pH, the lipid becomes (partially) neutral and further promotes its inclusion in a hydrophobic environment, such as the hydrophobic core of a nanoparticle. Alternatively, without being bound by theory, ionizable lipids may remain positively charged within the nanoparticle due to the action of the surface layer of the nanoparticle containing phospholipids, optionally sterols, and modified apolipoproteins, and / or due to the non-aqueous environment within the nanoparticle, even when the pH of the surrounding aqueous solution is raised to physiological pH, such as about 7.4. Furthermore, ionizable cationic lipids are theorized to promote endosomal escape of nucleic acids in target cells, where the ionizable cationic lipid is protonated due to the low pH.

[0147] Non-limiting examples of ionizable cationic lipids are DLin-DMA (2-[2,2-bis(octadeca-9,12-dienyl)-1,3-dioxolan-4-yl]-N,N-dimethylethanamine), DLin-KC2-DMA (2-[2,2-bis[(9Z,12Z)-octadeca-9,12-dienyl]-1,3-dioxolan-4-yl]-N,N-dimethylethanamine) and the following Formula 1:

[0148]

Chemical formula

[0149] Indeed, various series of ionizable cationic lipids have been developed and reported in the literature, so a wide range of ionizable cationic lipids (including lipidoids) can be used to prepare the nanoparticles of the present invention. Further non-limiting examples include the molecules cKK-E12, C12-200, L319, Acuitas-A9, Moderna-L5, TT3, and ssPalmE (e.g., as described in Witzigmann et al., Advanced Drug Delivery Reviews 159 (2020) 344-363; doi.org / 10.1016 / j.addr.2020.06.026).

[0150] The ionizable lipid may further be an ionizable triglyceride. A non-limiting example is of formula 2:

[0151]

Chemical formula

[0152] The ionizable lipid may further be a cholesterol ester (also called cholesteryl ester). A non-limiting example is of formula 3:

[0153]

Chemical formula

[0154] As used herein, the term "cationic lipid" refers to a lipid that is positively charged at physiological pH (e.g., pH 7.4). Non-limiting examples of cationic lipids are DOTMA (1,2-di-O-octadecyl-3-trimethylammonium propane), DOGS (2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecyl)amino]-2-oxoethyl]pentanamide), DOSPA (2-[3-[4-(3-aminopropylamino)butylamino]propylcarbamoylamino]ethyl-[2,3-bis[[(Z)-octadec-9-enoyl]oxy]propyl]-dimethylazanium), and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane). As another example, any ionizable cationic lipid molecule can be mentioned, where the tertiary amine moiety is converted to a quaternary ammonium moiety by alkylation such as methylation (-Me), ethylization (-Et), benzylation (-Bn), or ethoxylation (-CH2CH2-OH). The resulting quaternary ammonium molecule has a permanent positive (cationic) charge and thus also carries a counteranion, such as a chloride anion.

[0155] In one embodiment, only ionizable cationic lipids are used to prepare nucleic acid-containing nanoparticles as taught herein. Thus, in one embodiment, the nanoparticles as taught herein do not contain cationic lipids.

[0156] In one embodiment, only cationic lipids are used to prepare nucleic acid-containing nanoparticles as taught herein. Thus, in one embodiment, the nanoparticles as taught herein do not contain ionizable cationic lipids.

[0157] In one embodiment, a combination of ionizable cationic lipids and cationic lipids is used to prepare nucleic acid-containing nanoparticles as taught herein.

[0158] When referring to nucleic acid-containing nanoparticles as taught herein, the term "payload" specifically refers to a nucleic acid, preferably in combination with a cationic and / or ionizable cationic lipid.

[0159] The term "lipid" is well known in the art and, as used herein, is considered to include both lipids, i.e., naturally occurring hydrophobic biomolecules such as, for example, fatty acids, monoglycerides, diglycerides or triglycerides, sterols (derivatives) or phospholipids, and lipid-like biomolecules. The cationic lipids or ionizable cationic lipids (or lipidoids) described herein are usually not lipids within the narrowest interpretation of the term, i.e., naturally occurring hydrophobic biomolecules such as, for example, fatty acids, monoglycerides, diglycerides or triglycerides, sterols (derivatives) or phospholipids, but rather lipid-like biomolecules similar to lipid biomolecules, i.e., they preferably contain biocompatible groups (such as esters or amides, etc.) and / or are constructed using naturally occurring components (such as fatty acids, glycerol, cholesterol). It should be noted that.

[0160] In certain embodiments, the cationic or ionizable cationic lipid is selected from ionizable cationic esters of long-chain alcohols, ionizable cationic esters of diglycerides or ionizable cationic esters of sterols or combinations thereof.

[0161] The ionizable cationic ester of a long-chain alcohol is a tertiary amine having a carboxy group, for example, of the formula (CH3)2N(CH2) nA compound having COOH (wherein n is an integer of 1 or greater, for example, n is from 1 to 12), for example, an ester of 3-dimethylamino-propionic acid or 4-dimethylamino-butyric acid or 5-dimethylamino-pentanoic acid. The ester is formed using a long-chain alcohol. The long-chain alcohol is preferably a primary or secondary alcohol having 8 or more carbon atoms, for example, a straight-chain or branched-chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

[0162] The ionizable cationic ester of diglyceride is preferably a tertiary amine having a carboxy group, for example, the formula (CH3)2N(CH2) n A compound having COOH (wherein n is an integer of 1 or greater, for example, n is from 1 to 12), for example, a diacylglycerol (i.e., diglyceride) coupled at the 1- or 2-position with 3-dimethylamino-propionic acid or 4-dimethylamino-butyric acid or 5-dimethylamino-pentanoic acid. The diacylglycerol may include medium-chain or long-chain saturated or unsaturated fatty acids or their derivatives or modified forms.

[0163] The ionizable cationic ester of sterol is preferably a tertiary amine having a carboxy group, for example, the formula (CH3)2N(CH2) n An ester of sterol coupled with a hydroxyl group to a compound having COOH (wherein n is an integer of 1 or greater, for example, n is from 1 to 12), for example, 3-dimethylamino-propionic acid or 4-dimethylamino-butyric acid or 5-dimethylamino-pentanoic acid. The sterol can be cholesterol, stigmasterol or β-sitosterol.

[0164] In the above, the carboxy compound is represented by the formula (CH3)2N(CH2) n COOH (wherein n is an integer of 1 or greater). Instead of this compound, the formula NH2-(C=NH)-NH-(CH2)n Alternative compounds having COOH (where n is an integer of 1 or greater, for example, n is from 1 to 12) can be used. This carboxy compound contains a guanidine group instead of a tertiary amine group.

[0165] Ionizable cationic lipids can be selected, for example, from molecules according to formulas (I) - (V).

[0166]

Chemical formula

[0167] Formula (I) represents a triglyceride, where the ionizable cationic group (ICG) is included at the 1-position.

[0168] Formula (II) represents the same type of triglyceride as represented by formula (I), provided that the molecule is in the naturally occurring configuration, i.e., stereospecifically defined as in the case where it is found in phospholipids: the ICG group is present at the same position as the phosphate group is present in the phospholipid.

[0169] Formula (III) represents a triglyceride, where the ionizable cationic group (ICG) is included at the 2-position.

[0170] Formula (IV) represents a diester (or triester), where the ionizable cationic group (ICG) is bonded via an amide functionality.

[0171] Formula (V) represents a cholesteryl ester, where the ionizable cationic group (ICG) is bonded via an ester functionality.

[0172] The ionizable cationic group (ICG) is bonded via a wavy bond to the remainder of the molecule with respect to any of formulas (I) - (V), where ICG can represent a tertiary amine (ICG type A, or ICG - A), or ICG can represent guanidine (ICG type B, or ICG - B).

[0173] In formulas (I) to (IV), R1 can be independently selected for any position, and R1 represents a linear or branched C1-C19 alkyl, linear or branched C1-C19 alkenyl, aryl, arylene-alkyl or alkylene-aryl group, wherein the alkyl or alkenyl group optionally contains 5 heteroatoms independently selected from O and N. Preferably, any R1 group within a specific molecule according to any of formulas (I) to (IV) is the same R1 group. Preferably, the R1 group is a linear or branched C5-C19 alkyl group, or a linear or branched C5-C19 alkenyl group. When R1 is an alkenyl group, this group preferably has only a single double bond. More preferably, the R1 group is a linear or branched C9-C17 alkyl group or a linear or branched C5-C17 alkenyl group. Preferably, R1 is a linear C5-C15 alkyl group or a linear C17-C19 alkenyl. The carboxylic acid derived from R1, i.e., R1-COOH, is preferably a naturally occurring fatty acid molecule such as capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid or linoleic acid. C10-C16 saturated fatty acids and oleic acid (C18, unsaturated) are preferred.

[0174] The integer p is a discrete number and not an average value. p can be from 0 to 11. Preferably, p is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9. More preferably, p is 1, 2, 3 or 4.

[0175] The R2 group in formula (IV) is hydrogen, methyl, ethyl and -CH2-O-C(O)-R 1a group (wherein R 1a has the same meaning as R1 defined above) and can be selected from. Preferably, R2 is hydrogen, methyl or -CH2-O-C(O)-R1 group. More preferably, R2 is methyl.

[0176] The R3 group in formula (IV) can be selected from hydrogen, aryl, arylene-alkyl, alkylene-aryl or a linear C1-C6 alkyl group. Preferably, R3 is hydrogen or methyl. More preferably, R3 is hydrogen.

[0177] The R in ICG-A x groups can be independently selected for any position and are selected from methyl, ethyl, propyl and ethylene-hydroxy (-CH2-CH2-OH) groups. Preferably, the R in ICG-A x group is a methyl group. Preferably, the R in ICG-A x groups are both the same group, and they are preferably methyl groups.

[0178] The R in ICG-B y groups can be independently selected from hydrogen, linear or branched C1-C18 alkyl, aryl, arylene-alkyl or alkylene-aryl groups for any position, where the alkyl group optionally contains up to 5 heteroatoms independently selected from O and N. Preferably, the R y groups are selected from hydrogen and linear C1-C6 alkyl groups. More preferably, the R y group is hydrogen. Preferably, the four R y groups in ICG-B are all the same group, and they are preferably hydrogen.

[0179] From formulas (I)-(V), formulas (I), (II) and (IV) are preferred. Formulas (I) and (II) are more preferred.

[0180] From ICG-A and ICG-B of ICG, ICG-A is preferred, that is, an ionizable cationic lipid of a tertiary amine is preferred.

[0181] Ionizable cationic lipid molecules such as those according to any one of formulas (I) to (V) have a molecular weight greater than 250 Daltons, preferably greater than 350 Daltons, more preferably greater than 450 Daltons. The ionizable cationic lipid molecules have a molecular weight of less than 3000 Daltons, preferably less than 1800 Daltons, more preferably less than 1200 Daltons.

