Nucleic Acid Containing Nanoparticles

Abstract

Disclosed herein are nanoparticles comprising phospholipids, apolipoproteins and / or apolipoprotein mimetics, sterols, cationic lipids or ionizable cationic lipids and nucleic acids, as well as compositions comprising such nanoparticles and methods for preparing such nanoparticles. The nanoparticles can be used as pharmaceuticals, such as in the treatment of diseases by stimulating or inhibiting the innate immune response.

Description

[Technical field]

[0001] The present invention relates to the field of nucleic acid therapeutics and provides novel and innovative nanoparticles for intracellular delivery of nucleic acids at target sites.The present invention further relates to therapeutic methods using the nanoparticles, for example in the treatment of diseases by stimulating or inhibiting the innate immune response.The present invention further relates to in vivo, in vitro or ex vivo methods for introducing nucleic acids into cells using nanoparticles. [Background technology]

[0002] Nucleic acid therapeutics (NATs), such as small antisense oligonucleotides (ASOs), small interfering RNA (siRNA), messenger RNA (mRNA) and other types, are an innovative new class of drugs that have the potential to regulate gene expression. In recent years, several nucleic acid-based formulations have been approved for in vivo applications, including ASOs, N-acetylgalactosamine (GalNAc)-siRNA conjugates, lipid nanoparticles (LNPs) containing siRNA or mRNA, and several viral vectors containing plasmid DNA (pDNA). In addition, there are several NATs in late-stage clinical trials. Furthermore, several genetically engineered ex vivo cell therapeutic formulations have been approved.

[0003] The therapeutic application of nucleic acids after parenteral administration is difficult. Different types of nucleic acids have different sizes and physicochemical properties, but the common features are that they are large in size, polymeric, and negatively charged. As a result, upon systemic administration, nucleic acids are rapidly removed from the circulation by renal filtration and nuclease degradation. In addition, NAT acts intracellularly, but cannot easily cross cell membranes. Finally, administration of exogenous nucleic acids induces an immune response. Although this can be advantageous (e.g., for vaccine development), it usually contributes to the rapid clearance and adverse effects of nucleic acids.

[0004] To overcome these challenges, all nucleic acid therapeutics rely on chemical modifications and / or nanotechnology-based delivery systems. All approved NATs rely on chemical modifications and / or nanotechnology platforms to facilitate intracellular delivery and subsequently induce a therapeutic effect after parenteral administration. 1) ASOs are extensively chemically modified to increase stability, reduce immune stimulatory effects, and increase efficacy. ASOs are administered subcutaneously to target liver cells or intrathecally to target cells of the central nervous system. 2) GalNAc-siRNA conjugates are modified similarly to ASOs and administered subcutaneously, with the GalNAc moiety ensuring asialoglycoprotein receptor-mediated uptake in hepatocytes. 3) Lipid nanoparticles (LNPs) are approximately 50-100 nm in diameter and can be administered systemically, intradermally or intramuscularly. After systemic administration, LNPs efficiently accumulate in hepatocytes, providing an opportunity for gene silencing (siRNA) or protein production (mRNA). After intradermal or intramuscular administration, LNPs are taken up by immune cells, e.g., antigen-presenting cells, which can be exploited for vaccine purposes. LNPs are the current gold standard for mRNA therapeutics and are likely to also become the standard delivery platform for in vivo gene editing applications. LNPs contain synthetic polyethylene glycol (PEG)-conjugated lipids, which have been associated with hypersensitivity reactions and / or anaphylaxis. 4) Viral delivery systems, such as adenovirus, lentivirus or adeno-associated virus (AAV) vectors, are effective vehicles for delivering DNA. Viral vectors are characterized by their limited payload capacity and immunogenicity. However, in immune-privileged tissues such as the eye, viral vectors constitute the current gold standard for NAT. Viral vectors are widely used for ex vivo therapeutics (e.g., CAR T) or for vaccine purposes, administered intravenously to target cells in the liver, intravitreal / subretinal to target cells in the retina, or intramuscularly.

[0005] Except for viral vector or LNP-mRNA-based vaccines, the majority of approved nucleic acid therapeutics are developed for indications other than immunotherapy. Therefore, it remains difficult to deliver therapeutic nucleic acids to bone marrow compartments. Moreover, chemical modification or viral delivery of nucleic acid molecules inherently carries the risk of undesired activation of the immune system, resulting in degradation or clearance of NAT.

[0006] For example, nanoparticles carrying nucleic acid are described in, for example, WO2009 / 127060, which describes the use of cationic lipids in combination with non-cationic lipids and nucleic acid.The cationic lipids neutralize nucleic acid, allowing the formation of nanoparticles that can be used for the non-targeted delivery of nucleic acid in subjects.The disadvantage of these nanoparticles is that they cannot target bone marrow compartments.

[0007] Other systems, such as WO 2019 / 103998, describe nanobiological preparations capable of targeting the bone marrow compartment, which contain phospholipids and ApoA1 and small molecule drugs. A drawback of these nanobiological preparations is that due to their hydrophobic core, they do not allow the uptake of polar structures, such as nucleic acids, such as DNA and RNA.

[0008] Thus, there is a need for improved delivery systems for therapeutic nucleic acids to the bone marrow compartment. Summary of the Invention

[0009] The present inventors are the first to develop nanoparticles that allow delivery of nucleic acid cargo to the bone marrow compartment. More specifically, the present inventors have developed stable lipid-based nano-sized formulations (approximately 10-200 nm in diameter) that contain apolipoproteins and / or apolipoprotein mimetics, phospholipids, sterols, cationic or ionizable cationic lipids, and nucleic acids, such as siRNA or mRNA. Without wishing to be bound by any theory, the present inventors believe that the core of the nanoparticles comprises an assembly of nucleic acids that interact with (ionizable) cationic lipids, which are packaged and embedded within an outer protective surface or lipid shell that comprises apolipoproteins and / or apolipoprotein mimetics, phospholipids, and sterols, which act as a surface barrier.

[0010] Nucleic acids are suitably and stably incorporated into the nanoparticles of the invention without the need for synthetic (non-natural) hydrophilic polymers or (lipid) conjugates of polymers such as polyethylene-glycol (PEG).

[0011] Moreover, the nanoparticles of the present invention also do not aggregate and / or fuse uncontrollably, even in the absence of such synthetic (non-natural) hydrophilic polymers or (lipid) conjugates of such polymers.

[0012] In addition, the nanoparticles of the present invention have targeting capabilities to myeloid cells and other cells associated with the immune system as a result of the presence of apolipoproteins and / or apolipoprotein mimetics on the outer surface of the nanoparticles.

[0013] Moreover, the nanoparticles taught herein are stable, have low or non-toxic toxicity, and have high nucleic acid retention and activity.

[0014] The present inventors have further developed a controlled formulation process for successfully incorporating nucleic acids into apolipoprotein and / or apolipoprotein mimetic-based nanoparticles.

[0015] Thus, a first aspect of the present invention provides a nanoparticle comprising a core surrounded by a surface layer, the core comprises a nucleic acid and a cationic or ionizable cationic lipid; The surface layer is Phospholipids, Sterols, and Apolipoproteins or apolipoprotein mimetics or combinations thereof Includes.

[0016] The present invention further relates to a composition comprising the nanoparticles according to the invention and a physiologically acceptable carrier. The present invention further relates to the nanoparticles or the composition according to the invention for use as a medicament.

[0017] The present invention further relates to the nanoparticles or the composition according to the invention for use in the treatment of a disease by stimulating or inhibiting the innate immune response.

[0018] The present invention further provides a method for preparing nanoparticles, comprising the steps of: a) rapidly mixing lipid components in an organic solvent with nucleic acid in an aqueous buffer to prepare lipid nanoparticles, the lipid components comprising phospholipids, sterols, cationic lipids or ionizable cationic lipids, and optionally a bulking material, preferably a triglyceride, at a pH of 5.0 or less; b) rapidly mixing the lipid nanoparticles prepared under (a) with an apolipoprotein, an apolipoprotein mimetic, or a combination thereof to prepare nanoparticles at a pH of 6.0 to 8.0; The present invention relates to a method comprising the steps of:

[0019] The present invention further relates to an in vitro or ex vivo method for introducing a nucleic acid into a cell, comprising contacting the cell with the nanoparticles or the composition according to the invention.

[0020] The present invention further relates to nanoparticles according to the invention obtainable or obtainable by the process according to the invention.

[0021] The present invention further relates to an in vivo method for introducing a nucleic acid into a cell, comprising contacting the cell with a nanoparticle or a composition according to the invention.

[0022] The present invention further relates to a nanoparticle or a composition according to the invention for use in the in vivo delivery of a nucleic acid to a subject.

[0023] The present invention further relates to a method for in vivo delivery of a nucleic acid, comprising administering to a subject a nanoparticle or a composition according to the invention.

[0024] The present invention further relates to a method for treating a disease or disorder in a subject in need of treatment by stimulating or inhibiting the innate immune response, comprising administering to the subject a therapeutically effective amount of the nanoparticles or a composition according to the invention. [Brief description of the drawings]

[0025] [Figure 1] Schematic diagram of apolipoprotein lipid nanoparticle (aNP) platform technology according to certain embodiments of the present invention for nucleic acid delivery. Without wishing to be bound by any theory, such RNA-aNPs are believed to be composed of a hydrophobic core containing a nucleic acid such as RNA complexed with optional filler materials (e.g. triglycerides) and (ionizable) cationic lipids. The hydrophobic core is encapsulated and shielded by a surface layer or barrier, possibly a monolayer, containing phospholipids and sterols. The surface of the lipid nanoparticle also contains apolipoproteins for structural integrity to prevent aggregation, provide particle stability, provide natural stealth, and / or promote interaction with immune cells.

[0026] [Diagram 2]FIG. 1 is a schematic diagram of an exemplary method according to certain embodiments of the invention for preparing apolipoprotein lipid nanoparticles (aNPs) containing nucleic acids, such as RNA, as described herein.

[0027] [Diagram 3] Figure 1 shows the instability of siRNA retention in apolipoprotein nanoparticles (aNPs) according to certain embodiments of the present invention and comparative nanoparticles (NPs) without apolipoprotein. (A) Representative siRNA-containing aNPs (siRNA-aNPs) 18 and 34 were prepared according to the preparation procedure shown in Figure 2 (white bars). Additionally, comparative NPs were prepared by omitting the second step of the procedure where apolipoprotein A1 is incorporated into the formulation (black bars). RNA retention was determined using Ribogreen assay one day after formulation. (B) Representative image of comparative siRNA-NP formulation 18 without apolipoprotein A1 incorporated. (C) Representative cryo-transmission electron micrograph of comparative siRNA-NP formulation 18 (scale bar 50 nm).

[0028] [Figure 4]The lipid composition of siRNA-containing apolipoprotein lipid nanoparticles (aNPs) (siRNA-aNPs) according to certain embodiments of the invention influences their physicochemical properties and can be optimized to obtain siRNA-aNPs with optimal properties. (A) One day after formulation, the physicochemical properties of individual siRNA-aNP formulations of the library were determined according to (i) particle size (z-average) and (ii) particle size dispersity as assessed using dynamic light scattering (DLS), (iii) siRNA retention using the Ribogreen assay, (iv) apolipoprotein A1 (apo-A1) using a colorimetric protein quantification assay, and (v) cholesterol and (vi) phospholipid recovery using standard colorimetric assays. Data are shown for both formulation types using either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). (B) Analysis of individual siRNA-aNP formulations of the library by (i) particle size (number average) and (ii) particle size dispersity using dynamic light scattering (DLS) one day after formulation as indicated by the triglyceride content of the formulation. (C) Analysis of individual siRNA-aNP formulations of the library by (i) particle size (number average) and (ii) particle size dispersity using dynamic light scattering (DLS) one day after formulation as indicated by the N / P ratio of the formulation. The N / P ratio is the ratio used of positively chargeable amine (N=nitrogen) groups of the ionizable cationic material to negatively charged nucleic acid phosphate (P) groups.

[0029] [Diagram 5] Representative cryo-transmission electron micrographs showing that the lipid composition of siRNA-containing apolipoprotein lipid nanoparticles (aNPs) (siRNA-aNPs) according to certain embodiments of the present invention can be used to influence the morphology and size of these aNPs. All individual siRNA-aNP formulations of the library were subjected to cryo-transmission electron microscopy using an FEI TITAN 300 kV to determine particle size, morphology and formulation uniformity (scale bar 50 nm).

[0030] [Figure 6] Apolipoprotein lipid nanoparticles (aNPs) according to certain embodiments of the invention containing firefly luciferase siRNA (siRNA-aNPs) induce robust reporter gene expression knockdown in vitro. (A) Mouse RAW264.7 macrophages transfected with pmirGLO plasmid (Promega) for stable dual reporter luciferase expression (firefly and Renilla luciferase) were exposed for 48 hours to individual siRNA-aNP formulations of a library containing firefly luciferase (Fluc) siRNA. Luminescence assays were performed according to the manufacturer's protocol (Dual-Glo Luciferase Assay System, Promega). Data are corrected for control siRNA-aNP formulations containing non-specific siRNA. (B) Firefly luciferase expression knockdown data shown according to phospholipid type and triglyceride content of individual siRNA-aNP formulations of the library. (C) Firefly luciferase expression knockdown data shown according to phospholipid type and N / P ratio of individual siRNA-aNP formulations of the library. The N / P ratio is the ratio used of the positively chargeable amine (N=nitrogen) groups of the ionizable cationic material to the negatively charged nucleic acid phosphate (P) groups.

[0031] [Figure 7]Apolipoprotein lipid nanoparticles (aNPs) according to certain embodiments of the invention containing radiolabeled siRNA (siRNA-aNPs) localize to hematopoietic tissues including spleen and bone marrow after intravenous administration in mice. (A) Biodistribution of siRNA-aNPs after intravenous administration in mice. C57BL / 6 mice (n=6 / formulation) were intravenously injected with the siRNA-aNP formulations of the invention or comparative LNP formulations containing Zirconium-89 radiolabeled non-specific siRNA at a dose of 2 mg / kg siRNA. 24 hours after injection, mice were sacrificed and organs were collected for quantitative analysis by gamma counting. Data are presented as mean ± SD of % injected dose per gram of tissue (%ID / g) and analyzed by two-way ANOVA with Tukey's post-hoc test. * indicates p-value <0.05 and **** indicates p-value <0.0001. (B) Biodistribution results are shown as bone marrow vs. liver ratio of % injected dose per gram of tissue (%ID / g). #LNP-siRNA Comparative Example is composed of Dlin-MC3-DMA, DSPC, cholesterol and PEG-DMG (50:38.5:10:1.5 mol %) containing siRNA.