[0182] The molecules represented by formulas (I) to (V) may exist in various isomeric forms such as rotamers, tautomers, stereoisomers or regiomers, and all of these are included within the scope of the present invention.

[0183] The ionizable cationic lipid according to any one of formulas (I) to (V) is preferably a single compound, i.e., not a mixture of compounds. Therefore, the purity of the ionizable cationic lipid of formulas (I) to (V) is preferably 50% or higher, preferably 80% or higher, more preferably 90% or higher, and most preferably 95% or higher. When the ionizable cationic lipid is a mixture of compounds, this is preferably due only to the presence of undefined stereocenters in the molecule. One example is the use of a branched alkyl chain in an ionizable cationic lipid having a racemic origin. Another example is a triglyceride in which the substitution pattern across the three hydroxy groups in the glycerol entity is not stereospecifically defined.

[0184] Therefore, in certain embodiments, the ionizable cationic lipid is of formula (I), (II), (III), (IV) or (V)

[0185]

Chemical formula

[0186] (wherein ICG is

[0187]

Chemical formula

[0188] The term "alkyl", by itself or as part of another substituent, has the formula C n H 2n+1Refers to a hydrocarbyl group (wherein n is a number greater than or equal to 1). The alkyl group may be linear or branched and may be substituted as shown herein. Generally, the alkyl groups of the present invention contain from 1 to 18 carbon atoms, preferably from 1 to 17 carbon atoms, preferably from 1 to 15 carbon atoms, preferably from 1 to 6 carbon atoms, preferably from 1 to 5 carbon atoms, preferably from 1 to 4 carbon atoms, more preferably from 1 to 3 carbon atoms, and even more preferably from 1 to 2 carbon atoms. When a subscript is used following a carbon atom herein, the subscript refers to the number of carbon atoms that the named group may contain. For example, the term "C 1~6 alkyl" refers to a hydrocarbyl group of the formula -C n H 2n+1 (wherein n is a number in the range of 1 to 6) as a group or part of a group. Thus, for example, "C 1~6 alkyl" encompasses all linear or branched alkyl groups having from 1 to 6 carbon atoms, and thus includes methyl, ethyl, n-propyl, i-propyl, butyl and its isomers (e.g., n-butyl, i-butyl and t-butyl), pentyl and its isomers, hexyl and its isomers. For example, "C 1~5 alkyl" encompasses all linear or branched alkyl groups having from 1 to 5 carbon atoms, and thus includes methyl, ethyl, n-propyl, i-propyl, butyl and its isomers (e.g., n-butyl, i-butyl and t-butyl), pentyl and its isomers. For example, "C 1~4 alkyl" encompasses all linear or branched alkyl groups having from 1 to 4 carbon atoms, and thus includes methyl, ethyl, n-propyl, i-propyl, butyl and its isomers (e.g., n-butyl, i-butyl and t-butyl). For example, "C 1~3 alkyl" encompasses all linear or branched alkyl groups having from 1 to 3 carbon atoms, and thus includes methyl, ethyl, n-propyl, i-propyl.

[0189] When the suffix "ene" is used in combination with an alkyl group, i.e., in the case of "alkylene", this means that the alkyl group as defined herein has two single bonds as attachment points to other groups. As used herein, "C 1~6 alkylene" refers to a divalent, i.e., having two single bonds for attachment to two other groups, C 1~6 alkyl group, either by itself or as part of another substituent. The alkylene group may be straight-chain or branched and may be substituted as shown herein. Non-limiting examples of alkylene groups include methylene (-CH2-), ethylene (-CH2-CH2-), methylmethylene (-CH(CH3)-), 1-methyl-ethylene (-CH(CH3)-CH2-), n-propylene (-CH2-CH2-CH2-), 2-methylpropylene (-CH2-CH(CH3)-CH2-), 3-methylpropylene (-CH2-CH2-CH(CH3)-), n-butylene (-CH2-CH2-CH2-CH2-), 2-methylbutylene (-CH2-CH(CH3)-CH2-CH2-), 4-methylbutylene (-CH2-CH2-CH2-CH(CH3)-), pentylene and its chain isomers, hexylene and its chain isomers.

[0190] The term "alkenyl" refers to an unsaturated hydrocarbyl group that can be straight-chain or branched and contains one or more carbon-carbon double bonds, either as a group or as part of a group. When a subscript is used following a carbon atom herein, the subscript refers to the number of carbon atoms that the named group may contain. For example, the term "C 2~6 alkenyl" refers to an unsaturated hydrocarbyl group that can be straight-chain or branched, contains one or more carbon-carbon double bonds, and contains 2 to 6 carbon atoms. For example, C 2~4 alkenyl includes all straight-chain or branched alkenyl groups having 2 to 4 carbon atoms. C 2~6 Examples of alkenyl groups are ethenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl and its isomers, 2-hexenyl and its isomers, 2,4-pentadienyl, etc.

[0191] The term "aryl", as a group or part of a group, has a single ring (i.e., phenyl) or fused together (e.g., naphthyl), or a plurality of aromatic rings that are covalently bonded, and usually contains 6 to 24 carbon atoms, preferably 6 to 12 carbon atoms, preferably 6 to 10 carbon atoms, and is a polyunsaturated aromatic hydrocarbyl group in which at least one ring is aromatic. Examples of suitable aryls include C 6~10 aryl, more preferably C 6~8 aryl. Examples of C 6~12 aryl include phenyl; biphenylyl; biphenylene; or 1- or 2-naphthalenyl; 1-, 2-, 3-, 4-, 5- or 6-tetralinyl (also known as "1,2,3,4-tetrahydronaphthalene"); 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-azulenyl, 4-, 5-, 6 or 7-indenyl, 4- or 5-indanyl, 5-, 6-, 7- or 8-tetrahydronaphthyl; 1,2,3,4-tetrahydronaphthyl; and 1,4-dihydronaphthyl; 1-, 2-, 3-, 4- or 5-pyrenyl. When the suffix "ene" is used in combination with an aryl group, i.e., in the case of arylene, this means an aryl group as defined herein having two single bonds as points of attachment to other groups. Suitable "C 6~12 arylene" groups include 1,4-phenylene, 1,2-phenylene, 1,3-phenylene, biphenylene, naphthylene, indenylene, 1-, 2-, 5- or 6-tetralinylene, etc. When at least one carbon atom in the aryl group is replaced by a heteroatom, the resulting ring is referred to herein as a heteroaryl ring. The heteroatom can be selected from the group consisting of O, N, P, and S, preferably O or N.

[0192] The term "alkylene-aryl" means alkylene as defined herein, where at least one hydrogen atom is replaced, as a group or part of a group, by at least one aryl as defined herein. Alkylene-aryl groups typically contain from 7 to 25 carbon atoms. Non-limiting examples of alkylene-aryl groups include benzyl, phenethyl, dibenzylmethyl, methylphenylmethyl, 3-(2-naphthyl)-butyl, and the like. The term "arylene-alkyl" means arylene as defined herein, where at least one hydrogen atom is replaced, as a group or part of a group, by at least one alkyl group as defined herein. Arylene-alkyl groups typically contain from 7 to 25 carbon atoms.

[0193] Ester, amide, carboxylic acid, and alcohol groups are defined hereinafter, where Rp represents a hydrogen atom or a cyclic, straight-chain, or branched alkyl or alkylene group. In groups containing more than one Rp element, these elements can be selected independently. An ester (functional) group or moiety as shown in this document should be understood as a group represented by the formula -C(O)-O-. An amide (functional) group or moiety as shown in this document should be understood as a group represented by the formula -NRp-C(O)-. A carboxylic acid (functional) group or moiety as shown in this document should be understood as a moiety or group represented by the formula -C(O)OH. An alcohol (or hydroxy) functional group or moiety as shown in this document should be understood as a group represented by the formula -OH.

[0194] The ionizable cationic lipid according to any one of formulas (I) to (V) can be prepared by synthetic methods known in the art, such as (but not limited to) those described in WO 2022 / 268913 pamphlet, particularly in the Examples section of WO 2022 / 268913 pamphlet, particularly Example 9.

[0195] (Ionizable) cationic lipids can preferably be processed from solutions. Thus, (ionizable) cationic lipids are preferably soluble in solvents with various polarities. Thus, (ionizable) cationic lipids are preferably soluble in tricaprylin, ethanol, or isopropanol, more preferably in all three of these solvents. Solubility can be confirmed by evaluating whether about 20 mg of (ionizable) cationic lipid is stirred into about 1 gram of tricaprylin, ethanol, or isopropanol and all the materials spontaneously dissolve to create a clear / transparent solution with a concentration of about 2 w / w%. The test can be performed at about 20 °C (room temperature) or at about 37 °C. Preferably, the (ionizable) cationic lipid is soluble at room temperature.

[0196] (Ionizable) cationic lipids are preferably non-toxic or may have limited low toxicity when alone, or when bound to nucleic acids, or tested with nucleic acids, or assayed in nanoparticles as taught herein. Toxicity cell assays can be performed by methods known in the art, such as the cell viability MTT assay, or by similar or comparable assays.

[0197] In certain embodiments, the amount of modified apolipoprotein, particularly the cumulative amount in the case of two or more modified apolipoproteins, ranges from 0.08 to 2.0 mol%, such as from 0.10 to 2.0 mol%, and / or the amount of phospholipid ranges from 5 to 90 mol%, such as from 15 to 90 mol%, and / or the amount of sterol ranges from 2.5 to 65 mol%, such as from 2.5 to 50 mol%, and / or the amount of cationic or ionizable cationic lipid ranges from 5.0 to 80 mol%, such as from 8.0 to 80 mol%, Here, the mole percent is based only on the combined amount of modified apolipoprotein, phospholipid, sterol, and cationic or ionizable cationic lipid in the lipid nanoparticle.

[0198] When the nanoparticles contain two or more different apolipoprotein components, such as apolipoprotein and modified apolipoprotein, or two or more modified apolipoproteins and optionally one or more apolipoproteins, and the quantitative relationship with other components of the nanoparticles is discussed, the cumulative amount of these apolipoprotein components is considered.

[0199] In certain embodiments of the nucleic acid-containing nanoparticles as taught herein, the amount of modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) ranges from 0.05 to 2.0 mol%, such as from 0.10 to 2.0 mol% or from 0.08 to 0.5 mol%, and / or the amount of phospholipid ranges from 5 to 90 mol%, such as from 15 to 90 mol% or from 8.0 to 50 mol%, and / or the amount of sterol ranges from 2.5 to 65 mol%, such as from 2.5 to 50 mol% or from 4 to 65 mol%, and / or the amount of cationic or ionizable cationic lipid ranges from 5.0 to 80 mol%, such as from 8.0 to 80 mol% or from 5 to 65 mol%, where the mole percent is based only on the combined amount of modified apolipoprotein, phospholipid, sterol, and cationic and / or ionizable cationic lipid in the nanoparticle. These ranges contribute positively to the stability of the nanoparticles and their ability to incorporate nucleic acids.

[0200] In certain embodiments of the nucleic acid-containing nanoparticles as taught herein, The amount of the modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) is in the range of 0.01 to 2.0 mol%, for example 0.05 to 1.0 mol%, or 0.05 to 0.5 mol%, or 0.05 to 0.4 mol%, or 0.05 to 0.3 mol%, or 0.05 to 0.2 mol%, or 0.05 to 0.1 mol%, for example about 0.06, about 0.07, about 0.08, or about 0.09 mol%, and / or The amount of the phospholipid is in the range of 2 to 90 mol%, for example 2 to 80 mol%, or 2 to 70 mol%, or 2 to 60 mol%, or 2 to 50 mol%, or 2 to 40 mol%, or 2 to 30 mol%, or 2 to 20 mol%, for example 5 to 10 mol%, about 6, about 7, about 8, or about 9 mol%, and / or The amount of the sterol is in the range of 2.5 to 65 mol%, for example 2.5 to 50 mol%, or 5 to 50 mol%, or 5 to 40 mol%, or 5 to 30 mol%, or 10 to 30 mol%, for example 15 to 25 mol%, for example about 20, or about 21, or about 22 mol%, and / or The amount of the cationic or ionizable cationic lipid is in the range of 5.0 to 80 mol%, for example 10 to 80 mol%, or 10 to 70 mol%, or 10 to 60 mol%, or 10 to 50 mol%, or 10 to 40 mol%, or 10 to 30 mol%, or 15 to 25 mol%, for example about 18, or about 19, or about 21 mol%, and / or The amount of the lipid, for example preferably triglyceride, is in the range of 0 to 95 mol%, or 0 to 90 mol%, for example 1 to 95 mol%, or 1 to 90 mol%, for example 10 to 90 mol%, or 20 to 80 mol%, or 30 to 70 mol%, or 40 to 60 mol%, or 45 to 55 mol%, for example about 50, or about 51, or 52 mol%, and / or The amount of nucleic acid, such as preferably RNA, such as mRNA, is in the range of 0.01 to 1.0 mol%, such as 0.01 to 0.1 mol%, or 0.01 to 0.05 mol%, such as about 0.02, about 0.03, or about 0.04 mol%, and here, the mole percent is based only on the combined amount of modified apolipoprotein, phospholipid, sterol, cationic and / or ionizable cationic lipid, lipid, such as preferably triglyceride, and nucleic acid, such as preferably RNA. These ranges contribute positively to the stability of the nanoparticles and their ability to incorporate nucleic acid.

[0201] In certain embodiments of the nucleic acid-containing nanoparticles as taught herein, the amount of modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) is in the range of 0.1 to 90 wt%, the amount of nucleic acid is in the range of 0.01 to 90 wt%, the amount of phospholipid is in the range of 0.1 to 95 wt%, the amount of sterol is in the range of 0.1 to 95 wt%, and / or the amount of cationic and / or ionizable cationic lipid is in the range of 0.1 to 95 wt%, here, these weight percents are based on the combined amount of modified apolipoprotein, nucleic acid, phospholipid, sterol and cationic and / or ionizable cationic lipid.

[0202] In one embodiment of the nucleic acid-containing nanoparticles as taught herein, the amount of apolipoprotein (cumulative in the case of two or more modified apolipoproteins) is in the range of 0.2 to 50 wt%, such as 3 to 20 wt% or 4 to 20 wt%, more preferably 0.5 to 30 wt%, more preferably 1 to 20 wt%.

[0203] In one embodiment of the nucleic acid-containing nanoparticles as taught herein, the amount of the nucleic acid ranges from 0.02 to 30% by weight, more preferably from 0.05 to 20% by weight, still more preferably from 0.1 to 15% by weight, for example from 0.5 to 5% by weight.

[0204] In one embodiment of the nucleic acid-containing nanoparticles as taught herein, the amount of the phospholipid ranges from 0.2 to 60% by weight, more preferably from 1 to 50% by weight, for example from 10 to 50% by weight, still more preferably from 3 to 40% by weight, for example from 10 to 40% by weight.

[0205] In one embodiment of the nucleic acid-containing nanoparticles as taught herein, the amount of the sterol ranges from 0.2 to 90% by weight, more preferably from 0.5 to 70% by weight, for example from 2 to 65% by weight, still more preferably from 1 to 50% by weight, for example from 2 to 45% by weight, from 10 to 45% by weight or from 10 to 20% by weight.

[0206] In one embodiment of the nucleic acid-containing nanoparticles as taught herein, the amount of the cationic and / or ionizable cationic lipid ranges from 0.2 to 90% by weight, more preferably from 0.5 to 80% by weight, still more preferably from 1 to 70% by weight, for example from 5 to 60% by weight, from 8 to 60% by weight, from 9 to 60% by weight, from 10 to 60% by weight, from 15 to 25% by weight, or from 20 to 60% by weight.

[0207] In one embodiment of the nucleic acid-containing nanoparticles as taught herein, the amount of the optional filler or filler molecule ranges from 0 to 90% by weight, more preferably from 0 to 80% by weight, still more preferably from 0 to 70% by weight, for example from 0 to 65% by weight.

[0208] In a particular embodiment of the nucleic acid-containing nanoparticles as taught herein, the amount of the optional filler or filler molecule ranges from 20 to 80% by weight, more preferably from 30 to 70% by weight, still more preferably from 30 to 65% by weight, for example from 40 to 65% by weight, from 45 to 55% by weight or from 30 to 60% by weight.

[0209] These weight percentages, as indicated above, are based on the combined amounts of the modified apolipoprotein, nucleic acid, phospholipid, sterol and cationic and / or ionizable cationic lipid, and optionally a filler material, i.e., the total of these five or six components amounts to 100% of the weight of the nanoparticles in the context of these descriptions. These weight percentage ranges contribute positively to the stability of the nanoparticles and the ability to incorporate nucleic acids.

[0210] In certain embodiments, the nanoparticles as taught herein do not contain a filler or filler molecule.

[0211] In certain embodiments, the ratio of modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) to phospholipid based on molar weight percentage is from 1:25 to 1:400, more preferably from 1:50 to 1:200, even more preferably from 1:75 to 1:150.

[0212] In certain embodiments, the ratio of modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) to phospholipid based on weight is from 2:1 to 1:10, more preferably from 1:1 to 1:5, even more preferably from 1:1.5 to 1:4. These ranges contribute positively to the stability of the nanoparticles.

[0213] Alternatively, the payload may be a small organic compound such as a small molecule drug. Generally, small organic compounds are synthesized. The therapeutic agent may be, for example, an anti-cancer therapy such as chemotherapy. Alternatively, the payload may be a biologic. As used herein, the term biologic is used to denote biopharmaceuticals, also known as biological (medical) products, and can be any pharmaceutical manufactured from, extracted from, or semi-synthesized from a biological source. A biologic can be composed of sugars, proteins, nucleic acids, or complex combinations of these substances, or can be living cells or tissues.

[0214] In certain embodiments, the lipid nanoparticles have an average size of 10 to 100 nm, such as 30 to 100 nm.

[0215] In certain embodiments, the ratio of modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) to phospholipid based on molar weight percent is 1:25 to 1:400, more preferably 1:50 to 1:200, even more preferably 1:75 to 1:150. In certain embodiments, the ratio of modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) as taught herein based on molar weight percent to phospholipid is 1:25 to 1:400, more preferably 1:50 to 1:200, even more preferably 1:75 to 1:150.

[0216] In certain embodiments, the ratio of modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) to phospholipid based on weight is 3:1 to 1:100. In certain embodiments, the ratio of modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) as taught herein based on weight to phospholipid is 3:1 to 1:100.

[0217] In certain embodiments, in lipid nanoparticles as taught herein, such as, but not limited to, nanoparticles that do not contain nucleic acid: the amount of modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) is in the range of 0.05 to 2.0 mol%, such as 0.1 to 1.0 mol%, such as 0.2 to 0.7 mol%, or 0.2 to 0.5 mol%, for example preferably about 0.3 mol%, and / or the amount of phospholipid is in the range of 5 to 90 mol%, such as 10 to 50 mol%, such as 10 to 40 mol%, 10 to 30 mol%, or 10 to 20 mol%, for example about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 19 mol%, and / or The amount of sterol is in the range of 2.5 to 65 mol%, such as 2.5 to 60 mol%, 2.5 to 50 mol%, 2.5 to 40 mol%, or 2.5 to 30 mol%, or 2.5 to 20 mol%, or 2.5 to 10 mol%, such as 2.5 to 5 mol%, such as about 3.0 or about 4.0, or about 5.0 mol%, and / or The amount of lipid, such as preferably triglyceride, is 1 to 95 mol%, such as 10 to 95 mol%, or 20 to 95 mol%, or 30 to 95 mol%, or 40 to 95 mol%, or 50 to 95 mol%, or 60 to 95 mol%, such as 1 to 90 mol%, such as 10 to 90 mol%, or 20 to 90 mol%, or 30 to 90 mol%, or 40 to 90 mol%, or 50 to 90 mol%, or 60 to 90 mol%, such as 60 to 80 mol%, or 70 to 80 mol%, in the range of, for example, about 65, about 70, or about 75 mol%, wherein the mole percent is based only on the combined amount of the modified apolipoprotein, phospholipid, sterol, and lipid, such as preferably triglyceride, in the lipid nanoparticles.

[0218] In a further aspect, the present invention is a method for producing lipid nanoparticles as described herein, comprising a1) expressing and isolating a modified apolipoprotein as disclosed herein to obtain an isolated modified apolipoprotein, and / or a2) chemically conjugating a targeting moiety to an apolipoprotein or apolipoprotein mimetic to obtain a modified apolipoprotein and isolating the modified apolipoprotein, b) combining the isolated modified apolipoprotein obtained in step a1 and / or step a2 with phospholipid, sterol and optionally lipid to obtain lipid nanoparticles and relates to a method comprising.

[0219] In one embodiment, step b) is b1) A step of mixing a lipid component in an organic solvent with nucleic acid in an aqueous buffer, preferably mixing rapidly, to produce nanoparticles, wherein the lipid component comprises a phospholipid, a sterol, a core component, and optionally a lipid, and the aqueous buffer has a pH of 5.5 or less, preferably 5.0 or less. b2) A step of mixing the lipid nanoparticles with a modified apolipoprotein at a pH of 5.5 to 9.0, preferably at a pH of 6.0 to 8.0, more preferably at a pH of 6.5 to 8.0, preferably mixing rapidly, to produce nanoparticles comprising the above.