[0032] [Figure 8A]Apolipoprotein lipid nanoparticles (aNPs) according to certain embodiments of the invention can encapsulate mRNA to obtain stable formulations and induce gene expression in vitro. (A) Firefly luciferase messenger RNA (mRNA)-containing aNP formulations were prepared using the method described in FIG. 2. mRNA-aNP formulations according to certain embodiments of the invention and LNP-mRNA comparative formulation # were characterized for their particle size and particle size dispersity using dynamic light scattering (DLS). Ribogreen assay was used to evaluate mRNA capture efficiency. (B) Representative mRNA-aNP cryo-transmission electron micrographs (scale bar 50 nm). (C) Human HEK293 cells were exposed to firefly mRNA-containing aNPs and comparative LNPs for 24 hours. Reporter gene expression (left) was determined by luminescence and cell viability (right) was determined by MTT assay, showing that mRNA-aNPs induce dose-dependent firefly luciferase expression without inducing toxicity in vitro. (D) Mouse RAW264.7 macrophages were exposed to firefly mRNA-containing aNPs for 24 hours. Gene expression was determined by luminescence, showing that mRNA-aNPs induce dose-dependent firefly luciferase expression in macrophage cell cultures. (E) Primary mouse bone marrow-derived macrophages were exposed to firefly mRNA-containing aNPs for 24 hours. Gene expression was determined by luminescence, showing that mRNA-aNPs induce dose-dependent firefly luciferase expression in primary cells. #LNP-mRNA comparative example is composed of Dlin-MC3-DMA, DSPC, cholesterol and PEG-DMG (50:38.5:10:1.5 mol%) with mRNA. [Figure 8B] Same as explanation for Figure 8A.

[0033] [Figure 9] Molecular structures of monovalent, ionizable cationic materials that can be used to complex RNA (or other nucleic acids) for incorporation into apolipoprotein lipid nanoparticles (aNPs) according to certain embodiments of the present invention. Examples 1-15 referenced in FIG. 9 are subexamples 1-15 of Example 9.

[0034] [Figure 10] Apolipoprotein lipid nanoparticles (aNPs) containing siRNA (siRNA-aNPs) according to certain embodiments of the invention can be prepared using a variety of ionizable cationic materials to obtain stable formulations. siRNA-aNP formulations containing phospholipids, cholesterol, ionizable cationic material as shown in FIG. 9 (ionizable cationic lipids 5, 16, 17, and 19 are molecules of Examples 10, 13, 9, and 8, respectively, as shown in FIG. 9), triglyceride, apolipoprotein A1, and siRNA were prepared using the procedure described in FIG. 2. One day after formulation, individual siRNA-aNP formulations of the library and LNP-siRNA comparative formulation # were analyzed for (A) particle size and (B) particle size dispersity using dynamic light scattering (DLS), and (C) siRNA retention using Ribogreen assay. #LNP-siRNA comparative formulation is composed of Dlin-MC3-DMA, DSPC, cholesterol, and PEG-DMG (50:38.5:10:1.5 mol %) with siRNA.

[0035] [Figure 11A] Table 1: Exemplary formulations of a library of 72 siRNA aNP formulations. [Figure 11B] Table 1: Exemplary formulations of a library of 72 siRNA aNP formulations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0037] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," "containing," or "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. This term also encompasses "constituted of," "consists in," "consisting of," and "consists of," as well as the terms "consisting essentially of," "consisting essentially in," and "consists essentially of," which enjoy well-established meanings in patent language.

[0038] The recitation of numerical ranges by endpoints includes all integers and, where appropriate, fractions subsumed within each range, as well as the recited endpoints. This applies to numerical ranges whether introduced by the phrase "from" or "between" or another phrase. Any numerical range recited herein is intended to include all subranges subsumed therein.

[0039] The terms "about" or "approximately" as used herein when referring to a measurable value, such as a parameter, amount, duration, etc., are meant to encompass variation of the specified value and variation from the specified value, e.g., variation of no more than + / - 10%, preferably no more than + / - 5%, more preferably no more than + / - 1%, and even more preferably no more than + / - 0.1% of the specified value, insofar as such variations are appropriate for the practice of the disclosed invention. It is to be understood that the values ​​to which the modifier "about" or "approximately" refers are themselves specifically, and preferably disclosed.

[0040] Moreover, the terms first, second, third, etc. in the specification and claims are used to distinguish between similar elements, unless otherwise specified, and are not necessarily intended to describe a sequential or chronological order. The terms so used are interchangeable under appropriate circumstances, with the understanding that the embodiments of the invention described herein may operate in sequences other than those described or illustrated herein.

[0041] The term "one or more" or "at least one", e.g., one or more members or at least one member of a group of members, will itself be clear with further illustration, but the term specifically encompasses reference to any one of the members, or any two or more of the members, e.g., any three or more, four or more, five or more, six or more, seven or more, etc., of the members, and all of the members. In other examples, "one or more" or "at least one" can refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0042] As used herein, the term "and / or," when used in a list of two or more items, means that any one of the listed items can be used alone, or any combination of two or more of the listed items can be used. For example, if a list is stated to include groups A, B, and / or C, the list can include A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0043] The discussion of the background of the invention herein is included to explain the context of the invention and should not be construed as an admission that any of the mentioned material was published, publicly known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0044] Throughout this disclosure, various publications, patents and published patent specifications are referenced by specific citations. All documents cited herein are incorporated by reference in their entirety. In particular, the teachings or sections of such documents that are specifically mentioned herein are incorporated by reference.

[0045] Unless otherwise defined, all terms used in this disclosure, including technical and scientific terms, have the meanings commonly understood by those skilled in the art to which this invention belongs. By way of further guidance, term definitions are included to better understand the teachings of the present invention. When a particular term is defined in relation to a particular aspect of the present invention or a particular embodiment of the present invention, such connotation or meaning is meant to apply throughout this specification, i.e., in the context of other aspects or embodiments of the present invention, unless otherwise defined.

[0046] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined can be combined with any other aspect or embodiment, unless expressly indicated otherwise. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature or features indicated as being preferred or advantageous.

[0047] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may. Furthermore, particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, some embodiments described herein include some features included in other embodiments but not other features, but it is meant that combinations of features of different embodiments form different embodiments within the scope of the invention and as would be understood by one of ordinary skill in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0048] Similarly, in describing exemplary embodiments of the invention, it will be understood that various features of the invention may be grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects.

[0049] The term "in vitro" is well understood in the art and can specifically refer to experiments or measurements performed using biological components that have been isolated from their natural state.

[0050] As used herein, the term "ex vivo" is well understood in the art and can specifically refer to experiments or measurements that are performed in or on the tissue of an organism in an external environment with minimal alteration of natural conditions.

[0051] The terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" are well understood in the art. With further guidance, the term typically refers to a polymer of any length, preferably a linear polymer, essentially composed of nucleoside units. Nucleoside units generally comprise a heterocyclic base and a sugar group. Heterocyclic bases can include, among others, purine and pyrimidine bases, such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), which are widely distributed in naturally occurring nucleic acids, other naturally occurring bases (e.g., xanthine, inosine, hypoxanthine), and chemically or biochemically modified (e.g., methylated), non-natural or derivatized bases. Exemplary modified nucleobases include, but are not limited to, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.In particular, 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability. Sugar groups can include, inter alia, pentose (pentofuranose) groups, such as ribose and / or 2-deoxyribose, preferably those common in naturally occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups (such as, but not limited to, 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-alkylene linked, e.g., 2'-O,4'-C-methylene linked or 2'-O,4'-C-ethylene linked sugars, e.g., ribose; 2'-fluoro-arabinose, etc.).The nucleoside units may be linked together 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 phosphorothioate, alkyl phosphorothioates, e.g. methyl phosphorothioate, phosphorodithioate, alkyl phosphonates, e.g. methyl phosphonates, alkyl phosphonothioates, phosphorotriesters, e.g. alkyl phosphotriesters, phosphoramidates, phosphoropiperazidates, phosphoromorpholidates, bridged phosphoramidates, bridged methylene phosphonates, bridged phosphorothioates; and further, siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate, e.g. 3'-N-carbamate, morpholino, borano, thioether, 3'-thioacetal and sulfone internucleoside linkages. Preferably, the internucleoside linkage may be a phosphate-based linkage, including modified phosphate-based linkages, such as more preferably phosphodiester, phosphorothioate or phosphorodithioate linkages, or combinations thereof. The term "nucleic acid" also includes, but is not limited to, peptide nucleic acid (PNA), peptide nucleic acid with phosphate group (PHONA), locked nucleic acid (LNA), morpholino phosphorodiamidate backbone nucleic acid (PMO), cyclohexene nucleic acid (CeNA), tricyclo-DNA (tcDNA), and any other nucleic acid base containing polymers with backbone moieties having alkyl linkers or amino linkers, such as nucleic acid mimetics (see, for example, Kurreck 2003 (Eur J Biochem 270:1628-1644)). "Alkyl" as used in this context specifically includes lower hydrocarbon moieties, such as C1 to C4 straight or branched chain saturated or unsaturated hydrocarbons, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl.

[0052] 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" further preferably encompasses DNA, RNA and DNA / RNA hybrid molecules, in particular 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. naturally occurring or isolated from nature, recombinant, i.e. produced by recombinant DNA technology, and / or partially or wholly chemically or biochemically synthesized. A "nucleic acid" may be double-stranded, partially double-stranded or single-stranded. If single-stranded, the nucleic acid may be the sense strand or the antisense strand. In addition, the nucleic acid may be circular or linear.

[0053] In certain embodiments, the term may be intended to include DNA molecules and RNA molecules, as well as locked nucleic acids (LNA), bridged nucleic acids (BNA), morpholinos or peptide nucleic acids (PNA). The nucleic acid (molecule) may be any nucleic acid (molecule), e.g., single-stranded or double-stranded.

[0054] The terms "subject" or "individual" or "animal" or "patient" or "mammal" may be used interchangeably and are well understood in the art, and may refer to any subject, particularly a mammalian subject, for which diagnosis, prognosis, or treatment is desired. The term may refer, for example, to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, even more preferably primates, and may specifically include human patients as well as non-human mammals and primates. A preferred patient is a human subject, including both sexes and all age categories thereof. Mammalian subjects include humans, farm animals, livestock, and zoo, sport or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bovines, bears, and the like. As defined herein, a subject may be living or dead. Samples may be taken post-mortem, i.e., from a subject after death, and / or samples may be taken from a living subject. Preferably, the subject is a human.

[0055] The term "treat" or "treatment" is well understood in the art and can include both therapeutic treatment of an already established disease or condition, as well as preventative or prophylactic measures, the purpose of which is to prevent or reduce the likelihood of an undesirable affliction occurring, such as preventing the onset, development and progression of a disease or disorder. Beneficial or desired clinical results can include, but are not limited to, alleviation of one or more symptoms or one or more biological markers, attenuation of the extent of a disease, stabilization (i.e., not worsening) of a disease, delay or slowing of disease progression, improvement or amelioration of a disease state, and the like. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0056] As used herein, the term "nanoparticle" refers specifically to small particles, e.g., in the range of about 10 nm to about 200 nm in diameter, that can be used to deliver a payload to a target, e.g., an organ or cell of a subject.

[0057] As used herein, the term "targeting" when referring to targeting a cell (e.g., a target cell, such as, but not limited to, a myeloid cell) or targeting a tissue or organ should be understood to mean bringing into close proximity to the intended cell, organ or tissue, or concentrating in close proximity to the intended cell, organ or tissue. This means that when targeting an intended cell, organ or tissue, on average, more nanoparticles are in close proximity to the intended cell, organ or tissue than would be expected based on the random or natural distribution of particles. Close proximity, as used herein, means that the nanoparticles are positioned such that they can interact with the cell (or tissue or organ) to deliver their payload (nucleic acid).

[0058] The term "myeloid cells" is well understood in the art and can refer to blood cells derived from a common precursor of megakaryocytes, granulocytes, monocytes, and erythrocytes, among others. Myeloid cells are the major cellular compartment of the immune system, including monocytes, dendritic cells, tissue macrophages, and granulocytes. As used herein, the term myeloid compartment refers to all myeloid cells in an organism.

[0059] The term “alkyl” by itself or as part of another substituent refers to a group of the formula C n H 2n+1 where n is a number equal to or greater than 1. The alkyl groups may be straight or branched chain and may be substituted as set forth herein. Generally, the alkyl groups of the present invention contain 1 to 18 carbon atoms, preferably 1 to 17 carbon atoms, preferably 1 to 15 carbon atoms, preferably 1 to 6 carbon atoms, preferably 1 to 5 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 3 carbon atoms, and even more preferably 1 to 2 carbon atoms. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the specified group may contain. For example, "C" as a group or part of a group 1~6 The term "alkyl" refers to a group of the formula -C, where n is a number ranging from 1 to 6. n H 2n+1Thus, for example, "C 1~6 "Alkyl" includes all straight or branched chain 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" includes all straight or branched chain 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" includes all straight or branched chain alkyl groups having from 1 to 4 carbon atoms, and thus includes methyl, ethyl, n-propyl, i-propyl, butyl and their isomers (e.g., n-butyl, i-butyl and t-butyl). For example, "C 1~3 "Alkyl" includes all straight or branched chain alkyl groups having one to three carbon atoms and thus includes methyl, ethyl, n-propyl, i-propyl.