[0220] It is understood that the modified apolipoprotein can be expressed as a chimeric fusion protein of an apolipoprotein and a targeting entity, or can be chemically conjugated to the targeting entity, or can be produced by a combination thereof. Expression of chimeric proteins is known to those skilled in the art and can be used when the targeting entity is a peptide or protein. For example, it is well within the knowledge of those skilled in the art to produce a nucleic acid encoding such a protein using molecular techniques by cloning a targeting entity coding sequence in-frame with an apolipoprotein (or analogue or derivative) coding sequence, for example, at the C- or N-terminal sequence encoding a nucleotide. The advantage of using chimeric protein expression is that all of the proteins expressed are fusion proteins.

[0221] Alternatively, chemical conjugation may be used. Methods suitable for chemical conjugation of the targeting entity to the apolipoprotein (or a mimetic thereof) are known to those skilled in the art. Non-limiting examples are strain-promoted cycloaddition, aminolysis, and Michael-type addition. For example, existing or introduced cysteine residues can be used in either the apolipoprotein or the targeting entity. Introduction of cysteine residues can be achieved by point mutation of nucleotides in the coding nucleotide sequence or by introduction of a cysteine-encoding codon. The advantage of chemical conjugation is that it is not limited to the use of peptide or protein sequences and can be applied to any type of organic molecule.

[0222] It is understood that modified apolipoproteins, phospholipids, sterols, and any components may be rapidly mixed to obtain lipid nanoparticles. Optionally, lipids and / or payloads as defined herein may be added.

[0223] The present invention also provides a method for producing lipid nanoparticles, comprising: a) rapidly mixing a lipid component in an organic solvent with a nucleic acid in an aqueous buffer to produce lipid nanoparticles, wherein the lipid component comprises a phospholipid, a sterol, a cationic lipid, or an ionizable cationic lipid, and the aqueous buffer has a pH of 5.0 or less; b) rapidly mixing the lipid nanoparticles with one or more modified apolipoproteins as taught herein at a pH of 5.5 to 8.0, preferably 6.0 to 8.0, to produce lipid nanoparticles and including.

[0224] According to the above method, a two-step reaction is carried out, where in the first step, nucleic acid-containing nanoparticles are formed, and in the next second step, the modified apolipoprotein is included in the nanoparticles. Preferably, the first step is carried out at a low pH and the second step is carried out at a physiological pH.

[0225] The organic solvent may be an alcohol such as ethanol, isopropanol, methanol, acetonitrile, dimethyl sulfoxide (DMSO), chloroform, or a combination thereof. Preferred organic solvents are miscible with water and non-toxic, such as ethanol and DMSO, or a combination thereof.

[0226] For example, the organic solvent can be 96% - 100% ethanol, preferably 100% ethanol.

[0227] Rapid mixing is known in the art and is described, for example, in Hirota et al. BIOTECHNIQUES VOL. 27, NO. 2, p286 - 289, Jeffs et al., Pharm Res 22, 362 - 372 (2005), Kulkarni et al., ACS Nano 2018, 12, 5, 4787 - 4795.

[0228] The aqueous buffer in step a) has a low pH to ensure that the ionizable cationic lipid is positively charged, enabling binding within the particles and inclusion of the nucleic acid / cationic lipid complex in the particles. For example, the buffer can have a pH of 5.0 or less, such as 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5 or less. The aqueous buffer can be any buffer that does not damage the nucleic acid. An exemplary buffer is sodium acetate at pH 4.0. Subsequently, the nanoparticles are incorporated into an aqueous buffer having a pH of approximately 6 - 8, preferably 7 - 8, more preferably approximately 7.4. This can be achieved, for example, by dialysis using an aqueous buffer in the indicated pH range. A non - limiting example of an aqueous buffer suitable for this step is 155 mM PBS at pH 7.4, but it is understood that any buffer that does not damage the nucleic acid can be used.

[0229] In step b), the nanoparticles in an aqueous buffer at pH 6 to 8, preferably pH 7 to 8, are rapidly mixed with the modified apolipoprotein in an aqueous buffer at pH 5.5 to 8, preferably 6 to 8, more preferably pH 7 to 8, to obtain the nanoparticles.

[0230] The two-step formulation process as taught hereinabove as described herein results in nucleic acid-containing nanoparticles having a wide range of desired beneficial characteristics (stability, low or no toxicity, high nucleic acid retention, nucleic acid activity, etc.). However, since other processes may also result in nanoparticles having beneficial features, the described formulation method is non-limiting.

[0231] A further aspect of the invention provides a pharmaceutical composition comprising a modified apolipoprotein as taught herein, a nucleic acid as taught herein or a lipid nanoparticle as taught herein, and a pharmaceutically acceptable carrier.

[0232] In a further aspect, the present invention relates to a modified apolipoprotein as defined herein, or a lipid nanoparticle as defined herein, or a lipid nanoparticle obtainable or obtained by a method as described herein, or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein, for use as a medicament. Either the modified apolipoprotein contained in the nanoparticle (e.g., by the action of the conjugated payload) or the payload is assumed to be useful for treating, ameliorating or alleviating a condition in a subject. Thus, one embodiment is a modified apolipoprotein according to a first aspect, or a lipid nanoparticle according to a second aspect, or a lipid nanoparticle obtainable or obtained by a method according to a third aspect of the invention, or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein, for use in the treatment or prevention of an immune-related disorder such as transplant rejection, graft-versus-host disease (GVHD), atherosclerosis, infectious disease, inflammation, autoimmunity, allergy, cancer, genetic disorder, metabolic disorder, neuropathy or tissue trauma, preferably an immune-related disorder such as transplant rejection, atherosclerosis, infectious disease, autoimmunity, cancer, genetic disorder, metabolic disorder, neuropathy or tissue trauma. A further embodiment is a method of treating an immune-related disorder in a subject in need of treatment, the method comprising administering to the subject in need of said treatment a therapeutically effective amount of a modified apolipoprotein according to a first aspect, or a lipid nanoparticle according to a second aspect, or a lipid nanoparticle obtainable or obtained by a method according to a third aspect of the invention, or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein. Also provided herein is the use of a modified apolipoprotein according to a first aspect, or a lipid nanoparticle according to a second aspect, or a lipid nanoparticle obtainable or obtained by a method according to a third aspect of the invention, or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein, for the manufacture of a medicament for the treatment of an immune-related disorder in a subject.

[0233] An immune-related disorder, as used herein, includes any disorder in which the immune system plays a role in the development of the disease. An immune-related disorder can refer to a disorder in which the immune system is suppressed or (over)activated. Examples of immune-related diseases are cancer, infectious diseases, sepsis, autoimmune diseases, and cardiovascular diseases. Examples of autoimmune diseases are type 1 diabetes, rheumatoid arthritis (RA), psoriasis / psoriatic arthritis, multiple sclerosis (MS), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, and celiac disease. In one embodiment, the immune-related disorder is selected from the group consisting of transplant rejection, graft-versus-host disease (GVH), atherosclerosis, infectious diseases, inflammation, autoimmunity, allergies, cancer, genetic disorders, metabolic disorders, neuropathies, and tissue trauma, preferably transplant rejection, atherosclerosis, infectious diseases, autoimmunity, cancer, genetic disorders, metabolic disorders, neuropathies, or tissue trauma.

[0234] In one embodiment, the immune-related disorder is selected from the group consisting of cancer, infectious diseases, sepsis, type 1 diabetes, rheumatoid arthritis (RA), psoriasis / psoriatic arthritis, multiple sclerosis (MS), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, and celiac disease.

[0235] In a further aspect, the present invention relates to the use of a modified apolipoprotein as described herein, or a lipid nanoparticle as described herein, or a lipid nanoparticle obtained or obtainable by a method as described herein, or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein, when delivering a payload (e.g., a compound) to a target, preferably, where the target is a cell, tissue, and / or organ, more preferably, the target is a lymphocyte, a myeloid cell, a tumor cell, or the targeting entity binds to a bacterial, viral, fungal or parasitic protein or antigen, preferably, the lymphocyte or myeloid cell is selected from monocytes, macrophages, M1-like macrophages, M2-like macrophages, eosinophils, basophils, mast cells, NK cells, B cells, plasma cells, regulatory T cells, hematopoietic stem cells, granulocytes, helper T cells such as Th1, Th2, Th17 or Th22, plasmacytoid dendritic cells, dendritic cells such as conventional DC1 or conventional DC2, or tumor-associated macrophages. In one embodiment, the use is ex vivo or in vitro use. In an alternative embodiment, the use is in vivo use. Typically, the apolipoprotein or targeting entity binds to a cell surface protein such as a receptor. Thus, the target may be a protein such as a receptor, a cell or cell type (expressing the above protein), a tissue or tissue type (expressing the above protein) or an organ (expressing the above protein). It is understood that by selecting or adapting the targeting entity, the modified apolipoprotein can be targeted to various proteins. For example, the receptor-binding domain of a ligand can be used to target a specific receptor. Alternatively, a known binding partner of a cell surface protein can be used to reroute a fusion protein (i.e., a modified apolipoprotein).

[0236] A further aspect of the invention is an in vitro or ex vivo method of introducing a nucleic acid into a cell, the method comprising contacting the cell with a nucleic acid-containing lipid nanoparticle as taught herein, or a nucleic acid-containing lipid nanoparticle obtainable or obtained by a method as taught herein.

[0237] A further aspect of the invention is an in vivo method of introducing a nucleic acid into a cell, the method comprising contacting the cell with a nucleic acid-containing lipid nanoparticle as taught herein, or a nucleic acid-containing lipid nanoparticle obtainable or obtained by a method as taught herein.

[0238] A further aspect of the invention provides a nucleic acid-containing lipid nanoparticle as taught herein, or a nucleic acid-containing lipid nanoparticle obtainable or obtained by a method as taught herein, for use in the in vivo delivery of a nucleic acid to a subject.

[0239] A further aspect of the invention is a method of in vivo delivery of a nucleic acid, the method comprising administering to a subject a nucleic acid-containing lipid nanoparticle as taught herein, or a nucleic acid-containing lipid nanoparticle obtainable or obtained by a method as taught herein.

[0240] A further aspect of the invention is a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid-containing lipid nanoparticle as taught herein, or a nucleic acid-containing lipid nanoparticle obtainable or obtained by a method as taught herein.

[0241] The disease can be any of the diseases as discussed herein, such as transplantation rejection, graft-versus-host disease (GVH), atherosclerosis, infectious diseases, inflammation, autoimmunity, allergy, cancer, genetic disorders, metabolic disorders, neuropathies or tissue trauma, preferably immune-related disorders such as transplantation rejection, atherosclerosis, infectious diseases, autoimmunity, cancer, genetic disorders, metabolic disorders, neuropathies or tissue trauma.