[0060] When the suffix "ene" is used in conjunction with an alkyl group, i.e., "alkylene," it is intended to mean an alkyl group, as defined herein, having two single bonds as points of attachment to other groups. 1~6 The term "alkylene" by itself or as part of another substituent refers to a C alkyl group that is divalent, i.e., has two single bonds for attachment to two other groups. 1~6It refers to an alkyl group. Alkylene groups may be straight or branched chain and may be substituted as indicated herein. Non-limiting examples of alkylene groups include methylene (-CH-), ethylene (-CH-CH-), methylmethylene (-CH(CH)-), 1-methyl-ethylene (-CH(CH)-CH-), n-propylene (-CH-CH-CH-), 2-methylpropylene (-CH-CH(CH)-CH-), 3-methylpropylene (-CH-CH-CH(CH)-), n-butylene (-CH-CH-CH-CH-), 2-methylbutylene (-CH-CH(CH)-CH-CH-), 4-methylbutylene (-CH-CH-CH-CH(CH)-), pentylene and its chain isomers, and hexylene and its chain isomers.

[0061] The term "alkenyl" as a group or part of a group refers to an unsaturated hydrocarbyl group that may be straight or branched chain containing one or more carbon-carbon double bonds. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the specified group may contain. For example, "C 2~6 The term "alkenyl" refers to an unsaturated hydrocarbyl group that contains one or more carbon-carbon double bonds and may be straight or branched chain containing 2 to 6 carbon atoms. For example, C 2~4 Alkenyl includes all straight or branched chain alkenyl groups having 2 to 4 carbon atoms. 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 and the like.

[0062] The term "aryl" as a group or part of a group refers to polyunsaturated aromatic hydrocarbyl groups having a single ring (i.e. phenyl) or multiple aromatic rings fused together (e.g. naphthyl) or covalently linked, typically containing 6 to 24 carbon atoms, preferably 6 to 12 atoms, with at least one ring being aromatic, preferably 6 to 10 atoms. Examples of suitable aryls include C 6~10Aryl, more preferably C 6~8 Aryl is an example. 6~12 Non-limiting examples of aryl include phenyl; biphenylyl; biphenylenyl; or 1- or 2-naphthalene; 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 conjunction with an aryl group, i.e., arylene, this is intended to mean an aryl group as defined herein having two single bonds as points of attachment to other groups. The appropriate "C 6~12 "Arylene" groups include 1,4-phenylene, 1,2-phenylene, 1,3-phenylene, biphenylylene, naphthylene, indenylene, 1-, 2-, 5-, or 6-tetralinylene, and the like. When at least one carbon atom in an aryl group is replaced with a heteroatom, the resulting ring is referred to herein as a heteroaryl ring. The heteroatom may be selected from the group consisting of O, N, P, and S, and is preferably O or N.

[0063] The term "alkylene-aryl" as a group or part of a group means an alkylene, as defined herein, in which at least one hydrogen atom has been replaced with at least one aryl, as defined herein. The alkylene-aryl group typically contains 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" as a group or part of a group means an arylene, as defined herein, in which at least one hydrogen atom has been replaced with at least one alkyl, as defined herein. The arylene-alkyl group typically contains 7 to 25 carbon atoms.

[0064] Ester, amide, carboxylic acid and alcohol groups are defined below, where Rp represents a hydrogen atom or a cyclic, linear 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 is to be understood as a group according to the formula -C(O)-O-. An amide (functional) group or moiety as shown in this document is to be understood as a group according to the formula -NRp-C(O)-. A carboxylic acid (functional) group or moiety as shown in this document is to be understood as a moiety or group according to the formula -C(O)OH. An alcohol (or hydroxy) functional group or moiety as shown in this document is to be understood as a group according to the formula -OH.

[0065] The present invention constitutes a nanoparticle platform technology suitable for NAT delivery to myeloid cell compartments. The nanoparticles described herein are (phospho)lipid-based nanoparticles stabilized by apolipoproteins and / or apolipoprotein mimetics that protect the NAT payload in circulation by preventing degradation and rapid clearance. At the same time, the nanoparticles reduce the immune-stimulating related adverse effects of NAT by limiting undesirable interactions with components in the blood. In addition, the present invention allows efficient delivery of nucleic acid therapeutics to myeloid cell compartments of lymphoid organs, such as bone marrow and spleen, for effective immunotherapy.

[0066] The nanoparticles described herein are lipid-based nano-sized formulations (approximately 10-200 nm in diameter, e.g., in certain embodiments, approximately 30-200 nm in diameter) having a hydrophobic core and apolipoproteins and / or apolipoprotein mimetics coating the outer surface. Without being bound by theory, the inventors believe that the core of the nanoparticle comprises an assembly of nucleic acids interacting with (ionizable) cationic lipids, which are packaged and embedded within an outer protective surface or lipid shell comprising apolipoproteins and / or apolipoprotein mimetics, phospholipids and sterols that can function as a surface layer or barrier. The apolipoproteins and / or apolipoprotein mimetics can interact with other components of the outer protective surface using hydrophobic and / or charged (ionic) interactions. The outer protective surface can also optionally include some (ionizable) cationic lipids that are not complexed with the nucleic acid component. Figure 1 shows a schematic representation of the impression of the nanoparticles of the present invention. Apolipoproteins are helical proteins that have an inherent affinity for lipid layers due to their amphiphilic nature. There are several classes of apolipoproteins, all of which can be used as structural components in nanoparticle formulations. The incorporation of apolipoproteins affects the physicochemical properties and shelf life of nanoparticles by providing structural stability. Furthermore, the presence of apolipoproteins modulates the biological behavior of nanoparticles. For example, apolipoprotein A1 interacts with cells via the scavenger receptor class B1 (SRB1) and the ATP-binding cassette transporter ABCA1. This increases the interaction of nanoparticles with myeloid cells in lymphoid organs.

[0067] Phospholipids in nanoparticle formulations, due to their amphiphilic nature, accumulate at the interface between the hydrophobic core and the aqueous solvent, effectively forming a lipid monolayer, or surface layer or barrier. For biological uses, single or multiple phospholipid types are used due to their inherent biocompatibility and net neutral charge. Optionally, a mole percentage (about 1-95 mole %; relative to the total amount of phospholipid used) of a charged lipid, such as 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) or 1,2-dioleoyl-sn-glycero-3-phosphate (18PA), can be added to impart specific charge characteristics to the overall formulation.

[0068] The nanoparticles taught herein are engineered to complex nucleic acids, which are hydrophilic in nature, and therefore require helper molecules to draw the nucleic acid 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 may be permanently charged or ionizable. Cationic helper molecules include a wide variety of molecules, either commercially available or synthesized in-house, but must adhere to two general criteria: 1) a positively charged group to enable complexation with the negatively charged sugar phosphate backbone; and 2) a hydrophobic portion to form a hydrophobic shell and allow incorporation into the nanoparticle core. The content of cationic material in the nanoparticle formulation may range from a cation to anion 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 to the number of negative charges (usually phosphate) in the nucleic acid payload. Thus, the N / P ratio is the ratio of the cumulative molar amount of cationic and / or ionizable groups (N) in the cationic or ionizable lipid component to the cumulative molar amount of phosphate groups (P) in the nucleic acid component. In certain embodiments, the N / P ratio of the nanoparticles taught herein is 1-25, 1-20, 1-15, 1-12, 1-9, 1-6, or 1-3. For example, the N / P ratio of the nanoparticles taught herein can be 3, 6, 9, or 12.

[0069] In addition to nucleic acids and cationic helper molecules, additional hydrophobic molecules (e.g., bulking materials (i.e., bulking agents or bulking molecules)) can be included in the core of the nanoparticle formulation. Their main use is to alter the physicochemical properties and / or improve the stability of the nanoparticles.

[0070] Nanoparticles containing therapeutic nucleic acids are expected to precisely regulate gene expression in the myeloid cell compartment, thereby regulating the immune response. The main advantage of the nanoparticle platform technology taught herein is the possibility to exchange the nucleic acid payload without changing the biological behavior and interactions of the aNP formulation. Thus, nanoparticles containing therapeutic nucleic acids can be implemented as immunotherapy to promote immune responses, for example to treat cancer or infectious diseases, or to attenuate immune responses, for example to treat autoimmune diseases or during organ transplantation.

[0071] Thus, in a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: - apolipoproteins and / or apolipoprotein mimetics; -phospholipids; -Sterols; - cationic lipids, ionizable cationic lipids, or combinations thereof; - nucleic acids; and Optionally, a filler material The present invention relates to nanoparticles comprising, consisting essentially of, or consisting of:

[0072] Without being bound by theory, the inventors believe that the nanoparticles described herein have an outer layer comprising primarily apolipoproteins and / or apolipoprotein mimetics, phospholipids and sterols, and a core comprising cationic or ionizable cationic lipids and cargo, i.e., nucleic acids. More specifically, as described elsewhere herein, the core of the nanoparticles taught herein comprises a collection of nucleic acids interacting with (ionizable) cationic lipids, and this core of the nanoparticles of the invention is surrounded by a lipid shell comprising, consisting essentially of, or consisting of apolipoproteins and / or apolipoprotein mimetics, phospholipids and sterols.

[0073] The nanoparticles can be used to deliver a cargo to an intended destination, such as a cell, tissue or organ. Preferably, the nucleic acid cargo is delivered intracellularly to the target cell, tissue or organ.

[0074] In certain embodiments, the nucleic acid is located internally (i.e., inside) the nanoparticle, in other words, in certain embodiments, the nucleic acid is not located on the outer surface of the nanoparticle and / or is not exposed to the periphery of the nanoparticle.

[0075] In certain embodiments, the apolipoprotein and / or apolipoprotein mimetic is located on the exterior surface of the nanoparticle and / or is exposed at the periphery of the nanoparticle.

[0076] In certain embodiments, the present invention relates to a nanoparticle comprising a core surrounded by a surface layer, the core comprises, consists essentially of, or consists of nucleic acid and cationic or ionizable cationic lipids; The surface layer is Phospholipids, Sterols, and Apolipoproteins or apolipoprotein mimetics or combinations thereof comprises, consists essentially of, or consists of.

[0077] It has been found that by using apolipoproteins and / or apolipoprotein mimetics, such as ApoA1, nanoparticles can be successfully targeted to bone marrow compartments in vitro, ex vivo and in vivo. This has the advantage that immune precursor cells can be targeted by drugs to stimulate or inhibit natural immune responses. There are several therapeutic applications in which such use may be beneficial, including but not limited to cancer, cardiovascular disease, autoimmune disorders and xenograft rejection.

[0078] Because the nanoparticles described herein have the same exterior as HDL particles, the nanoparticles do not elicit an immune response, which can result in early degradation or clearance of the nanoparticles by the immune system before they reach their intended target, e.g., the bone marrow compartment.

[0079] The present invention is based on the recognition that apolipoprotein-based nanoparticles or apolipoprotein mimetic-based nanoparticles can be successfully modified to accommodate nucleic acids. This is due to the combination of the following features: - using cationic or ionizable cationic lipids to neutralize the nucleic acid and load it into the hydrophobic core of the nanoparticles; This can be achieved by defining the range of structural components of the nanoparticles and / or their relative amounts, e.g. the amounts of apolipoproteins and / or apolipoprotein mimetics, sterols, phospholipids, cationic or ionizable cationic lipids, and optionally filler materials (e.g. triglycerides).

[0080] The nanoparticles of the present invention are different from any nanoparticles described in the art.

[0081] In certain embodiments, the nanoparticles of the present invention have low toxicity or are non-toxic.

[0082] In certain embodiments, the core of the nanoparticles of the invention is not surrounded by a lipid bilayer, such as those present in vesicle-like or liposomal particles, which have a lipid bilayer surrounding an aqueous core.

[0083] In certain embodiments, the nanoparticles of the invention do not contain synthetic (non-natural) hydrophilic polymers or (lipid) conjugates of such polymers, such as, most notably, polyethylene glycol (PEG), such that such nanoparticles do not induce undesirable immune responses, especially upon repeated administration.

[0084] In certain embodiments, the payload (i.e., nucleic acid) of the nanoparticles of the invention is not bound by ionic interactions at the outside (surface) of the particle. Binding of nucleic acid to the outside surface of the particle is undesirable as the nucleic acid remains exposed to the immediate surroundings, possibly making the particle more toxic and resulting in rapid (bio)degradation of the nucleic acid payload.

[0085] In certain embodiments, the nanoparticles of the present invention are substantially or completely biodegradable. In certain embodiments, the nanoparticles of the present invention are formed by natural or biocompatible components. For example, the nanoparticles of the present invention consist essentially of or consist of C, H, N, O, S and P atoms and additional counter cations and / or anions. In certain embodiments, the nanoparticles of the present invention do not contain minerals and / or metals (e.g., solid Au or Ag). Minerals and / or metals are not biodegradable or are poorly biodegradable and poorly compatible for in vivo use. In further particular embodiments, the core of the nanoparticles of the present invention does not contain minerals and / or metals (e.g., solid Au or Ag).

[0086] The core of the nanoparticle may be solid and may not have or support significant aqueous voids or reservoirs within the core, hi certain embodiments, the core of the nanoparticle is non-aqueous.

[0087] The inventors further developed a method to successfully incorporate nucleic acids into apolipoprotein and / or apolipoprotein mimetic-based nanoparticles, since the nanoparticles described herein cannot be obtained by simply mixing the individual components. It was found that it is essential to carry out a two-step reaction in which in a first step a nucleic acid-containing nanoparticle is formed, and then in a second step the apolipoprotein and / or apolipoprotein mimetic is included in the nanoparticle. Preferably, the first step is carried out at low pH and the second step is carried out at physiological pH. This finding makes it possible for the first time to include nucleic acids in apolipoprotein and / or apolipoprotein mimetic-based nanoparticles, thus enabling the delivery of said nucleic acids to the bone marrow compartment.

[0088] As used herein, nanoparticles refer to small particles, e.g., in the range of about 10 nm to about 200 nm in diameter, that can be used to deliver a payload to a target, e.g., an organ or cell of a subject.

[0089] As used herein, a subject may be a human or a non-human animal, such as a mammal, preferably a human.

[0090] Filler Material The nanoparticles described herein may further comprise a filler material (also referred to herein as "filler" or "filler molecule"), such as, but not limited to, a lipid, such as a triglyceride. Thus, in one embodiment, the nanoparticles further comprise a filler selected from triacylglycerides (also referred to simply as triglycerides) and cholesterol acyl esters (also referred to as cholesteryl esters) or a combination thereof, preferably the triacylglyceride 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. Further filler materials that may be applied are diglycerides or triglycerides or other esters derived from C1-C18 carboxylic acids, preferably C6-C18 fatty acids, these carboxylic acids and fatty acids may be saturated or unsaturated. Preferably, the filler is a triglyceride derived from C6-C18 fatty acids.

[0091] The nanoparticles described herein may form nanodiscs or nanospheres, i.e. particles of different shapes. The shape of the nanoparticles may depend on the presence or absence of filler materials. The filler may be, for example, triglycerides that are included in the core of the particle along with the payload (nucleic acid) and cationic or ionizable cationic lipids. It is understood that the inclusion of more filler may make the nanoparticles larger to a certain extent, which may lead to particle instability. Without being bound by theory, the inclusion of filler materials may contribute to the stabilization of the nanoparticles, or may stabilize the inclusion of the payload, or may modulate or enhance the delivery of the nucleic acid.

[0092] nucleic acid Many different types of RNA, DNA or synthetic oligonucleotides are used as nucleic acid therapeutics.The present invention is not limited to a specific type of nucleic acid, since it is envisioned that the present invention can work with any type that can be loaded using cationic or ionizable cationic lipid in nanoparticles.Therefore, in one embodiment, nucleic acid is RNA, or DNA or nucleic acid analogue.

[0093] In a preferred embodiment, the RNA is a microRNA (miRNA), a small interfering RNA (siRNA), a piwi-interacting RNA (piRNA), a small nuclear RNA (snoRNA), a transfer RNA (tRNA), a small tRNA-derived RNA (tsRNA), a small regulatory RNA (srRNA), a messenger RNA (mRNA), a modified mRNA, a ribosomal RNA (rRNA), a long non-coding RNA (lncRNA) or a guide RNA (gRNA), or a combination thereof and / or modifications thereof.

[0094] In certain embodiments, the antisense oligonucleotide is single-stranded DNA or RNA.

[0095] In a preferred embodiment, the DNA is single-stranded or double-stranded DNA.

[0096] In a preferred embodiment, the antisense oligonucleotide is a single-stranded DNA or RNA composed of nucleotide or nucleoside analogues containing a phosphodiester backbone or a 2' ribose modification, preferably the nucleotide or nucleoside analogues are selected from locked nucleic acids (LNA), bridged nucleic acids (BNA), morpholinos or peptide nucleic acids (PNAs).

[0097] In one embodiment of the present invention, nucleic acid is conjugated and the nucleic acid conjugate is incorporated into the nanoparticle of the present invention.Nucleic acid conjugates include, for example, phospholipids, or lipid conjugates with sterols, such as cholesterol, or hydrophobic alkyl chains.Nucleic acid conjugates also include conjugates with oligomers or polymers.Preferably, these oligomers or polymers are hydrophobic.

[0098] In one embodiment of the present invention, the nucleic acid is incorporated intact or "intact" into the nanoparticles of the present invention, meaning that the nucleic acid is not conjugated, presumably so that the nanoparticles of this embodiment behave in a favorable biocompatible manner.

[0099] Apolipoproteins As used herein, the term "apolipoprotein" refers to a protein that, together with lipids, forms a lipoprotein, i.e., a lipid-protein aggregate. The term encompasses wild-type apolipoproteins (e.g., particularly human wild-type apolipoproteins), as well as biologically active fragments thereof, biologically active variants of apolipoproteins or biologically active fragments thereof, including biologically active mutant (e.g., naturally occurring or non-naturally occurring mutant) apolipoproteins or biologically active fragments thereof. Apolipoproteins typically function to transport lipids and lipophilic substances in the blood. Apolipoproteins have been described, including, but not limited to, ApoA1, ApoA1-Milano, ApoA2, ApoA4, ApoA5, ​​ApoB48, ApoB100, ApoC-I, ApoC-II, ApoC-III, ApoC-IV, ApoD, ApoE, ApoF, ApoH, ApoL, and ApoM.

[0100] The term "fragment" as used throughout this specification with respect to a peptide, polypeptide or protein generally refers to a portion of the peptide, polypeptide or protein, such as a form in which the peptide, polypeptide or protein has been truncated, typically at the N-terminus and / or C-terminus. Preferably, a fragment comprises 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%, and even more preferably at least about 95% or even about 99% of the amino acid sequence length of the peptide, polypeptide or protein.

[0101] The term "variant" of a protein, polypeptide, peptide or nucleic acid generally refers to a protein, polypeptide or peptide, or nucleic acid, whose amino acid sequence or whose nucleotide sequence is substantially identical (i.e. largely but not completely identical) to the sequence of the protein, polypeptide or peptide, or nucleic acid, such as at least about 80% identical or at least about 85% identical, such as preferably at least about 90% identical, such as at least 91% identical, 92% identical, more preferably at least about 93% identical, such as at least 94% identical, even more preferably at least about 95% identical, such as at least 96% identical, even more preferably at least about 97% identical, such as at least 98% identical, and most preferably at least 99% identical. Preferably, the variant may show such a degree of identity to the recited protein, polypeptide, peptide or nucleic acid when the entire sequence of the recited protein, polypeptide, peptide or nucleic acid is queried in a sequence alignment (i.e. overall sequence identity). Sequence identity can be determined using suitable algorithms for performing sequence alignment and determination of sequence identity known per se.Exemplary, but non-limiting, algorithms include those based on the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215:403-10), such as the "Blast 2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174:247-250), e.g., using published default settings or other suitable settings (e.g., for the BLASTN algorithm: cost to open a gap=5, cost to extend a gap=2, penalty for mismatch=-2, reward for match=1, gapx_dropoff=50, expectation=10.0, wordsize=28; or for the BLASTP algorithm: matrix=Blosum62 (Henikoff et al. al., 1992, Proc. Natl. Acad. Sci., 89:10915-10919), cost to open a gap = 11, cost to extend a gap = 1, expectation = 10.0, word size = 3).

[0102] An exemplary procedure for determining the percent identity between a particular amino acid sequence and the amino acid sequence of a query polypeptide involves aligning the two amino acid sequences using the Blast 2 sequences (Bl2seq) algorithm, available at the NCBI website (www.ncbi.nlm.nih.gov) as a web application or as a stand-alone executable program (BLAST version 2.2.31+), using appropriate algorithm parameters.

[0103] A variant of a protein, polypeptide or peptide can include one or more amino acid additions, deletions or substitutions with respect to (ie, compared to) the corresponding protein or polypeptide.

[0104] The term "biologically active" is interchangeable with terms such as "functionally active" or "functional," which indicate that the fragments and / or variants retain at least partially the biological activity or intended functionality of the respective or corresponding peptide, polypeptide or protein. Reference to the "activity" of a peptide, polypeptide or protein can generally encompass any one or more aspects of the biological activity of the peptide, polypeptide or protein, including, but not limited to, any one or more aspects of its biochemical activity, enzymatic activity, signaling activity, interaction activity, ligand activity and / or structural activity, e.g., within a cell, tissue, organ or organism.

[0105] Preferably, a functionally active fragment or variant, e.g., a mutant, can retain at least about 20%, such as at least about 25%, or at least 30%, or at least about 40%, or at least about 50%, such as at least 60%, more preferably at least about 70%, such as at least 80%, even more preferably at least about 85%, even more preferably at least about 90%, and most preferably at least about 95%, or even about 100%, of the intended biological activity or functionality compared to the corresponding peptide, polypeptide or protein. In certain embodiments, a functionally active fragment or variant can exhibit greater biological activity or functionality compared to the corresponding peptide, polypeptide or protein, e.g., at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500% of the intended biological activity or functionality compared to the corresponding peptide, polypeptide or protein. According to examples in which the activity of a given peptide, polypeptide or protein can be readily measured in an assay with a quantitative output, such as an enzymatic assay or a signal transduction assay or a binding assay that generates a quantifiable signal, a functionally active fragment or variant of a peptide, polypeptide or protein can generate a signal that is at least about 20%, or at least about 25%, or at least 30%, or at least about 40%, or at least about 50%, or at least 60%, more preferably at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500% of the signal generated by the corresponding peptide, polypeptide or protein.

[0106] By way of example and not limitation, a biologically active fragment or variant of an apolipoprotein retains at least partially one or more aspects of the biological activity of the corresponding native or wild-type apolipoprotein. For example, reference to the biological activity of an apolipoprotein can refer, inter alia, to the ability to interact with components of the surface layer of a nanoparticle (e.g., phospholipids and sterols), to stabilize the nanoparticles taught herein, and / or to target the myeloid compartment, e.g., to myeloid cells.

[0107] As used herein, the term apolipoprotein can further refer to apolipoprotein mimetics. Apolipoprotein mimetics are short peptides, such as up to 50 amino acids, 18-mers or 36-mers, that mimic the properties of apolipoproteins. An example of an ApoA1 mimetic peptide is commonly referred to as "18A", which is a peptide with an unfunctionalized N-terminus and C-terminus and the amino sequence: DWLKAFYDKVAEKLKEAF (SEQ ID NO: 1). Another reported, more convenient and more active mimetic is the ApoA1 mimetic peptide "2F", which is Ac-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 2), i.e., the ApoA1 mimetic peptide 18A with an acetamide-capped N-terminus and an amide C-terminus. In Leman, LJ et al., J.Med.Chem.2014,57,2169-2196 (10.1021 / jm4005847), further examples of ApoA1 peptide mimetics are described, especially in Tables 2 and 3. Other ApoA1 peptide mimetics, such as dimeric, trimeric and tetrameric peptides, are shown in Zhou et al., J.Am.Chem.Soc.2013,135,13414-13424 (dx.doi.org / 10.1021 / ja404714a). Preferred ApoA1 peptide mimetics are 18A, 2F and 4F, as well as any multimers of these peptides. More preferred is 2F and any dimer or trimer of this peptide.

[0108] Apolipoproteins are proteins that bind lipids 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 the amphiphilic nature of lipoproteins, apolipoproteins and other amphiphilic molecules, such as phospholipids, can surround lipids to create lipoprotein particles that are themselves water-soluble and therefore can be transported through the aqueous circulation (i.e., blood, extracellular fluid, lymph). In addition to stabilizing lipoprotein structure and solubilizing lipid components, apolipoproteins interact with lipoprotein receptors and lipid transport proteins, thereby participating in the uptake and clearance of lipoproteins. Apolipoproteins also function as enzyme cofactors for certain enzymes involved in lipoprotein metabolism.

[0109] Apolipoprotein A1 is a protein that is encoded by the APOA1 gene in humans. In a particular embodiment, apolipoprotein A1 is human apolipoprotein A1. For further guidance, human apolipoprotein A1 precursor is annotated under UniProt accession number P02647.1 (www.uniprot.org). As the main component of HDL particles, human apolipoprotein A1 precursor has a specific role in lipid metabolism. As a component of HDL particles, this protein allows the efflux of fat molecules by accepting fat from within the cell (including macrophages in arterial walls that are overloaded with fat ingested from oxidized LDL particles) and transporting it elsewhere (in the water outside the cell) and then excreting it back to the LDL particle or liver.

[0110] It is envisaged that any apolipoprotein can be used in the nanoparticles.Thus, in one embodiment, the apolipoprotein is selected from ApoA1, ApoA1-Milano, ApoA2, ApoA4, ApoA5, ​​ApoB48, ApoB100, ApoC-I, ApoC-II, ApoC-III, ApoC-IV, ApoD, ApoE, ApoF, ApoH, ApoL and ApoM, preferably selected from ApoA1, ApoA2, ApoA4, ApoA5, ​​ApoB100, ApoC-I, ApoC-II, ApoC-III, ApoC-IV and ApoE, more preferably selected from ApoA1, ApoA4, ApoA5, ​​ApoB100, ApoC-III and ApoE, and even more preferably selected from ApoA1, ApoB100 and ApoE. In a particularly preferred embodiment, the apolipoprotein is ApoA1, since it allows highly efficient targeting of the nanoparticles to the bone marrow compartment. In another preferred embodiment, the apolipoprotein is ApoE, since it allows targeting of the nanoparticles to dendritic cells.

[0111] In certain embodiments, the apolipoprotein is a wild-type apolipoprotein or a fragment thereof, preferably a full-length wild-type apolipoprotein.

[0112] In certain embodiments, the apolipoprotein is a variant of an apolipoprotein or a fragment thereof or a mutant of an apolipoprotein or a fragment thereof.

[0113] Apolipoproteins can be produced and purified by methods known in the art, for example, recombinant protein expression from E. coli or other organisms, followed by the steps necessary to isolate the apolipoprotein, e.g., ApoA1, in (sufficiently) pure form. Apolipoproteins can also be isolated from blood by applying a series of purification methods known in the art, for example, the methods described in Chapman MJ, Goldstein S, Lagrange D, Laplaud PM. Density gradient ultracentrifugation for the isolation of major lipoprotein classes from human serum. J Lipid Res. 1981 Feb; 22(2): 339-58. PMID: 6787159. Apolipoprotein peptide mimetics can be synthesized according to peptide synthesis protocols and conjugation methods known in the art.

[0114] The inventors have found that there are several advantages associated with the use of apolipoproteins and / or apolipoprotein mimetics in nanoparticles for delivering nucleic acids to target sites. First, the apolipoproteins and / or apolipoprotein mimetics stabilize the nanoparticles by preventing aggregation during preparation and storage. For the nanoparticles to remain in a stable emulsion, it is essential that the nanoparticles do not aggregate or fuse, which may result in precipitation of the particles. The apolipoproteins and / or apolipoprotein mimetics help stabilize the particles and prevent aggregation. Furthermore, the apolipoproteins and / or apolipoprotein mimetics ensure the in vivo stability of the nanoparticles. Because the apolipoproteins and / or apolipoprotein mimetics are naturally present on lipid particles circulating in the bloodstream, e.g., LDL and HDL, they are not recognized as non-self by the immune system, thereby ensuring natural stealth, as opposed to chemical modification or other non-natural methods for improving stability. Finally, the use of the apolipoproteins and / or apolipoprotein mimetics promotes desirable interactions with immune cells, e.g., in the bone marrow compartment, for delivering nucleic acid cargo.

[0115] Thus, in one embodiment, the apolipoprotein and / or apolipoprotein mimetic in the nanoparticles is -To prevent aggregation during preparation and storage, To improve in vivo stability, -To provide natural stealth, and / or -To promote interaction with immune cells Used for.