[0242] The purpose of the nucleic acid-containing nanoparticles described herein is to deliver nucleic acids to cells or to deliver nucleic acid therapies to subjects. The nucleic acid may be, for example, mRNA encoding a peptide or protein of interest to be expressed in the cell, or may include short nucleic acids such as siRNA, shRNA, etc. that are intended to interfere with gene expression (e.g., gene silencing), or the nucleic acid may include components of the CRISPR-Cas or related gene editing system (e.g., gRNA) that induce mutations in the genome of the cell. Thus, generally, the mechanism of action of the nucleic acid (payload of the nanoparticle) is present in the cytoplasm or nucleus. Thus, the nanoparticles preferably have at least the following properties: 1) the nanoparticles enable targeting of the intended target cells, and 2) the nanoparticles enable delivery of the payload such that the nanoparticles can assert their action (thus, in most cases, in the cytoplasm or nucleus of the target cell).

[0243] It is understood that nucleic acid therapies comprising nanoparticles can be administered to a subject in need thereof. Depending on the target cell or tissue, the administration may be parenteral, such as intravenous, intramuscular or subcutaneous. The administration may further be oral, sublingual, topical, rectal, nasal (inhalation) or vaginal. Further, the targeting of the target tissue or cell is determined by the appropriate selection of modified apolipoproteins. In one embodiment, the use of the nanoparticles or composition comprises delivering the nucleic acid to the bone marrow compartment or spleen. This can be achieved, for example, by intravenous parenteral administration.

[0244] The nanoparticles of certain embodiments of the present invention can target tissues (spleen, bone marrow) associated with the presence of immune cells after systemic injection.

[0245] Certain embodiments are nucleic acid-containing nanoparticles as taught herein, or compositions as taught herein, for use in immunotherapy.

[0246] In one aspect, the present invention relates to nanoparticles comprising nucleic acid-containing nanoparticles as taught herein, or compositions comprising them, for use in the treatment of diseases by stimulating or inhibiting the innate immune response, preferably where the disease is one that would benefit from stimulating or inhibiting the innate immune response in a subject, e.g., a disease characterized by innate immune deficiency, and more preferably where the disease to be treated is cancer, cardiovascular disease, autoimmune disorder or xenograft rejection. Thus, the nanoparticles can be used in the treatment of any disease related to the immune system such as any immune disorder, or in the treatment of any disease or disorder where modulation of the immune response is considered an option for treatment.

[0247] In a further aspect, the present invention relates to a nucleic acid encoding a fusion protein (i.e., a modified apolipoprotein) as defined herein. The nucleic acid can be included in a vector such as a viral vector for stable integration in a cell, or an expression vector that allows transient expression.

[0248] It will be understood by those skilled in the art that numerous variations and / or modifications may be made to the above-described aspects and / or embodiments without departing from the broad general scope of the present invention. Accordingly, these aspects and / or embodiments are to be considered in all respects as illustrative and not restrictive. The present invention includes the following non-limiting examples.

Examples

[0249] [Example 1] Generation of Cell Marker-Specific Apolipoprotein-Nanobody Fusion Proteins aNP is a lipid-based nano-sized formulation (with a diameter of approximately 30 - 200 nm) having a hydrophobic core and an apolipoprotein covering the outer surface. The apolipoprotein is a helical protein that has an inherent affinity for the lipid layer due to its amphiphilicity. There are several types of apolipoproteins, all of which can be used as structural components of aNP formulations. The presence of apolipoproteins regulates the biological behavior of aNP. For example, apolipoprotein A1 interacts with cells via scavenger receptor class B type 1 (SRB1) and ATP-binding cassette transporter ABCA1. This increases the interaction between aNP and myeloid cells in lymphoid organs. However, it has been shown that therapeutically functionalized aNP has little effect on immune cell types when these mentioned receptors are not used. Furthermore, by enhancing the targeting specificity within the myeloid compartment, the efficiency of therapeutic agents can also be increased. Therefore, by rerouting aNP to specific cell types, this modular platform power is enhanced.

[0250] Nanobodies are single antigen-specific polypeptides consisting of the variable domain of the heavy chain (VH domain) of heavy-chain-only antibodies found in camelids, and are also called VHH domains or single-domain antibodies (sdAb). Nanobodies are low in complexity, very stable and soluble, and they are endowed with higher therapeutic potential compared to their human antibody equivalents. Nanobodies consist of a conserved framework of β-sheets and three variable complementarity-determining regions (CDR). The specificity of nanobodies results from the variation of these CDR loops. Nanobodies lack the hydrophobic patches present in the human antibody framework for enhancing the interaction between the VH domain and the VL domain. Nanobodies have four hallmark mutations in this region that are important for their stability and solubility. The conserved framework can be modified using point mutations so as to humanize the nanobody and limit its immunogenicity.

[0251] The apolipoprotein-nanobody fusion protein combines the particle-forming ability of apolipoprotein with the targeting ability of nanobody. The fusion protein is generated by molecular cloning of genes for apolipoprotein and nanobody that are fused using an amino acid linker appropriate to allow sufficient space between the apolipoprotein and the nanobody. Subsequently, the fusion protein is recombinantly expressed in Escherichia coli (E. coli) and purified using a purification tag such as a histidine tag. Next, by including the fusion protein during aNP production, cell-type specific aNPs are generated. The cell type to be targeted can be readily determined by changing the nanobody sequence to be specific to a desired cell marker.

[0252] Nanobodies specific to a desired cell marker are engineered via yeast surface display methods and directed evolution. Generative modeling under machine learning guidance is used to introduce variation in the directed evolution process and create nanobody sequences with higher affinity for its target. This process is repeated until a sequence with the desired binding affinity is selected.

[0253] [Example 2] Evaluating rerouted aNPs Perform an in vitro evaluation of cell-specific aNPs to analyze aNP-cell interactions. Incubate aNPs with human peripheral blood mononuclear cells (PBMCs) and use flow cytometry to evaluate aNP binding to immune cells. After uptake of the therapeutic compound, evaluate the therapeutic efficacy of aNPs in vitro.

[0254] Subsequently, evaluate the in vivo efficiency of cell targeting by rerouted aNPs in mice with a humanized immune system. Examine organ-specific and cell-specific biodistribution, as well as the toxicology and therapeutic efficacy of aNPs.

[0255] [Example 3] Practice The rerouted aNP may specifically deliver therapeutic compounds to the desired immune cell type, thereby regulating the immune response. The main advantage of the rerouted aNP platform technology is that the nanobody and the therapeutic payload can be easily exchanged. Therefore, rerouted aNP can be practiced, for example, as an immunotherapy to promote an immune response for treating cancer or infectious diseases, or as an immunotherapy to treat autoimmune diseases or weaken the immune response during organ transplantation.

[0256] [Example 4] Fusion proteins of apolipoproteins fused to rerouting proteins or peptides (targeting bodies) and their incorporation into lipid nanoparticles Materials and methods Expression and purification of VHHCD8-apoA1 fusion protein: A small culture of ClearColi cells transformed with pET20b-VHHCD8-apoA1 plasmid and pDiscoTune plasmid was started in LB medium with 100 μg / mL ampicillin. The next day, 40 mL of the small culture was diluted into 1 liter of 2YT medium to start a large culture, and L-rhamnose was added at a final concentration of 50 μM to induce T7 lysozyme with the pDiscoTune plasmid. The culture was grown at 37 °C and 150 rpm until the OD600 reached 0.6 - 0.8, then isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM to induce expression. The induced culture was incubated overnight at 18 °C and 150 rpm. The induced bacterial culture was pelleted, and the cells were resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, pH 7.9). Benzonase nuclease (Merck Millipore) and one cOmplete™ EDTA-free protease inhibitor cocktail tablet (Roche) per 50 mL of cell suspension were added, and the cell suspension was incubated at 4 °C with stirring. Subsequently, the suspension was homogenized 3 times at 15000 - 20000 psi using an Avestin Emulsiflex C3. The cell lysate was always kept on ice. After lysis, the cell lysate was centrifuged to pellet the insoluble cell debris, and the supernatant was passed through an immobilized metal affinity chromatography (IMAC) column containing immobilized nickel ions. The column was washed with 8 column volumes of buffer A (20 mM Tris, 500 mM NaCl, 10 mM imidazole, pH 7.9), followed by 8 column volumes of buffer A50 (20 mM Tris, 500 mM NaCl, 50 mM imidazole, pH 7.9). To elute VHHCD8-apoA1, 8 column volumes of buffer A500 (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.9) were applied to the column. All fractions of the purification steps were collected and analyzed by SDS-PAGE.The buffer of the fraction containing purified VHHCD8-apoA1 was changed to PBS using Amicon Ultracentrifugal Filters (Amicon). To store VHHCD8-apoA1, aliquots were snap-frozen in liquid nitrogen and stored at -70°C.

[0257] The VHHCD8-apoA1 fusion protein has a sequence as defined by SEQ ID NO: 1 or is encoded by a sequence as defined by SEQ ID NO: 2, which includes a linker between apoA1 containing cysteine and VHHCD8.

[0258] SEQ ID NO: 1

[0259]

Chem.

[0260] SEQ ID NO: 2

[0261]

Chem.

[0262] aNP formulation: All phospholipids were purchased from Avanti Polar Lipids Inc. For discoidal lipid nanoparticles: From a stock solution (10 mg / mL) in chloroform, DMPC (128 μL), cholesterol (Sigma-Aldrich) (7.5 μL), and for spherical lipid nanoparticles: POPC (66.5 μL), PHPC (17.5 μL), cholesterol (4.5 μL), and tricaprylin (Sigma-Aldrich) (2.8 μL from a 0.956 g / mL stock) were combined in a glass vial and dried under vacuum. The resulting thin film was redissolved in an acetonitrile / methanol mixture (95:5 wt%, total volume 900 μL). Separately, solutions of VHHCD8-apolipoprotein A1 protein in PBS (6.5 mL, 0.14 mg / mL) were prepared twice. Using a microfluidic pump fusion 100 (Chemyx Inc) at a flow rate of 0.8 mL / min for the lipid solution and 6 mL / min for the apolipoprotein A1 solution, both solutions were simultaneously injected into a Zeonor herringbone mixer (Microfluidic Chipshop). For discoidal, a 10 kDa MWCO was used, and for spherical aNPs Vivaspin tubes, a 50 kDa MWCO was used. The resulting solution was concentrated by centrifugal filtration at 4000 rpm to obtain a volume of 1 mL. PBS (5 mL) was added, and the solution was concentrated to 1 mL, and this was repeated twice. The washed solution was concentrated to approximately 1.5 mL and filtered through a 0.22 μm PES syringe filter to obtain the finished VHHCD8-apolipoprotein A1-containing nanoparticles.

[0263] DLS: The VHHCD8-apolipoprotein A1-containing nanoparticle formulation in PBS was filtered through a 0.22 μm PES syringe filter and analyzed by dynamic light scattering using a Malvern Zetasizer Nano ZS analyzer. Values are reported as the mean of the number-average size distribution.

[0264] Cryo-TEM: First, the surface of a 200-mesh lacey carbon-coated copper grid (Electron Microscopy Sciences) was plasma-treated for 40 seconds using a Cressington 208 carbon coater. Subsequently, 3 μl of VHHCD8-apoA1-containing nanoparticles (protein approximately 1 mg / ml) was applied onto the grid and vitrified into a thin film by plunge vitrification in liquid ethane using an automated robot (FEI Vitrobot Mark IV). Cryo-TEM imaging was performed on a cryoTITAN (Thermo Fisher Scientific) equipped with a field emission gun (FEG), a post-column Gatan imaging filter (model 2002), and a post-GIF 2k×2k Gatan CCD camera (model 794). Images were acquired at an acceleration voltage of 300 kV in bright-field TEM mode, using zero-loss energy filtering, at either a magnification of 6,500× (dose rate 1.64 electrons / Å2·s) or 24,000× (dose rate 11.8 electrons / Å2·s), and an acquisition time of 1 second.

[0265] In vitro binding of VHHCD8-apoA1 in mouse splenocytes: Spleens were obtained from mice, minced, and filtered through a 70-μm strainer (Corning) multiple times to obtain a splenocyte suspension. Cells were centrifuged at 1500 rpm for 10 minutes, the supernatant was removed, and the cells were resuspended in 2 mL of 1× red blood cell lysis buffer (Thermofisher). The suspension was incubated at room temperature for 5 minutes, 10 mL of Roswell Park Memorial Institute (RPMI) medium (Thermofisher) was added, and the cells were centrifuged again at 1500 rpm for 10 minutes. Next, the cells were resuspended in RPMI medium and plated at 150,000 cells / well in a 96-well plate.

[0266] The protein was labeled by adding sulfo-cyanine 5-maleimide (Lumiprobe) in 5-fold molar excess in dimethyl sulfoxide (DMSO). The mixture was incubated at room temperature for 2 hours. The excess dye was removed using a PD mini-trap G-25 desalting column (Cytiva). The fluorescently labeled aNP was formulated by adding 6.4 μg of DiI for discoidal formulations and 21 μg of DiI for spherical formulations. The fluorescently labeled fusion protein or aNP and controls were added to the wells and incubated at 4 °C (for proteins) or 37 °C (for aNP) for 30 minutes, after which the cells were harvested, washed, stained for CD3 and CD4, and measured on a Cytoflex (Beckman Coulter Inc.). Flow cytometry data were analyzed using FlowJo software (BD).

[0267] Results The VHHCD8-apoA1 fusion protein was successfully expressed in Clearcoli cells. Trace protein contaminants were present after IMAC purification [lane E1] (Figure 3). The most prominent band corresponded to the fusion protein with a molecular weight of 43.3 kDa (Figure 3). The exact mass was later confirmed by mass spectrometry (data not shown).

[0268] Discoidal apolipoprotein nanoparticles incorporating VHHCD8-apoA1 were formulated. Dynamic light scattering (DLS) was used to determine the particle size and polydispersity index (PDI). The particle size remained stable for 7 days (data not shown). By day 14, the size had increased slightly (data not shown). The PDI remained stable for 14 days (data not shown). Cryo-TEM images of the nanoparticles showed the expected discoidal shape (data not shown).

[0269] VHHCD8-apoA1 and apoA1 were fluorescently labeled and subsequently added to mouse splenocytes. For the VHHCD8-apoA1 fusion protein, a dose-dependent increase in mean fluorescence intensity (MFI) was observed, indicating binding of the fusion protein to the CD8 receptor (Figure 4, lower panel). The apoA1 condition did not show this dose-dependent behavior and had an MFI similar to the control sample.

[0270] Disc-shaped aNP and spherical aNP were formulated using VHHCD8-apoA1 and apoA1. A fluorescent dye was incorporated into the lipid structure of the particles. Mouse splenocytes were incubated with the nanoparticles. Both VHHCD8-apoA1 particles showed a dose-dependent increase in MFI, indicating binding of the nanoparticles to the CD8 receptor (Figure 5). The disc-shaped nanoparticles showed a greater increase than the spherical nanoparticles. For the apoA1 nanoparticle condition, no increase in MFI was observed.

[0271] [Example 5] In Vivo Biodistribution of Lipid Nanoparticles Containing a Fusion Protein of an Apolipoprotein Fusion Protein with a Rerouting Protein or Peptide Materials and Methods Formulation of Spherical VHHCD8-aNP All phospholipids were purchased from Avanti Polar Lipids Inc. For spherical aNP, POPC (66.5 μL), PHPC (17.5 μL), cholesterol (4.5 μL), and tricaprylin (from Sigma-Aldrich) (2.8 μL from a 0.956 g / mL stock) were combined in a glass vial and dried under vacuum. The resulting thin film was redissolved in an acetonitrile / methanol mixture (95:5%, total volume 900 μL).

[0272] Separately, two solutions of the VHHCD8-apoA1 protein (SEQ ID NO: 1) in PBS (6.5 mL, 0.14 mg / mL) were prepared.

[0273] For the lipid solution, at a flow rate of 0.8 mL / min, and for the apoA1 solution, at a rate of 6 mL / min, both solutions were simultaneously injected into a Zeonor herringbone mixer (Microfluidic Chipshop) using a microfluidic pump fusion 100 (Chemyx Inc). The resulting solution was concentrated by centrifugal filtration at 4000 rpm using a globular aNPs Vivaspin tube with a 50 kDa MWCO to obtain a volume of 1 mL. PBS (5 mL) was added, and the solution was concentrated to 1 mL, and this was repeated twice. The washed solution was concentrated to approximately 1.5 mL and filtered through a 0.22 μm PES syringe filter to obtain the finished aNP.

[0274]

Table 1

[0275] In vivo biodistribution of VHHCD8-apoA1 in mice (Figure 7) Globular VHHCD8-aNP with 2 mol% DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) was intravenously injected into C57BL / 6 mice at a dose of 1 mg / kg of DiR. After 24 hours, the mice were sacrificed, blood was collected, and after PBS perfusion, other tissues of interest (bone marrow, spleen, lymph nodes) were collected. The tissues were homogenized into single cell suspensions and stained for myeloid (CD115, F4 / 80, Ly6C, CD11b, CD45, Ly6G, CD11c), lymphoid (CD45, CD11b, CD3, CD19, CD4, CD8a), and progenitor (CD117, CD34, Ly-6A / E, CD135, CD16 / 32, CD48, CD41, CD150, CD3, CD11b, CD45R / B220, Ly-76, Ly6G, Ly6C) cell markers and analyzed by Cytoflex (Beckman Coulter Inc.). Flow cytometry data were analyzed using FlowJo software (BD).

[0276] Results Spherical lipid nanoparticles containing VHHCD8-apolipoprotein A1 were characterized using DLS to determine their hydrodynamic size (number average) and polydispersity index (Pdi), and cryo-electron microscopy was used to determine their size and morphology (Figure 6).

[0277] In vivo biodistribution data indicate that VHHCD8-apolipoprotein A1 fusion protein nanoparticles associated significantly less with bone marrow, spleen, and bone marrow cells in blood compared to control apolipoprotein A1 protein nanoparticles (Figure 8A). VHHCD8-apolipoprotein A1 fusion protein nanoparticles associated significantly more with CD8+ T cells in bone marrow, spleen, and blood compared to control apolipoprotein A1 protein nanoparticles (Figure 8B). VHHCD8-apolipoprotein A1 fusion protein nanoparticles associated less with CD4+ T cells in bone marrow, spleen, and blood compared to control apolipoprotein A1 protein nanoparticles (Figure 8C). Furthermore, VHHCD8-apolipoprotein A1 fusion protein nanoparticles associated more with CD3+ T cells in bone marrow, spleen, and blood compared to control apolipoprotein A1 protein nanoparticles (Figure 8D). In lymph nodes, VHHCD8-apolipoprotein A1 fusion protein nanoparticles associated more with CD8+ or CD3+ compared to control apolipoprotein A1 protein nanoparticles, and VHHCD8-apolipoprotein A1 fusion protein nanoparticles associated less with CD4+ T cells in lymph nodes compared to control apolipoprotein A1 protein nanoparticles (Figure 9).

[0278] VHHCD8-apolipoprotein A1 fusion protein nanoparticles associated with more CD8+ T cells in bone marrow, blood, spleen, and lymph nodes compared to control apolipoprotein A1 protein nanoparticles (Figure 10). No difference was seen in the binding of CD4+ T cells between treatment groups (Figure 10).

[0279] [Example 6] Lipid nanoparticles comprising a fusion protein of an apolipoprotein fused to a routing peptide or protein and / or a fusion protein of an apolipoprotein fused to an immunomodulatory biomolecule Materials and Methods ApoA1-IL-2 was reduced by incubation in 1 mM DTT for 2 hours at room temperature to cleave the disulfide bonds between cysteines in the protein. ApoA1-IL-2 and VHHCD8-apoA1 were fluorescently labeled with cy3 and cy5, respectively, as described in Example 4.

[0280] The apoA1-IL-2 fusion protein has a sequence as defined by SEQ ID NO: 3 or SEQ ID NO: 5, or is encoded by a sequence as defined by SEQ ID NO: 4 or SEQ ID NO: 6.

[0281] [Table 2]

[0282] Disc-shaped aNPs were formulated using 513 μg of apoA1-IL-2 and 467 μg of VHHCD8-apoA1. Spherical aNPs were formulated in two sizes, where only the triglyceride content was changed. The large spheres were formulated as described in Example 5, and the small spheres were formulated using half of the triglyceride content of the large spheres. Spheres containing only apoA1-IL-2 (donor) contained 1025 μg of apoA1-IL-2. Spheres containing only VHHCD8-apoA1 (acceptor) contained 921 μg of VHHCD8-apoA1. Large and small spheres with a 1:1 mix of donor and acceptor contained 513 μg of apoA1-IL-2 and 467 μg of VHHCD8-apoA1.

[0283] aNP in PBS was pipetted into a black 96-well plate (Thermo Fisher Scientific - Nunclon) at 100 μL per well. For the 1:1 mix of donor-only spheres and acceptor-only spheres, 50 μL of each formulation was placed in the well. A 1:1 mix (molar ratio) of fluorescently labeled apoA1-IL-2 and VHHCD8-apoA1 was included as a control. Samples were excited at 520 nm and luminescence was detected in a Spark plate reader (Tecan) at 5 nm step sizes between 560 nm and 780 nm. Data was normalized by dividing the intensity of one sample at each wavelength by the intensity of that sample at 565 (donor emission maximum).