[0116] Cationic lipids and ionizable cationic lipids As used herein, the term ionizable cationic lipid refers to lipids that have a neutral charge at physiological pH (e.g., pH 7-7.5, preferably pH 7.3-7.5, e.g., at pH 7.4) and a protonated or positive charge at lower pH (e.g., pH 1-5, preferably pH 1-4, e.g., at pH 4). It is understood that ionizable cationic lipids are particularly useful because they can be protonated at low pH, thus facilitating binding to hydrophilic nucleic acids. By subsequently increasing the pH, the lipids can be (partially) neutralized, further facilitating encapsulation in a hydrophobic environment, e.g., in the hydrophobic core of a nanoparticle. Alternatively, without being bound by theory, the ionizable lipids may remain positively charged within the nanoparticle even when the pH of the surrounding aqueous solution is increased to a physiological pH, such as about 7.4, due to the action of the surface layer of the nanoparticles, which includes phospholipid sterols and apolipoproteins and / or apolipoprotein mimetics, and / or due to the non-aqueous environment within the nanoparticle. Additionally, ionizable cationic lipids are theorized to facilitate endosomal escape of nucleic acids in target cells, where the ionizable cationic lipids become protonated due to the low pH.

[0117] 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 DLin-MC3-DMA ([(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butanoate), represented by the following formula 1: formula 1 [ka]

[0118] Indeed, a wide range of ionizable cationic lipids (including lipidoids) can be used in the preparation of the nanoparticles of the present invention, as a diverse series of ionizable cationic lipids have been developed and reported in the literature. Further non-limiting examples include the molecules cKK-E12, C12-200, L319, Acuitas-A9, Moderna-L5, TT3 and ssPalmE (described, for example, in Witzigmann et al., Advanced Drug Delivery Reviews 159 (2020) 344-363; doi.org / 10.1016 / j.addr.2020.06.026).

[0119] The ionizable lipid may also be an ionizable triglyceride. A non-limiting example is the compound shown in Formula 2. formula 2 [ka]

[0120] The ionizable lipid may be a cholesterol ester (also called a cholesteryl ester). A non-limiting example is the compound shown in formula 3. formula 3 [ka]

[0121] 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-octadecenyl-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). Other examples include any ionizable cationic lipid molecule in which a tertiary amine moiety has been converted to a quaternary ammonium moiety, for example by alkylation, such as methylation (-Me), ethylation (-Et), benzylation (-Bn) or ethoxylation (-CHCH-OH). The resulting quaternary ammonium molecule has a permanent positive (cationic) charge and therefore also has a counter anion, for example a chloride anion.

[0122] In one embodiment, only ionizable cationic lipids are used to prepare the nanoparticles of the present invention. Thus, in one embodiment, the nanoparticles taught herein do not contain cationic lipids.

[0123] In one embodiment, only cationic lipids are used to prepare the nanoparticles of the invention. Thus, in one embodiment, the nanoparticles taught herein do not include ionizable cationic lipids.

[0124] In one embodiment, a combination of ionizable cationic lipids and cationic lipids is used to prepare the nanoparticles of the present invention.

[0125] As used herein, the term "payload" generally refers to a substance contained in a particle and delivered to a target site. When referring to the nanoparticles of the present invention, the term "payload" preferably refers to a nucleic acid in combination with a cationic and / or ionizable cationic lipid.

[0126] The term "lipid" is well known in the art and, as used herein, can be considered to include, in particular, both lipids, i.e. naturally occurring hydrophobic biomolecules, such as fatty acids, mono-, di- or tri-glycerides of fatty acids, sterol (derivatives) or phospholipids, and lipid-like biomolecules. It should be noted that the cationic lipids or ionizable cationic lipids (or lipidoids) described herein are typically not lipids within the narrowest interpretation of this term, i.e. naturally occurring hydrophobic biomolecules, such as fatty acids, mono-, di- or tri-glycerides of fatty acids, sterol (derivatives) or phospholipids, but are lipid-like biomolecules similar to lipid biomolecules, i.e. preferably contain biocompatible groups (such as esters or amides) and / or contain groups constructed using naturally occurring building blocks (such as fatty acids, glycerol, cholesterol). In one embodiment, 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.

[0127] Ionizable cationic esters of long chain alcohols include tertiary amines having a carboxy group, such as those of the formula (CH3)2N(CH2), where n is an integer of 1 or greater, e.g., n is 1 to 12. nIt may be an ester of a compound having a COOH, for example, 3-dimethylamino-propionic acid or 4-dimethylamino-butyric acid or 5-dimethylamino-pentanoic acid. The ester is formed with a long-chain alcohol. The long-chain alcohol is preferably a primary or secondary alcohol having a linear or branched chain length of 8 or more carbon atoms, for example 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

[0128] The ionizable cationic ester of a diglyceride is preferably a tertiary amine having a carboxy group, such as a carboxyl group having the formula (CH3)2N(CH2), where n is an integer of 1 or more, e.g., n is 1 to 12. n A compound having a COOH group, such as a diacylglycerol coupled at the 1- or 2-position with 3-dimethylamino-propionic acid or 4-dimethylamino-butyric acid or 5-dimethylamino-pentanoic acid (i.e., a di-glyceride). The diacylglycerol can contain medium or long chain saturated or unsaturated fatty acids or derivatives or modifications thereof.

[0129] The ionizable cationic ester of a sterol is preferably a tertiary amine having a carboxy group, such as a ester of the formula (CH3)2N(CH2), where n is an integer of 1 or more, e.g., n is 1 to 12. n It is an ester of a sterol linked at a hydroxyl group to a compound having a COOH, such as 3-dimethylamino-propionic acid or 4-dimethylamino-butyric acid or 5-dimethylamino-pentanoic acid. The sterol may be cholesterol, stigmasterol or β-sitosterol.

[0130] In the above, the carboxy compound has the formula (CH3)2N(CH2), where n is an integer of 1 or more. n Alternatively, the compound may be represented by the formula NH2-(C=NH)-NH-(CH2), where n is an integer of 1 or more, for example, n is 1 to 12. nAlternative compounds having COOH can be used, which contain a guanidine group instead of a tertiary amine group.

[0131] For preparing the nanoparticles of the invention, the ionizable cationic lipids can be selected, for example, from the molecules according to formulae (I) to (V). [ka]

[0132] Formula (I) represents a triglyceride, containing an ionizable cationic group (ICG) at the 1-position.

[0133] Formula (II) represents the same type of triglyceride represented by formula (I), but the molecule is stereospecifically defined in its naturally occurring configuration, i.e., in phospholipids, the ICG group is defined in the same position as the phosphate group in the phosphate group.

[0134] Formula (III) represents a triglyceride, in which an ionizable cationic group (ICG) is included at the 2-position.

[0135] Formula (IV) represents a diester (or triester) in which the ionizable cationic group (ICG) is attached via an amide functionality.

[0136] Formula (V) represents a cholesteryl ester, in which an ionizable cationic group (ICG) is attached via the ester functionality.

[0137] The ionizable cationic group (ICG) is attached to the remainder of the molecule of any of formulas (I)-(V) via a wavy bond, and ICG can represent a tertiary amine (ICG type A, or ICG-A) or a guanidine (ICG type B, or ICG-B).

[0138] In formulae (I)-(IV), R1 may be independently selected at all positions and represents a linear or branched C1-C19 alkyl, linear or branched C1-C19 alkenyl, aryl, arylene-alkyl or alkylene-aryl group, the alkyl or alkenyl group optionally containing 5 heteroatoms and independently selected from O and N. Preferably, all R1 groups in a particular molecule according to any of formulae (I)-(IV) are 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, it is preferred that this group only has 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 group. 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, palmitic acid, oleic acid or linoleic acid. C10-C16 saturated fatty acids as well as oleic acid (C18, unsaturated) are preferred.

[0139] The integer p is a random number and is not an average value, and 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.

[0140] The R group in formula (IV) is hydrogen, methyl, ethyl, and -CH-OC(O)-R 1a group (in the formula, R 1a has the same meaning as R1 defined above). Preferably, R2 is hydrogen, methyl or a -CH2-OC(O)-R1 group. More preferably, R2 is methyl.

[0141] The R3 group in formula (IV) can be selected from hydrogen, aryl, arylene-alkyl, alkylene-aryl or linear C1-C6 alkyl groups. Preferably, R3 is hydrogen or methyl. More preferably, R3 is hydrogen.

[0142] R in ICG-A x The groups can be independently selected for all positions and are selected from methyl, ethyl, propyl and ethylene-hydroxy (-CH-CH-OH) groups, preferably methyl groups. x The groups are the same, preferably methyl groups.

[0143] R in ICG-B y The groups may be independently selected at any position from hydrogen, straight or branched C1-C18 alkyl, aryl, arylene-alkyl or alkylene-aryl groups, the alkyl groups optionally containing up to five heteroatoms independently selected from O and N. Preferably, R y The group is selected from hydrogen and linear C1-C6 alkyl groups. Even more preferably, R y Preferably, the four R groups in ICG-B are hydrogen. y The groups are all the same group, and they are preferably hydrogen.

[0144] Of the formulae (I) to (V), the formulae (I), (II) and (IV) are preferred, and the formulae (I) and (II) are more preferred.

[0145] Of ICG-A and ICG-B, ICG-A is preferred, ie, a tertiary amine ionizable cationic lipid.

[0146] The ionizable cationic lipid molecule according to any one of formulas (I)-(V) has a molecular weight of more than 250 Daltons, preferably more than 350 Daltons, more preferably more than 450 Daltons. It has a molecular weight of less than 3000 Daltons, preferably less than 1800 Daltons, more preferably less than 1200 Daltons.

[0147] The molecules represented by formulas (I)-(V) may exist in various isomeric forms, such as rotamers, tautomers, stereoisomers, or positional isomers, all of which are included within the scope of the present invention.

[0148] The ionizable cationic lipid according to any one of formulas (I)-(V) is preferably a single compound, i.e. not a mixture of compounds. Thus, the purity of the ionizable cationic lipid of formulas (I)-(V) is preferably 50% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more. If the ionizable cationic lipid is a mixture of compounds, this is preferably only due to the presence of undefined stereocenters in the molecule. An example is the use of branched alkyl chains in ionizable cationic lipids of racemic origin. Another example is triglycerides, where the substitution pattern for the three hydroxyl groups in the glycerol entity is not stereospecifically defined.

[0149] The ionizable cationic lipid according to any one of formulas (I)-(V) can be prepared by synthetic methods known in the art. In the Examples section of this application, more specifically in Example 9, various non-limiting syntheses of ionizable cationic lipids are presented.

[0150] The (ionizable) cationic lipid can preferably be processed from solution. Thus, the (ionizable) cationic lipid is preferably soluble in a range of solvents of polarity. Thus, the (ionizable) cationic lipid is preferably soluble in tricaprylin, ethanol or isopropanol, more preferably in all three of these solvents. The solubility can be checked by stirring about 20 mg of the (ionizable) cationic lipid in about 1 gram of tricaprylin, ethanol or isopropanol and evaluating whether all the material spontaneously dissolves to produce a clear / transparent solution with a concentration of about 2% by weight. The test can be carried out at about 20°C (room temperature) or about 37°C. Preferably, the (ionizable) cationic lipid is soluble at room temperature.

[0151] The (ionizable) cationic lipids are preferably non-toxic or may have limited low toxicity, either by themselves, or when tested in combination with or together with nucleic acids, or when assayed with the nanoparticles of the present invention. Toxicity cell testing can be performed by methods known in the art, such as cell viability MTT assays, or similar or equivalent tests.

[0152] A further embodiment provides an ionizable cationic lipid molecule according to any one of formulae (I)-(V), as specified in more detail above. A further embodiment provides the use of an ionizable cationic lipid molecule according to any one of formulae (I)-(V) in the preparation of nanoparticles, such as nucleic acid-containing nanoparticles, e.g., the ionizable cationic lipid molecule is used to complex with a nucleic acid.

[0153] Sterols As used herein, the term sterol refers to compounds derived from sterol (2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17-hexadecahydro-1H-cyclopenta[a]phenanthren-3-ol) by replacing some of the hydrogen atoms with other chemical groups or by modifying the bonds in the ring. Sterols and related compounds play essential roles in eukaryotic physiology. For example, cholesterol forms part of animal cell membranes, affects cell membrane fluidity, and acts as a secondary messenger in developmental signaling. As used herein, sterol can refer to a sterol selected from the group consisting of, for example, cholesterol, desmosterol, stigmasterol, β-sitosterol, ergosterol, hopanoids, hydroxysteroids, phytosterols, steroids, hydrogenated cholesterol, campesterol, zoosterol, or combinations thereof. In nanoparticles, the sterol maintains or regulates membrane fluidity (i.e., at the (mono)layer barrier at the phospholipid surface of the nanoparticle). In one embodiment, the sterol is selected from cholesterol, stigmasterol or β-sitosterol, or a combination thereof. In one embodiment, the sterol is cholesterol, ergosterol, hopanoid, hydroxysteroid, phytosterol, steroid, zoosterol, stigmasterol or β-sitosterol. In a preferred embodiment, the sterol is or comprises cholesterol.

[0154] Phospholipids Phospholipids, also known as phosphatides, are a type of lipid whose molecule has a hydrophilic head containing a phosphate group and two hydrophobic tails derived from fatty acids, linked by a glycerol molecule.

[0155] When the phospholipid is a marine phospholipid, the phospholipid typically has the omega-3 fatty acids EPA and DHA incorporated as part of the phospholipid molecule. Simple organic molecules such as choline, ethanolamine or serine can be used to modify the phosphate group.

[0156] Phospholipids are important components of all cell membranes. Phospholipids are able to form lipid bilayers due to their amphipathic characteristics. In eukaryotes, cell membranes also contain another type of lipid, sterols (especially cholesterol), interspersed between the phospholipids. This combination provides two-dimensional fluidity combined with mechanical strength against rupture.

[0157] Thus, in one embodiment, the phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol, or combinations thereof.

[0158] The acyl groups in the phospholipids may be individually medium or long chain fatty acids. In one embodiment, at least one, preferably both, of the acyl groups in the phospholipids are long chain fatty acids, preferably the long chain fatty acids are selected from C14, C16 and C18 chains, i.e., myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, linoleic acid and oleic acid, or combinations thereof.

[0159] In a particularly preferred embodiment, the phospholipid is a neutral phospholipid, meaning that it is zwitterionic (having a net neutral charge) at physiological pH. Thus, in a preferred embodiment, the phospholipid is a phosphatidylcholine (PC) or a phosphatidylethanolamine (PE).

[0160] Thus, non-limiting examples of phospholipids that can be used include dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG ...lauroylphosphatidylglycerol (DLPG), dilauroylphosphatidylglycerol (DLPG), dilauroylphosphatidylglycerol (DLPG), dilauroylphosphatidylglycerol (DMPG), dilauroylphosphatidylglycerol (DLPG), dilauroylphosphatidylglycerol (DLPG), dilauroylphosphatidylglycerol (DLPG), dilauroylphosphatidylglycerol ( dilauroyl phosphatidylethanolamine (DLPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE), dilauroyl phosphatidylserine (DLPS), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylserine (DSPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and mixtures thereof.

[0161] Lysophospholipids are phospholipids in which one of the acyl groups has been removed by hydrolysis, leaving an alcohol group. Thus, these molecules have one fatty acid chain instead of two. These phospholipids can also be applied, for example, to modulate the shape, function and fluidity of the outer layer of the nanoparticles taught herein. As lysophospholipids, 1-myristoyl-2-hydroxy-sn-glycerophosphocholine (MHPC), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHPC) and 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (SHPC), or mixtures thereof, can be used.

[0162] In one embodiment, all phospholipids used to prepare the nanoparticles of the present invention are of natural origin, which means that they are found in any kind of natural environment, for example (a) in certain cell membranes.Therefore, these phospholipids are biocompatible and biodegradable.Natural phospholipids can be isolated and purified from natural sources (soybean, milk, rapeseed, chicken eggs, sunflower, etc.), but can also be prepared and purified by (semi) synthetic means.

[0163] Nanoparticle Characteristics The nanoparticles of the present embodiments comprise, consist essentially of, or consist of nucleic acids, cationic and / or ionizable cationic lipids, phospholipids, sterols, apolipoproteins and / or apolipoprotein mimetics, and optionally filler materials; the amount of apolipoprotein and / or apolipoprotein mimetic is in the range of 0.1-90% by weight; and / or the amount of nucleic acid is in the range of 0.01 to 90% by weight, and / or the amount of phospholipids is in the range of 0.1 to 95% by weight, and / or the amount of sterol is in the range of 0.1 to 95% by weight, and / or The amount of cationic and / or ionizable cationic lipid is in the range of 0.1 to 95% by weight; The amount of filler material optionally present ranges from 0 to 95% by weight, these weight percentages being based on the apolipoproteins and / or apolipoprotein mimetics, nucleic acids, phospholipids, sterols and cationic and / or ionizable cationic lipids plus any filler materials, i.e., the total amount of these five or six components being 100% of the nanoparticle weight.

[0164] In one embodiment, the amount of apolipoprotein and / or apolipoprotein mimetic is in the range of 0.2-50% by weight, such as 3-20% or 4-20% by weight, more preferably 0.5-30% by weight, more preferably 1-20% by weight.

[0165] In one embodiment, the amount of nucleic acid is in the range of 0.02 to 30% by weight, more preferably 0.05 to 20% by weight, more preferably 0.1 to 15% by weight, for example, 0.5 to 5% by weight.

[0166] In one embodiment, the amount of phospholipid is in the range of 0.2 to 60% by weight, more preferably 1 to 50% by weight, for example 10 to 50% by weight, more preferably 3 to 40% by weight, for example 10 to 40% by weight.

[0167] In one embodiment, the amount of sterol is in the range of 0.2-90% by weight, more preferably 0.5-70% by weight, for example 2-65% by weight, more preferably 1-50% by weight, for example 2-45% by weight, 10-45% by weight or 10-20% by weight.

[0168] In one embodiment, the amount of cationic and / or ionizable cationic lipid is in the range of 0.2-90% by weight, more preferably 0.5-80% by weight, more preferably 1-70% by weight, such as 5-60% by weight, 8-60% by weight, 9-60% by weight, 10-60% by weight, 15-25% by weight, or 20-60% by weight.

[0169] In one embodiment, the amount of any filler or filler molecules ranges from 0 to 90% by weight, more preferably 0 to 80% by weight, more preferably 0 to 70% by weight, for example 0 to 65% by weight.

[0170] In certain embodiments, the amount of any filler or filler molecules ranges from 20 to 80% by weight, more preferably from 30 to 70% by weight, even more preferably from 30 to 65% by weight, such as from 40 to 65% by weight, 45 to 55% by weight, or 30 to 60% by weight.

[0171] In certain embodiments, the nanoparticles taught herein do not include a filler or filler molecules.

[0172] These weight percentages as above are based on the amounts of apolipoproteins and / or apolipoprotein mimetics, nucleic acids, phospholipids, sterols and cationic and / or ionizable cationic lipids, and optionally filler materials, i.e., the total amount of these five or six components, which in the context of these descriptions, amounts to 100% of the weight of the nanoparticles.

[0173] It has been found that nanoparticles constructed from apolipoproteins and / or apolipoprotein mimetics, phospholipids, sterols and cationic and / or ionizable cationic lipids within these ranges are stable and can successfully incorporate nucleic acids. The outer layer of the nanoparticle is composed of phospholipids, apolipoproteins and / or apolipoprotein mimetics, and sterols. In a preferred embodiment, the ratio of apolipoproteins and / or apolipoprotein mimetics to phospholipids by weight is 2:1 to 1:10, which allows for the assembly of stable nanoparticles. Thus, in one embodiment, the ratio of apolipoproteins and / or apolipoprotein mimetics to phospholipids by weight used is 2:1 to 1:10, more preferably 1:1 to 1:5, even more preferably 1:1.5 to 1:4.

[0174] In one embodiment, the relative amounts of the components in the nanoparticles relate to the ratios used to prepare the nanoparticles.

[0175] After formulation and optional purification of the nanoparticles of the present invention, the retention (or recovery or capture) of the various nanoparticle components can be evaluated. This can be done by methods known in the art. For example, RNA retention can be determined using the Ribogreen assay, and Apolipoprotein A1 (Apo-A1) recovery can be evaluated using colorimetric protein quantification assays. Cholesterol and lipid recovery can be determined using standard colorimetric assays. The recovery of the various components of the nanoparticles of the present invention is high.

[0176] In one embodiment, the relative amounts of components in the nanoparticles relate to the determined incorporation levels of the components in the nanoparticles after formulation and optionally purification.

[0177] In one embodiment, the retention rate of nucleic acid (e.g., siRNA or mRNA) in the nanoparticles is preferably 1% or more, preferably 5% or more, more preferably 20% or more, such as 40% or more, even more preferably 50% or more, such as 60% or more, 70% or more, or 80% or more.

[0178] In one embodiment, the recovery of sterol (e.g., cholesterol) in the nanoparticles is at least 1%, preferably at least 10%, more preferably at least 30%, such as at least 40%, even more preferably at least 50%, such as at least 60%, at least 70%, at least 80%, or at least 85%.

[0179] In one embodiment, the phospholipid recovery in the nanoparticles is at least 1%, preferably at least 10%, more preferably at least 30%, such as at least 40%, even more preferably at least 50%, such as at least 60%, at least 70%, or at least 80%.

[0180] In one embodiment, the recovery of apolipoprotein (e.g., Apo-A1) and / or apolipoprotein mimetic in the nanoparticles is 1% or more, preferably 5% or more, more preferably 10% or more, even more preferably 20% or more, such as 30% or more or 35% or more.

[0181] In one embodiment, the amount of apolipoprotein and / or apolipoprotein mimetic is in the range of 0.05-2.0 mol%, such as 0.10-2.0 mol% or 0.08-0.5 mol%, and / or the amount of phospholipid is in the range of 5-90 mol%, such as 15-90 mol% or 8.0-50 mol%, and / or the amount of sterol is in the range of 2.5-65 mol%, such as 2.5-50 mol% or 4-65 mol%, and / or the amount of cationic or ionizable cationic lipid is in the range of 5.0-80 mol%, such as 8.0-80 mol% or 5-65 mol%, wherein the molar percentages are based only on the total amount of apolipoprotein and / or apolipoprotein mimetic, phospholipid, sterol and cationic and / or ionizable cationic lipid in the nanoparticles. It has been found that nanoparticles constructed from apolipoproteins and / or apolipoprotein mimetics, phospholipids, sterols and cationic and / or ionizable cationic lipids within these ranges are stable and can successfully incorporate nucleic acids.

[0182] The outer layer of the nanoparticle is composed of phospholipids, apolipoproteins and / or apolipoprotein mimetics, and sterols. To assemble a stable nanoparticle, the ratio of apolipoproteins to phospholipids based on molar weight percentage is preferably 1:25 to 1:400. Thus, in one embodiment, the ratio of apolipoproteins and / or apolipoprotein mimetics to phospholipids 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.

[0183] The nanoparticles according to the present invention have been found to have a relatively defined and constant size. In other words, the nanoparticles according to the present invention are uniform in size. It is believed that the average size is determined primarily by the amount and type of the core component, i.e., the amount of cationic and / or ionizable cationic lipids, and the amount of filler. It is understood that the filler is optional, and that the particle size can possibly be increased by increasing the amount of filler. In one embodiment, the nanoparticles according to the present invention have an average size of about 10 to about 200 nm, about 20 to about 200 nm, or about 30 to about 200 nm, preferably about 30 to about 100 nm, and preferably the average size refers to the particle size.

[0184] In certain embodiments, the size is a z-average size or a number average size.

[0185] The size of the nanoparticles of the present invention can be evaluated by methods known in the art.For example, dynamic light scattering (DLS) can be used to measure the diameter of nanoparticles.Cryo-TEM measurement can also be used for this purpose.Both techniques can also be used to evaluate the diameter distribution (or dispersity) of the nanoparticle preparations prepared.

[0186] In certain embodiments, the particle size dispersity within the nanoparticles taught herein is between 0 and 0.5, preferably between 0 and 0.4, more preferably between 0 and 0.3, and most preferably between 0 and 0.2.

[0187] The shape and properties of the nanoparticles of the present invention can be evaluated, for example, by cryo-TEM measurements. The particles may be spherical or approximately spherical in shape. The particles may also be ellipsoidal or helical in shape. The particles may be discoidal. Preferably, the particles are spherical, approximately spherical, and / or somewhat ellipsoidal in shape. Preferably, the particles are not discoidal in shape. Preferably, the particles appear essentially solid, i.e., no significant or large internal aqueous compartments can be observed within the particles. Particles observed in cryo-TEM do not need to be completely homogeneous, i.e., the electron density can vary within the particle. Preferably, the particles of the present invention have similar sizes and shapes, i.e., there is no large distribution in size and shape. Nanoparticles as defined herein comprise a hydrophobic core and a hydrophilic surface, and therefore can be dissolved in water or aqueous solutions, such as saline or buffer solutions. Due to the inherent properties resulting from the constituents of the nanoparticles defined by the present invention, the nanoparticles are stable in suspension for several months, for example, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, or at least 12 months. Suitable aqueous buffer solutions are known in the art, such as phosphate buffered saline (PBS), Tris buffered saline (TBS). Suitable saline solutions are known, non-limiting examples include aqueous solutions of NaCl or KCl. If the nanoparticles are intended to be administered to a subject, they must be suspended in a carrier that is physiologically acceptable for that purpose. For example, if the nanoparticles are intended for intravenous delivery, the physiologically acceptable carrier is typically a fluid that is isotonic with blood. For example, a solution of sodium chloride at a concentration of 0.9% by weight / volume, dextrose solution at 5% by weight / volume, Ringer's solution, lactated Ringer's solution, or acetated Ringer's solution may be used, although other suitable carriers are known.

[0188] Thus, in one aspect, the present invention relates to a composition comprising the nanoparticles according to the present invention and a physiologically acceptable carrier. In one embodiment, the composition is a pharmaceutical composition. It is understood that the composition can further comprise additional components, such as, but not limited to, pharmaceuticals or biopharmaceuticals. This may be an attractive option for combination therapy of nucleic acids (contained in nanoparticles) and drugs. The drug may be, but is not limited to, a small molecule compound, an antibody or an antigen-binding fragment, a further nanoparticle.

[0189] use The purpose of the nanoparticles described herein is to deliver nucleic acids to cells or to deliver nucleic acid therapy to a subject. The nucleic acid may be, for example, an mRNA that codes for a peptide or protein of interest to be expressed in the cell, or it may contain short nucleic acids, such as siRNA, shRNA, intended to disrupt gene expression (e.g., gene silencing), or may contain components of CRISPR-Cas or related systems (e.g., gRNA) that induce mutations in the genome of the cell. Thus, in general, the mode of action of the nucleic acid (the payload of the nanoparticle) is in the cytoplasm or nucleus. Thus, the nanoparticle preferably has at least the following properties: 1) it allows targeting of the intended target cell, and 2) it can deliver the payload to the place where it can exert its action (thus, in most cases, in the cytoplasm or nucleus of the target cell).

[0190] A further aspect of the invention relates to the nanoparticles according to the invention or the composition according to the invention for use as a medicament.

[0191] It is understood that the nucleic acid therapy comprising the nanoparticles can be administered to a subject in need thereof. Depending on the target cell or tissue, administration can be parenteral, for example, intravenous, intramuscular or subcutaneous. Administration can also be oral, sublingual, topical, rectal, nasal (inhalation) or vaginal. Furthermore, the targeting of the target tissue or cell is determined by the appropriate selection of the apolipoprotein and / or apolipoprotein mimetic. In one embodiment, the use of the nanoparticles or compositions according to the present invention includes delivering the nucleic acid to the bone marrow compartment or the spleen. This delivery can be achieved, for example, by intravenous parenteral administration. Preferably, the apolipoprotein and / or apolipoprotein mimetic is an apolipoprotein that targets the bone marrow compartment, for example, ApoA1.

[0192] The inventors have found that the nanoparticles taught herein enable efficient delivery of nucleic acid therapeutics to lymphoid organs, including but not limited to the myeloid cell compartment of the bone marrow and spleen, for effective immunotherapy. Indeed, the inventors have found that the nanoparticles of the present invention can target tissues associated with the presence of immune cells (spleen, bone marrow) after systemic injection.

[0193] A further aspect provides a nanoparticle as taught herein or a composition as taught herein for use in immunotherapy.