[0284] Results The results of FRET show that both VHHCD8-apolipoprotein A1 and IL-2-apolipoprotein A1 fusion proteins can be readily and stably incorporated into one lipid nanoparticle formulation (Figure 11).

[0285] [Example 7] Production, Analysis, and Evaluation of mRNA-Containing Apolipoprotein Nanoparticles Targeting CD8+ T Cells Materials and Methods Production of Apolipoprotein Nanoparticles CD8-aNP-mRNA is prepared by rapid mixing with a T-junction mixer. Cationic ionizable lipid ALC-0315, dimyristoylphosphatidylcholine (DMPC), cholesterol, and tricaprylin are dissolved in ethanol at an appropriate molar ratio (Table 2) to a final concentration of 10 mM total lipid. mRNA is dissolved in 25 mM sodium acetate buffer at pH 4.0 to obtain a final mixture with a defined nucleic acid to lipid weight / μmol ratio of 0.0278 (N / P 6). In the first mixing step, the organic and aqueous solutions are mixed at a flow rate ratio of 1:3 (v:v) and a total flow rate of 28 mL / min. The resulting mixture is dialyzed overnight against 1000-fold volume of phosphate buffered saline (PBS) pH 7.4. In the second mixing step, the lipid nanoparticles containing mRNA and CD8VHH-apolipoprotein A1 fusion protein are mixed at a flow rate ratio of 1:3 (v:v) and a total flow rate of 28 mL / min. The resulting CD8-aNP-mRNA is sterile filtered (0.2 μm) and concentrated using centrifugal flow filtration.

[0286]

Table 3

[0287] Physicochemical analysis of apolipoprotein nanoparticles CD8-aNP-mRNA is characterized according to size distribution by dynamic light scattering, zeta potential by laser Doppler electrophoresis, mRNA encapsulation efficiency by Ribogreen assay, and cholesterol / phospholipid content using a dedicated colorimetric assay.

[0288] Ex vivo evaluation of apolipoprotein nanoparticles Obtain the spleen from a mouse, cut it finely, filter it through a 70 μm strainer (Corning) multiple times to obtain a spleen cell suspension. Centrifuge the cells at 1500 rpm for 10 minutes, remove the supernatant, and dissolve the cells in 2 mL of 1× red blood cell lysis buffer (Thermofisher). Incubate the cell suspension at room temperature for 5 minutes, add 10 mL of RPMI medium (Thermofisher), and centrifuge the cells again at 1500 rpm for 10 minutes. Next, redissolve the cells in RPMI medium and plate culture them at 150,000 cells / well in a 96-well plate. Add CD8-aNP-mRNA encoding mCherry and a control formulation to the wells at appropriate concentrations, incubate at 37 °C for 6 hours, then collect the cells, wash them, stain them for CD3 and CD4, and measure them with a Cytoflex (Beckman Coulter Inc.). Flow cytometry data is analyzed using FlowJo software (BD).

[0289] In vivo evaluation of apolipoprotein nanoparticles Intravenously inject CD8-aNP-mRNA encoding mCherry and a control formulation into C57BL / 6 mice at a dose of 0.5 mg / kg of mRNA. After 6 hours, sacrifice the mice, collect the blood, and after PBS perfusion, collect other tissues of interest (bone marrow, spleen, and lymph nodes). Homogenize the tissues into single cell suspensions and stain them for myeloid (CD115, F4 / 80, Ly6C, CD11b, CD45, Ly6G, CD11c), lymphoid (CD45, CD11b, CD3, CD19, CD4, CD8a), and progenitor (CD117, CD34, Ly-6A / E, CD135, CD16 / 32, CD48, CD41, CD150, CD3, CD11b, CD45R / B220, Ly-76, Ly6G, Ly6C) cell markers, and analyze them with a Cytoflex (Beckman Coulter Inc.). Flow cytometry data is analyzed using FlowJo software (BD).

[0290] Results Flow cytometry analysis of mouse splenocytes incubated ex vivo with apoA1 aNP, VHHCD8-apoA1 aNP, and VHHGFP-apoA1 aNP containing mRNA encoding the fluorescent mCherry protein is shown in Fig. 12 (left panel: CD3+CD4-mCherry+ T cells, right panel: CD11b+mCherry+ myeloid cells). The results show that VHHCD8-apoA1 aNP induces functional mCherry reporter gene expression in significantly more CD3+CD4- T cells when compared to apoA1 aNP, VHHGFP-apoA1 aNP, and the Lipofectamine control. ApoA1 aNP induces functional mCherry reporter gene expression in significantly more CD11b+ myeloid cells when compared to VHHCD8-apoA1 aNP, VHHGFP-apoA1 aNP, and the Lipofectamine control. These results indicate that apoA1 aNP has a natural tendency to interact with myeloid cells and that these aNPs can be rerouted to other immune cells (such as CD8+ T cells) by incorporating fusion proteins of apolipoproteins and targeting moieties.

Claims

1. A modified apolipoprotein comprising an apolipoprotein, an apolipoprotein derivative, an apolipoprotein pseudo-molecule, or an apolipoprotein pseudo-molecule derivative bound to a targeting molecule, wherein the targeting molecule is a molecule capable of binding to molecules on the cell surface of a target cell.

2. The aforementioned targeting body, - Antibodies or antigen-binding fragments of antibodies, - Protein ligand, protein-binding domain, or protein-binding fragment thereof - Peptides, - Peptide pseudobody, or - Glycopolymer A modified apolipoprotein according to claim 1, selected from the above.

3. The modified apolipoprotein according to claim 2, wherein the antibody or its antigen-binding fragment is selected from Fab, Fab2, scFv, scFv-Fc, dAb-Fc, free light chain antibody, half antibody, bispecific Fab2, Fab3, triplicate Fab3, diabody, bispecific diabody, triabody, triplicate triabody, minibody, IgG, IgGNAR, monovalent IgG, VhH, nanobody, or VNAR, or antigen-binding fragments thereof, preferably camelid or shark VhH or derivatives thereof or antigen-binding fragments thereof.

4. The targeting organism binds to proteins on the surface of lymphocytes, bone marrow cells, tumor cells, endothelial cells, hematopoietic stem cell progenitor cells (HSPCs), hematopoietic stem cells (HSCs), pluripotent progenitor cells (MPPs), and common myeloid progenitor cells (CMPs), or the targeting organism binds to bacterial, viral, fungal, or parasitic proteins or antigens. Preferably, the modified apolipoprotein according to claim 1, wherein the lymphocytes or bone marrow cells are selected from monocytes, macrophages, M1-like macrophages, M2-like macrophages, eosinophils, basophils, mast cells, NK cells, B cells, plasma cells, regulatory T cells, hematopoietic stem cells, granulocytes, helper T cells such as Th1, Th2, Th17 or Th22, plasmacytoid dendritic cells, dendritic cells such as conventional DC1 or conventional DC2, or tumor-associated macrophages.

5. The targeting bodies are CD1a, CD1b, CD1c, CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD15u, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD 40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RO, CD45RA, C D45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, C D51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, C D66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79α, CD79β, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, C D111, CD112, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, C D121a, CDw121b, CD122, CD123, CD124, CD125, CD126, CD127, CDw128, CD129, CD130, CDw131, CD132, CD133, CD134, CD135, CDw136, CDw137, CD138, CD139 , CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148,CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CD157、CD158、CD158a、CD158b、CD159a、CD160、CD161、CD162、CD162R、CD163、CD164、CD165、CD166、CD167a、CD168、CD169、CD170、CD171、CD172a、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD183、CD184、CD195、CDw197、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207(CLEC4K)、CD208、CD209(CLEC4L)、CDw210、CD212、CD213a1、CD213a2、CDw217、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD236R、CD238、CD239、CD240CE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CXCR1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6、CXCR7、CCR1、CCR2、CCR3、CCR4、CCR5、CCR7、CCR8、CCR9、CLEC1A、CLEC1B、CLEC2A、CLEC2B、CLEC3A、CLEC3B、CLEC4A、CLEC4C(CD303)、CLEC4D、CLEC4J CLEC4E、CLEC4F、CLEC4G、ASGR1(CLEC4H1)、ASGR2(CLEC4H2)、FCER2()、CLEC4M、CLEC5A、CLEC6A、CLEC7A、OLR1(CLEC8A)、CLEC9A、CLEC10A、CLEC11A、CLEC12A、CLEC12B、CD302(CLEC13A)、LY75(CLEC13B)、PLA2R、 １ (CLEC13C), MRC1 (CLEC13D), MRC2 (CLEC13E), CLEC14A, CLEC16A, CLEC17A, KLRA1, KLRB1 (CLEC5B), KLRC1, KLRC2, KLRC3, KLRC4, KLRD1, KLRF1 (CLEC5C), KLRG1 (CLEC1 5A), KLRG2 (CLEC15B), KLRK1, AGC1, ATRNL1, BCAN, CD248, CHODL, CL-K1-Ia, CL-K1 -Ib, CL-K1-Ic, CLECSF5, COLEC10, COLEC11, COLEC12, CSPG3, FCER2, FREM1, HBXBP, A modified apolipoprotein according to claim 1, selected from LAYN, LOC348174, LOC728276, MAFA, MBL2, MGC34761, MICL, MRC1L1, PAP, PKD1, PKD1L2, PRG2, PRG3, REG1A, REG1B, REG3A, REG3G, REG4, SELE, SELL, SELP, SFTPA1, SFTPA2, SFTPA2B, SFTPD, SRCL, THBD, VCAN, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelial tumor antigen (ETA), tyrosinase, and melanoma-associated antigen (MAGE).

6. The apolipoprotein, apolipoprotein derivative, apolipoprotein pseudobody, or apolipoprotein pseudobody derivative is selected from apoA1, apoA-1 Milano, apoA2, apoA4, apoA5, ​​apoB, apoB48, apoB100, apoC-I, apoC-VO, apoC-IV, apoC-IV, apoD, apoE, apoF, apoH, apoL, apoL1, apoL2, apoL3, apoL4, apoL5, apoL6, apoLD1, apoO, apoOL, and apoM, or combinations thereof, or pseudobody or derivative thereof. Preferably, selected from apoA1, apoA2, apoA4, apoA5, ​​apoB48, apoB100, apoC-I, apoC-VO, apoC-IVO, apoC-IV, apoD, apoE, apoF, apoH, apoL, and apoM or their pseudo-forms or derivatives, More preferably, selected from apoA1, apoA2, apoA4, apoA5, ​​apoB100, apoC-I, apoC-II, apoC-III, apoC-IV and apoE or their pseudo-forms or derivatives, More preferably, apoA1, apoA4, apoA5, ​​apoB100, apoC-III and apoE or their pseudo-forms or derivatives are selected. Most preferably, the modified apolipoprotein according to claim 1, selected from apoA1, apoB100, and apoE or their pseudo-forms or derivatives.