[0194] In one aspect, the invention relates to the nanoparticles according to the invention or the composition according to the invention for use in the treatment of a disease by stimulating or inhibiting the innate immune response, preferably the disease is a disease that benefits from stimulating or inhibiting the innate immune response in a subject, such as a disease characterized by a defective innate immune response, more preferably the disease to be treated is cancer, a cardiovascular disease, an autoimmune disorder or a xenograft rejection. Thus, the nanoparticles according to the invention 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 in which modulation of the immune response is considered a viable therapeutic option.

[0195] In a further aspect, the present invention relates to a method for in vivo delivery of a nucleic acid, comprising administering to a subject the nanoparticles according to the invention or the composition according to the invention.

[0196] In a further aspect, the present invention relates to a method for treating a disease or disorder in a subject in need of treatment by stimulating or inhibiting the innate immune response, comprising administering a therapeutically effective amount of the nanoparticles according to the invention or the composition according to the invention to the subject. In a particular embodiment, the disease or disorder is a disease or disorder characterized by a defective innate immune response. In one embodiment, the disease or disorder is selected from cancer, cardiovascular disease, autoimmune disorder or xenograft rejection.

[0197] By targeting the bone marrow compartment, nucleic acid therapy can be successfully delivered to the precursor cells of different blood cell types, as opposed to already differentiated cells present in blood and tissues, such as T cells and macrophages. In this way, the natural immune response can be modulated, for example stimulated or inhibited, by nucleic acid therapy, depending on the desired outcome. For example, in autoimmune disorders, cardiovascular disease or (prevention of) xenograft rejection, inhibition of autoimmune response is desired, while in cancer, stimulation of immune response against target cancer cells is desired.

[0198] Preparation of nanoparticle formulations - aNPs The present invention provides apolipoprotein and / or apolipoprotein mimetic-based nanoparticles containing nucleic acids (herein, these particles of the present invention may be referred to as aNPs). Until now, it has not been possible to include nucleic acids in such nanoparticles because the core of such particles is hydrophobic and therefore not suitable for the uptake of nucleic acids, which are hydrophilic. The use of ionizable cationic lipids together with nucleic acids has been described as a tool for intracellular delivery of nucleic acids, but simply combining ionizable cationic lipids and nucleic acids with other lipid components does not result in the formation of lipid nanoparticles as described herein. For example, mixing nucleic acids (e.g., siRNA or mRNA) with liposomal formulations prepares particles in which the nucleic acid is exposed to the aqueous surroundings of the particle and in which the nucleic acid is subject to rapid degradation. Furthermore, these particles are unstable and exhibit the formation of large, poorly defined aggregates. In another example, the simple addition of apolipoproteins and / or apolipoprotein mimetics to liposomal formulations does not result in nanoparticle formulations with defined, desired characteristics.

[0199] Surprisingly, without the use of PEG, PEG conjugates or another synthetic polymeric stabilizer material, the inventors have found a controlled formulation process to prepare stable and / or non-toxic aNPs with well-defined size and shape, with encapsulated and shielded nucleic acid payloads, with adequate recovery for the components used, and with nucleic acid payloads that are active when exposed to (various) cell lines. Furthermore, a wide range of compositions (e.g., various types and / or levels of apolipoproteins and / or apolipoprotein mimetics, phospholipids, sterols, cationic and / or ionizable cationic lipids, nucleic acids and optional bulking agents) can be used to prepare these aNPs.

[0200] Thus, the present invention also revolves around the recognition that nucleic acids can be incorporated into nanoparticles by using a two-step formulation process.

[0201] Thus, in one aspect, the present invention provides a method for preparing nanoparticles, comprising the steps of: a) preparing lipid nanoparticles by mixing, preferably rapidly mixing, lipid components in an organic solvent with nucleic acid in an aqueous buffer, the lipid components comprising phospholipids, sterols, cationic lipids or ionizable cationic lipids, and optionally a bulking material (e.g., triglycerides), and the aqueous buffer has a pH of 5.0 or less; b) mixing, preferably rapidly mixing, the lipid nanoparticles prepared under (a) with apolipoproteins and / or apolipoprotein mimetics to prepare the nanoparticles of the invention at a pH of 6.0 to 8.0; The present invention relates to a method comprising the steps of:

[0202] The organic solvent may be an alcohol, such as ethanol, isopropanol, methanol, acetonitrile, dimethylsulfoxide (DMSO), chloroform, or a combination thereof. Preferred organic solvents are water-miscible and non-toxic, such as ethanol and DMSO, or a combination thereof.

[0203] For example, the organic solvent may be 96% to 100% ethanol, preferably 100% ethanol.

[0204] 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.

[0205] The aqueous buffer of step a) is at a low pH, ensuring that the ionizable cationic lipids are positively charged to allow binding and encapsulation of the nucleic acid / cationic lipid complexes within the particle. For example, the pH of the buffer may be 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 may be any buffer that does not damage the nucleic acid. An exemplary buffer is sodium acetate at pH 4.0. The nanoparticles are then taken up in an aqueous buffer having a pH of about 6-8, preferably 7-8, more preferably about 7.4. This can be achieved, for example, by dialysis with 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, although it is understood that any buffer that does not damage the nucleic acid may be used.

[0206] In step b), the nanoparticles in an aqueous buffer of pH 6-8, preferably pH 7-8, are rapidly mixed with an apolipoprotein and / or an apolipoprotein mimetic in an aqueous buffer of pH 6-8, preferably pH 7-8, to obtain the nanoparticles according to the invention.

[0207] The above two-step formulation process taught herein results in aNPs with a wide range of desirable beneficial characteristics (stability, low or no toxicity, high nucleic acid retention, nucleic acid activity, etc.) However, the formulation methods described are non-limiting, as aNPs with beneficial characteristics can be obtained by other processes.

[0208] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: a) preparing lipid nanoparticles by mixing, preferably rapidly mixing, lipid components in an organic solvent with nucleic acid in an aqueous buffer, the lipid components comprising phospholipids, sterols, cationic lipids or ionizable cationic lipids, and optionally a bulking material (e.g., triglycerides), and the aqueous buffer has a pH of 5.0 or less; b) mixing, preferably rapidly mixing, the lipid nanoparticles prepared under (a) with apolipoproteins and / or apolipoprotein mimetics to prepare the nanoparticles of the invention at a pH of 6.0 to 8.0; The present invention relates to nanoparticles obtainable or obtained by a process comprising the steps of:

[0209] Further aspects of the invention It is understood that the nanoparticles according to the invention can deliver nucleic acid to target cells or tissues. The target cells or tissues can be in a subject or in vitro or ex vivo. Thus, in one aspect, the invention relates to an in vivo, in vitro or ex vivo method for introducing nucleic acid into cells, comprising contacting the nanoparticles according to the invention or the composition according to the invention with the cells. In a particular embodiment, the cells are cells of the bone marrow compartment or myeloid cells. The present application also provides aspects and embodiments that are described in the following description.

[0210] Description 1. A nanoparticle, - apolipoproteins, -phospholipids, -sterols, cationic or ionizable cationic lipids, and -Nucleic acid Nanoparticles comprising:

[0211] Statement 2. The nanoparticles according to statement 1, wherein the nanoparticles further comprise a filler selected from triacylglycerides and cholesterol acyl esters or combinations thereof, preferably the triacylglyceride is tricaprylin and / or the cholesterol acyl ester is cholesterol caprylate and / or cholesterol oleate.

[0212] Statement 3. The nucleic acid is RNA, DNA or a nucleic acid analogue; Preferably, the RNA is a microRNA (miRNA), a small interfering RNA (siRNA), a piwi-interacting RNA (piRNA), a small nuclear RNA (snoRNA), a transfer RNA (tRNA), a small tRNA-derived RNA (tsRNA), a small regulatory RNA (srRNA), a messenger RNA (mRNA), a modified mRNA, a ribosomal RNA (rRNA), a long non-coding RNA (lncRNA) or a guide RNA (gRNA), or a combination thereof and / or a modification thereof, or Preferably, the DNA is single-stranded or double-stranded DNA, or Preferably, the antisense oligonucleotide is a single stranded DNA or RNA consisting of nucleotide or nucleoside analogues containing a phosphodiester backbone or a 2' ribose modification, more preferably the nucleotide or nucleoside analogues are selected from locked nucleic acid (LNA), bridged nucleic acid (BNA), morpholino or peptide nucleic acid (PNA). 3. A nanoparticle according to any one of statements 1 and 2.

[0213] Statement 4. The apolipoprotein is selected from ApoA1, ApoA2, ApoA4, ApoA5, ​​ApoB48, ApoB100, ApoC-I, ApoC-II, ApoC-III, ApoC-IV, ApoD, ApoE, ApoF, ApoH, ApoL, and ApoM; Preferably selected from ApoA1, ApoA2, ApoA4, ApoA5, ​​ApoB100, ApoC-I, ApoC-II, ApoC-III, ApoC-IV and ApoE, More preferably, it is selected from ApoA1, ApoA4, ApoA5, ​​ApoB100, ApoC-III and ApoE; Most preferably selected from ApoA1, ApoB100 and ApoE. 4. A nanoparticle according to any one of statements 1 to 3.

[0214] Statement 5. Apolipoproteins in nanoparticles -To prevent aggregation during preparation and storage, To improve in vivo stability, -To provide natural stealth, and / or -Used to promote interaction with immune cells, 6. A nanoparticle according to any one of statements 1 to 5.

[0215] Statement 6. The nanoparticle of any one of statements 1 to 5, wherein 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.

[0216] Statement 7. The nanoparticle of any one of statements 1 to 6, wherein the sterol is selected from cholesterol, stigmasterol or β-sitosterol, or a combination thereof.

[0217] Statement 8. The phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol or a combination thereof, preferably at least one, more preferably both, of the acyl groups in the phospholipid is a long chain fatty acid, even more preferably the long chain fatty acid is selected from myristoleic acid, palmitoleic acid and oleic acid or a combination thereof; 8. A nanoparticle according to any one of statements 1 to 7.

[0218] Statement 9. The amount of apolipoprotein is in the range of 0.10 to 2.0 mol %; and / or the amount of phospholipids is in the range of 15-90 mol %; and / or the amount of sterol is in the range of 2.5 to 50 mol %; and / or The amount of cationic or ionizable cationic lipid is in the range of 8.0-80 mol%, the molar percentage being based only on the total amount of apolipoprotein, phospholipid, sterol and cationic or ionizable cationic lipid in the nanoparticle; 9. A nanoparticle according to any one of statements 1 to 8.

[0219] Statement 10. Nanoparticles according to any one of statements 1 to 9, wherein the ratio of apolipoprotein to phospholipid based on molar weight percentage is between 1:25 and 1:400, more preferably between 1:50 and 1:200, even more preferably between 1:75 and 1:150.

[0220] Statement 11. The nanoparticles of any one of statements 1 to 10, having an average size of 30 to 100 nm.

[0221] Statement 12. A composition comprising nanoparticles according to any one of statements 1 to 11 and a physiologically acceptable carrier, said composition being preferably a pharmaceutical composition.

[0222] Statement 13. A nanoparticle according to any one of statements 1 to 11 or a composition according to statement 12 for use as a medicament.

[0223] Statement 14. The nanoparticle or composition for use according to statement 13, wherein the use comprises delivering the nucleic acid to a bone marrow compartment or to the spleen.

[0224] Statement 15. A nanoparticle according to any one of statements 1 to 11 or a composition according to statement 12 for use in the treatment of a disease by stimulating or inhibiting the innate immune response, preferably wherein the disease is cancer, a cardiovascular disease, an autoimmune disorder or xenograft rejection.

[0225] Statement 16. A method for preparing nanoparticles, comprising: a) preparing lipid nanoparticles by mixing, preferably rapidly mixing, lipid components in an organic solvent with nucleic acid in an aqueous buffer, the lipid components comprising phospholipids, sterols, triglycerides (optional) and cationic lipids or ionizable cationic lipids, and nucleic acid, the aqueous buffer having a pH of 5.0 or less; b) mixing, preferably rapidly mixing, lipid nanoparticles and apolipoprotein to prepare nanoparticles of the present invention at a pH of 6.0 to 8.0; A method comprising:

[0226] Statement 17. An in vivo, in vitro or ex vivo method for introducing a nucleic acid into a cell, comprising contacting the cell with a nanoparticle according to any one of statements 1 to 11 or a composition according to statement 12.

[0227] Statement 18. A method for in vivo delivery of a nucleic acid, comprising administering to a subject a nanoparticle according to any one of statements 1 to 11 or a composition according to statement 12.

[0228] Statement 19. A method for treating a disease or disorder in a subject in need of treatment by stimulating or inhibiting an innate immune response, comprising administering to the subject a therapeutically effective amount of a nanoparticle according to statements 1 to 11 or a composition according to statement 12.

[0229] Statement 20. The method of statement 19, wherein the disease is selected from cancer, cardiovascular disease, an autoimmune disorder, or xenograft rejection. While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0230] The presently disclosed aspects and embodiments of the present invention are further supported by the following non-limiting examples. EXAMPLES

[0231] Example 1. General description of formulation and characterization Nanoparticle formulations self-assemble based on ionic and hydrophobic interactions. The components are prepared at the desired concentrations in their respective organic solvents (lipids and other structural components) or aqueous buffers (nucleic acid payload). The solutions are then brought together by rapid mixing techniques, including microfluidic or T-junction mixing.

[0232] An excess of aqueous buffer is essential for the formation process. As used herein, excess aqueous buffer refers to a ratio (by volume) of (aqueous buffer):(organic solvent) of at least 2:1 or more, such as 2.2:1, 2.5:1, 2.8:1, or 3:1 or more.

[0233] After the initial mixing, a small amount of organic solvent is removed, for example, by dialysis or centrifugal filtration. These steps result in lipid nanoparticles, and in the next step, the apolipoprotein and / or apolipoprotein mimetic is added by a rapid mixing technique, such as dropwise addition. After the addition and processing of the apolipoprotein and / or apolipoprotein mimetic, the remaining protein must be removed by dialysis or centrifugal filtration. Finally, the sample is concentrated to the desired concentration (see Figure 2).

[0234] Therefore, the nucleic acid nanoparticles aNP are (a) Nucleic acid, (b) (ionizable) cationic molecules; (c) apolipoproteins, (d) phospholipids, (d) sterols, and (e) optionally, a triglyceride or a derivative thereof; Includes.