7. A lipid nanoparticle comprising an outer layer and a core, wherein the outer layer is - Phospholipids, - Sterols, and - Modified apolipoprotein according to claim 1 Lipid nanoparticles comprising, wherein the core comprises at least one core component selected from lipids, cationic lipids, or polyvalent molecules.

8. The lipid nanoparticle according to claim 7, wherein the nanoparticles further comprise a payload, preferably the payload is contained in the core, the phospholipid layer, or the payload is bound to a component of the outer layer.

9. The lipid nanoparticle according to claim 8, wherein the payload is selected from nucleic acids or nucleic acid analogs, therapeutic agents, biologics, cytokines, chemokines, hormones, growth factors, or combinations thereof.

10. The lipid nanoparticles according to claim 9, wherein the lipid nanoparticles comprise nucleic acids and cationic or ionizable cationic lipids.

11. The lipid nanoparticle according to claim 10, wherein the nucleic acid and the cationic or ionizable cationic lipid are contained in the core, and the modified apolipoprotein and the phospholipid are contained in the outer layer.

12. A lipid nanoparticle comprising an outer layer and a core, wherein the outer layer is - Phospholipids, - Sterols, and - Modified apolipoprotein according to claim 1 Lipid nanoparticles comprising a core comprising nucleic acids and cationic or ionizable cationic lipids.

13. The lipid nanoparticle according to claim 10, wherein the nanoparticle core further comprises a filler, preferably selected from triacylglycerides and cholesterol acyl esters, or a combination thereof, for example, the triacylglyceride being tricaprylin and / or the cholesterol acyl ester being cholesteryl caprylate and / or cholesteryl oleate.

14. The lipid nanoparticle according to claim 9, wherein the nucleic acid is RNA, DNA, or a nucleic acid analog.

15. The lipid nanoparticle according to claim 14, wherein the RNA is a microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), nuclear small RNA (snRNA), nucleolar small RNA (snoRNA), transfer RNA (tRNA), tRNA-derived small RNA (tsRNA), small regulatory RNA (srRNA), messenger RNA (mRNA), modified mRNA, ribosomal RNA (rRNA), long non-coding RNA (lncRNA), or guide RNA (gRNA), or a combination thereof and / or a modification thereof.

16. The lipid nanoparticle according to claim 14, wherein the DNA is single-stranded or double-stranded DNA.

17. The lipid nanoparticle according to claim 9, wherein the nucleic acid is an antisense oligonucleotide, and the antisense oligonucleotide is a single-stranded DNA or RNA comprising a nucleotide or nucleoside analog containing a phosphodiester backbone or 2'-ribose modification.

18. The lipid nanoparticle according to claim 17, wherein the nucleotide or nucleoside analog is selected from loc nucleic acid (LNA), cross-linked nucleic acid (BNA), morpholino or peptide nucleic acid (PNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), hexitol nucleic acid (HNA), or mixtures or combinations thereof.

19. The lipid nanoparticle according to claim 10, wherein the cationic or ionizable cationic lipid is selected from ionizable cationic esters of long-chain alcohols, ionizable cationic esters of diglycerides, ionizable cationic esters of sterols, or a combination thereof.

20. The ionizable cationic lipid is of formula (I), (II), (III), (IV), or (V) 【Chemistry 1】 (In the formula, ICG is, 【Chemistry 2】 The dashed lines indicate the bonding points to the compound of formula (I), (II), (III), (IV), or (V). p is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11. R １ Each of these is independently selected from the group consisting of linear or branched C1-C19 alkyl, linear or branched C1-C19 alkenyl, aryl, arylene-alkyl, and alkylene-aryl groups, and the alkyl or alkenyl group optionally contains up to five heteroatoms independently selected from O and N. R ２ These are hydrogen, methyl, ethyl and -CH ２ -O-C(O)-R １ａ Selected from the group consisting of, R ３ This is selected from the group consisting of hydrogen, aryl, arylene-alkyl, alkylene-aryl, and linear C1-C6 alkyl groups. R １ａ This is selected from the group consisting of linear or branched C1-C19 alkyl, linear or branched C1-C19 alkenyl, aryl, arylene-alkyl, and alkylene-aryl groups, and the alkyl or alkenyl group optionally contains up to five heteroatoms independently selected from O and N. R ｘ are each independently selected from the group consisting of methyl, ethyl, propyl and -CH ２ -CH ２ -OH, R ｙ Each group is independently selected from the group consisting of hydrogen, linear or branched C1-C18 alkyl, aryl, arylene-alkyl, or alkylene-aryl groups, and the alkyl group optionally contains up to five heteroatoms independently selected from O and N. Lipid nanoparticles according to claim 10, which are molecules comprising any one of the above, or rotational isomers, tautomers, stereoisomers or positional isomers thereof.

21. The amount of the modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) is in the range of 0.08 to 2.0 mol%, for example, 0.10 to 2.0 mol%, and / or The amount of phospholipids is in the range of 5 to 90 mol%, for example, 15 to 90 mol%, and / or The amount of sterols is in the range of 2.5 to 65 mol%, for example, 2.5 to 50 mol%, and / or The amount of cationic or ionizable cationic lipids is in the range of 5.0 to 80 mol%, for example, 8.0 to 80 mol%, The lipid nanoparticles according to claim 10, wherein the mole percentage is based solely on the combined amount of the modified apolipoprotein, phospholipid, sterol and cationic or ionizable cationic lipid in the lipid nanoparticles.

22. The amount of the modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) is in the range of 0.1 to 90% by weight. The amount of nucleic acid is in the range of 0.01 to 90% by weight. The amount of phospholipids is in the range of 0.1 to 95% by weight. The amount of sterols is in the range of 0.1 to 95% by weight, and / or The amount of cationic and / or ionizable cationic lipids is in the range of 0.1 to 95% by weight. Lipid nanoparticles according to claim 10, wherein these weight percentages are based on the combined amounts of the modified apolipoprotein, the nucleic acid, the phospholipid, the sterol, and the cationic and / or ionizable cationic lipid.

23. The lipid nanoparticles according to claim 10, wherein the ratio of the modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) to the phospholipid, based on molar weight percentage, is 1:25 to 1:400, more preferably 1:50 to 1:200, and even more preferably 1:75 to 1:

150.

24. The lipid nanoparticles according to claim 10, wherein the weight-based ratio of the modified apolipoprotein (cumulative in the case of two or more modified apolipoproteins) to the phospholipid is 2:1 to 1:10, more preferably 1:1 to 1:5, and even more preferably 1:1.5 to 1:

4.

25. A method for producing lipid nanoparticles according to claim 7, a1) The step of expressing and isolating the modified apolipoprotein described in claim 1 to obtain an isolated modified apolipoprotein, and / or a2) A step of chemically conjugating the targeting compound to an apolipoprotein or apolipoprotein pseudo-compound to obtain a modified apolipoprotein, and then isolating the modified apolipoprotein. b) A step to obtain lipid nanoparticles by combining the isolated modified apolipoprotein obtained in step a1 and / or step a2 with phospholipids, sterols and optionally lipids. A method that includes this.

26. Step b) is, b1) A step of producing nanoparticles by mixing a lipid component in an organic solvent with nucleic acid in an aqueous buffer, preferably rapidly, wherein the lipid component comprises phospholipids, sterols, core components, and optionally lipids, and the aqueous buffer has a pH of 5.5 or less, preferably 5.0 or less. b2) A step to produce the nanoparticles by mixing the lipid nanoparticles with the modified apolipoprotein at a pH of 5.5 to 9.0, preferably at a pH of 6.0 to 8.0, more preferably at a pH of 6.5 to 8.0, preferably rapidly. The method according to claim 25, including the following.

27. A method for producing lipid nanoparticles, a) A step of rapidly mixing a lipid component in an organic solvent with nucleic acid in an aqueous buffer to produce lipid nanoparticles, wherein the lipid component comprises phospholipids, sterols, cationic lipids, or ionizable cationic lipids, and the aqueous buffer has a pH of 5.0 or less. b) A step of producing lipid nanoparticles by rapidly mixing the lipid nanoparticles with one or more modified apolipoproteins according to claim 1 at a pH of 5.5 to 8.0, preferably 6.0 to 8.

0. A method that includes [this].

28. A modified apolipoprotein according to claim 1, for use as a pharmaceutical.

29. Lipid nanoparticles according to claim 7 for use as a pharmaceutical.

30. The modified apolipoprotein according to claim 1, for use in the treatment or prevention of immune-related disorders such as transplant rejection, graft-versus-host disease (GVH), atherosclerosis, infection, inflammation, autoimmunity, allergy, cancer, genetic disorders, metabolic disorders, neurological disorders, or tissue trauma.

31. Lipid nanoparticles according to claim 7 for use in treating or preventing immune-related disorders such as transplant rejection, graft-versus-host disease (GVH), atherosclerosis, infection, inflammation, autoimmunity, allergy, cancer, genetic disorders, metabolic disorders, neurological disorders, or tissue trauma.

32. The use of the modified apolipoprotein according to claim 1 in vitro or ex vivo for delivering a compound to a target, wherein the target is preferably a cell, tissue, and / or organ, and more preferably the target is a lymphocyte, myeloid cell, tumor cell, endothelial cell, hematopoietic stem cell progenitor cell (HSPC), hematopoietic stem cell (HSC), pluripotent progenitor cell (MPP), common myeloid progenitor cell (CMP), or the targeting body is bound to a bacterial, viral, fungal, or parasitic protein or antigen. Preferably, the lymphocytes or bone marrow cells used are selected from monocytes, macrophages, M1-like macrophages, M2-like macrophages, eosinophils, basophils, mast cells, NK cells, B cells, plasma cells, regulatory T cells, hematopoietic stem cells, helper T cells such as Th1, Th2, Th17, or Th22, plasmacytoid dendritic cells, dendritic cells such as conventional DC1 or conventional DC2, or tumor-associated macrophages.

33. In vitro or ex vivo use of the lipid nanoparticles according to claim 7 for delivering a compound to a target, wherein the target is preferably a cell, tissue, and / or organ, and more preferably the target is a lymphocyte, bone marrow cell, tumor cell, endothelial cell, hematopoietic stem cell progenitor cell (HSPC), hematopoietic stem cell (HSC), pluripotent progenitor cell (MPP), common myeloid progenitor cell (CMP), or the targeting body is bound to a bacterial, viral, fungal, or parasitic protein or antigen. Preferably, the lymphocytes or bone marrow cells used are selected from monocytes, macrophages, M1-like macrophages, M2-like macrophages, eosinophils, basophils, mast cells, NK cells, B cells, plasma cells, regulatory T cells, hematopoietic stem cells, helper T cells such as Th1, Th2, Th17, or Th22, plasmacytoid dendritic cells, dendritic cells such as conventional DC1 or conventional DC2, or tumor-associated macrophages.

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