[0235] After the aNPs are formulated, the physicochemical properties of the nanoparticle formulations are determined. These properties may vary depending on the specific composition of the formulation. The size and dispersity of the nanoparticles are determined by dynamic light scattering (DLS) and electron microscopy (e.g., cryo-TEM). Electron microscopy is also used to evaluate nanoparticle morphology. In addition, the recovery of input material components, such as nucleic acids, apolipoproteins and / or apolipoprotein mimetics, phospholipids, and cholesterol, is determined by various commercially available assays known in the art. Shelf life is evaluated by determining the physicochemical properties of the formulations over an extended period (1 month) while stored in buffer at 4°C. A large number of nucleic acid nanoparticle formulations (approximately 150) have been characterized for their physicochemical properties and shelf life. Specific formulations were used to investigate reproducibility and stability under physiological conditions.

[0236] The following molar percentage ranges of components were tested and the prepared aNPs were found to be stable, the molar percentages being based on the total amount of apolipoprotein (Apo-A1), phospholipids, sterol (cholesterol) and cationic or ionizable cationic lipids only, excluding bulking agents, nucleic acids and any other components: the amount of apolipoprotein Apo-A1 is in the range of 0.08 mol % to 2.0 mol %, for example in the range of 0.10 mol % to 2.0 mol %, and / or the amount of phospholipid is in the range of 5 to 90 mol %, for example 15 to 90 mol %, and / or the amount of sterol 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 lipid is in the range of 5.0 to 80 mol %, for example, 8.0 to 80 mol %.

[0237] Outside these ranges, the nanoparticles may be unstable. Additionally, filler materials such as triglycerides may be added in the range of 0-95 mole %, with the mole percentage being based on the total amount of apolipoproteins, phospholipids, sterols, and cationic or ionizable cationic lipids only.

[0238] Example 2. An exemplary method for preparing apolipoprotein lipid nanoparticles (aNPs) containing nucleic acids, such as RNA, described herein (FIG. 2). In a first step, phospholipids, sterols, e.g. cholesterol, ionizable cationic lipids, and optional filler materials (e.g. triglycerides) were dissolved in a water-miscible organic solvent, e.g. 96%-100% ethanol (e.g. 2.33 mL), and the solution was rapidly mixed (at the specified flow rates and ratios) with an aqueous solution containing nucleic acids maintained at a lower pH (e.g. 7 mL of 25 mM sodium acetate, pH 4). For mixing, a T-junction mixing device, e.g. 28 mL / min, was used. Other microfluidic-based mixing methods, e.g. mixing in chips with staggered herringbone structures, can also be used. The resulting lipid nanoparticles were dialyzed at physiological pH (e.g. dialysis at 4°C overnight, 2x, 155 mM PBS, pH 7.4), and then in a second step rapidly mixed at physiological pH with apolipoproteins, e.g. apolipoprotein A1, to obtain nanoparticles (aNPs) according to the invention. Apolipoprotein A1 can be present in 155 mM PBS, pH 4. Alternatively, a peptidomimetic of an apolipoprotein can be used in the second mixing step. For mixing, a T-junction mixer can be used, for example at 13.3 mL / min. After mixing, the resulting nucleic acid nanobiological agent can be incubated for 1 hour. Optionally, the nanoparticles can be filtered and concentrated (e.g., 0.2 μm filtration followed by 100 kDa centrifugal filtration). The aNPs of the present invention can also be processed by other methods.

[0239] Example 3. siRNA retention in apolipoprotein nanoparticles (aNPs) and instability of comparative nanoparticles (NPs) containing no apolipoprotein (Figure 3). Following the preparation procedure according to Example 2 (FIG. 2), two representative siRNA-containing aNPs (siRNA-aNPs) (aNPs 18 and 34, the formulations of which are shown in Table 1 (FIG. 11)) were prepared. siRNA-aNP formulations 18 and 34 are formulations according to certain embodiments of the present invention, and contain various amounts of phospholipids (i.e., 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)), cholesterol, ionizable cationic lipid (i.e., Dlin-MC3-DMA), triglyceride, apolipoprotein A1 (ApoA1), and siRNA.

[0240] Additionally, comparative NPs were prepared by omitting the second step of the procedure, where apolipoprotein A1 is incorporated into the formulation. RNA retention was determined using the Ribogreen assay (ThermoFisher-R11490) one day after NP formulation.

[0241] Only the aNPs according to the invention convincingly captured the siRNA payload (FIG. 3A). Comparative example NPs without apolipoprotein retained little or no siRNA (FIG. 3A).

[0242] Figure 3B shows a representative image of comparative siRNA-NP formulation 18, in which apolipoprotein A1 was not incorporated, showing large, poorly defined precipitates / aggregates in a cloudy solution, indicating an inability to form a stable (clear) formulation.

[0243] FIG. 3C shows a representative cryo-transmission electron micrograph of comparative siRNA-NP formulation 18 showing large, poorly defined aggregates (scale bar 50 nm).

[0244] In conclusion, these data indicate that apolipoproteins are important and essential structural components for the formation and stability of apolipoprotein lipid nanoparticles (aNPs) containing nucleic acids.

[0245] Purification of ApoA1 Small-scale cultures of ClearColi cells transformed with the pET20b-apoA1 plasmid were started in LB medium containing 100 μg / mL ampicillin. The next day, 20 mL of the small culture was diluted into 1 liter of 2YT medium to start a large-scale culture. The cultures were grown at 37 °C and 150 rpm to an OD600 of 0.6-0.8, and then expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. The induced cultures were incubated overnight at 20 °C and 150 rpm. The induced bacterial cultures were pelleted, and the cells were chemically lysed by resuspending the pellet in 5 mL of BugBuster Protein Extraction Reagent (Novagen) per gram of pellet. Benzonase nuclease (Merck Millipore) was added to the cells resuspended in BugBuster, followed by incubation at room temperature with shaking. The cell lysates were kept on ice at all times. After lysis, the cell lysate was centrifuged to pellet insoluble cell debris and the supernatant was loaded onto an 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) and then with 8 column volumes of buffer A50 (20 mM Tris, 500 mM NaCl, 50 mM imidazole, pH 7.9). To elute apoA1, 8 column volumes of buffer A500 (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.9) were added to the column. All fractions of the purification step were collected and analyzed by SDS-PAGE. The buffer of the fractions containing purified apoA1 was changed to PBS using an Amicon ultracentrifugal filter (Amicon). To preserve apoA1, aliquots were flash frozen in liquid nitrogen and stored at -70°C.

[0246] Example 4. The lipid composition of siRNA-containing apolipoprotein lipid nanoparticles (aNPs) (siRNA-aNPs) affects their physicochemical properties and can be optimized to obtain siRNA-aNPs with optimal properties (Figure 4). A library of 72 siRNA-aNP formulations was established and physicochemical parameters were analyzed. The siRNA-aNP formulations contained 8-52 mol% phospholipid (i.e., 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)), 4-62 mol% cholesterol, 5-62 mol% ionizable cationic lipid (i.e., Dlin-MC3-DMA), 0-76 mol% triglyceride, 0.08-0.5 mol% apolipoprotein A1 (prepared as described in Example 3) and 0.03-0.18% nonspecific (Integrated DNA technologies-51-01-14-03) or firefly luciferase (Integrated DNA technologies-custom sequence) siRNA. The exact formulations of formulations 1, 3, 6, 7, 8, 9, 10, 11, 12, 14, 18, 19, 20, 21, 22, 23, 24, 29, 31, 32, 33, 34, 35, 39, 42, 43, 44, 45, 46, 47, 48, 50, 54, 55, 56, 59, 60, 67, 68, 71 and 72 are shown in Table 1 (Figure 11).

[0247] siRNA-aNP formulations were prepared using the procedure described in Example 2.

[0248] One day after formulation, the physicochemical properties of the individual siRNA-aNP formulations of the library were determined according to (i) particle size (z-average) and (ii) particle size dispersity as assessed using dynamic light scattering (DLS), (iii) siRNA retention using the Ribogreen assay, (iv) apolipoprotein A1 (apo-A1) using a colorimetric protein quantification assay, and (v) cholesterol and (vi) phospholipid recovery using standard colorimetric assays (Figure 4A). Data are shown in Figure 4A for both formulation types using either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) as the phospholipid. The results in Figure 4A show that the aNPs were optimized by varying the composition to obtain stable and uniform formulations of approximately 100 nm that effectively encapsulated the siRNA. The results in Figure 4A also show that apolipoprotein A1, cholesterol and phospholipids were effectively incorporated into the formulation.

[0249] Individual siRNA-aNP formulations from the library were further analyzed according to (i) particle size (number average) and (ii) particle size dispersity using dynamic light scattering (DLS) one day after manufacture, as indicated by the triglyceride content of the formulation. The results in Figure 4B show that the addition of triglycerides as bulking molecules increased siRNA-aNP size and uniformity.

[0250] Individual siRNA-aNP formulations of the library were also analyzed according to (i) particle size (number average) and (ii) particle size dispersity using dynamic light scattering (DLS) one day after manufacture, as indicated by the formulation's N / P ratio. The N / P ratio is the ratio used of the positively chargeable amine (N=nitrogen) groups of the ionizable cationic material to the negatively charged nucleic acid phosphate (P) groups in the nucleic acid component, as described elsewhere herein. The results in Figure 4C show that siRNA-aNPs were prepared with a variety of N / P ratios without affecting particle size or dispersity.

[0251] Example 5. Representative cryo-transmission electron micrographs showing that the lipid composition of siRNA-containing apolipoprotein lipid nanoparticles (aNPs) (siRNA-aNPs) can be used to influence the morphology and size of these aNPs (Figure 5). All individual siRNA-aNP formulations of the library were subjected to cryo-electron transmission electron microscopy using a FEI TITAN 300 kV to determine particle size, morphology and formulation uniformity (scale bar 50 nm).

[0252] Cryo-transmission electron microscopy (cryo-TEM) images were taken from all individual formulations of the library containing 72 siRNA-aNPs. The formulations of the 72 siRNA-aNPs were as described in Example 4. The results show that the formulations had a spherical appearance and their morphology, internal structure, size and uniformity depended on the formulation composition. For example, while formulations containing low amounts of cholesterol and triglycerides, such as formulation 1, appeared to have an internal structure containing multiple concentric rings, formulations containing high amounts of cholesterol and triglycerides, such as formulation 72, appeared to have an electron-dense core surrounded by a surface barrier. Inspection of the images shows that the particles contained a (distinct) surface barrier layer, although not necessarily a single layer, likely composed of phospholipids, cholesterol and apolipoproteins. Without wishing to be bound by any theory, the inventors believe that the layer shields and protects the siRNA embedded in the core by ionizable cationic lipids.

[0253] Example 6. Apolipoprotein lipid nanoparticles (aNPs) containing firefly luciferase siRNA (siRNA-aNPs) induce potent reporter gene expression knockdown in vitro (Figure 6). The functional effect of 72 individual formulations of the siRNA-aNP library was determined by measuring firefly luciferase knockdown in mouse RAW264.7 macrophages. More specifically, mouse RAW264.7 macrophages were transfected with pmirGLO plasmid (Promega, E1330) for stable dual reporter luciferase expression (firefly and Renilla luciferase) and subsequently exposed to individual siRNA-aNP formulations of the library containing firefly luciferase (Fluc) siRNA for 48 hours. Luminescence assays were performed according to the manufacturer's protocol (Dual-Glo Luciferase Assay System, Promega, E2920). Data were corrected for a control siRNA-aNP formulation containing a non-specific siRNA. The formulations of the 72 siRNA-aNPs are as described in Example 4.

[0254] The results show that, depending on the composition of the formulation, firefly luciferase siRNA-aNPs induced strong gene silencing compared to nonspecific siRNA-aNPs. Figure 6A shows several representative formulations resulting in silencing of over 40% and even up to 100%. The results in Figure 6B show that adding triglycerides to the formulations can affect their functional efficacy, regardless of the formulation's phospholipid type. The results in Figure 6C show that increasing the N / P ratio from 3 to 9 appeared to improve the functional efficacy, regardless of the formulation's phospholipid type.

[0255] Example 7. Apolipoprotein lipid nanoparticles (aNPs) containing radiolabeled siRNA (siRNA-aNPs) localize to hematopoietic tissues including spleen and bone marrow after intravenous administration in mice (Figure 7). siRNA-aNP formulations 3, 39, 14, 55, 22, and 72 are all formulations according to certain embodiments of the present invention and contained various amounts of phospholipid (i.e., 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)), cholesterol, ionizable cationic lipid (i.e., Dlin-MC3-DMA), triglyceride, apolipoprotein A1 (prepared as described in Example 3), and siRNA. The formulations of siRNA-aNP are as described in Table 1 (Figure 11).

[0256] 7A shows the biodistribution of siRNA-aNP following intravenous administration in mice. An exemplary siRNA-aNP formulation of the invention or a comparative LNP formulation containing zirconium-89 radiolabeled non-specific siRNA was administered to C57BL / 6 mice (n=6 / formulation). # were injected intravenously at a dose of 2 mg / kg siRNA. The LNP control formulation contained PEGylated lipid. 24 hours after injection, mice were sacrificed and organs were collected for quantitative analysis by gamma counting. Data are presented as mean ± SD of % injected dose per gram of tissue (%ID / g) and analyzed by two-way ANOVA with Tukey's post-hoc test. * indicates p value < 0.05, **** indicates p-value <0.0001.

[0257] FIG. 7B shows the biodistribution results expressed as bone marrow to liver ratio of percent injected dose per gram of tissue (%ID / g). # The LNP-siRNA comparative example consisted of Dlin-MC3-DMA, DSPC, cholesterol, and PEG-DMG (50:38.5:10:1.5 mol %) containing siRNA.

[0258] In conclusion, the data show that aNPs containing siRNA as a payload according to certain embodiments of the present invention were able to target tissues associated with the presence of immune cells after systemic injection. Furthermore, the composition of the siRNA-aNPs can be used to guide the targeting and subsequent biodistribution.

[0259] Example 8. Apolipoprotein lipid nanoparticles (aNPs) can encapsulate mRNA to obtain stable formulations and induce gene expression in vitro (Figure 8). Firefly luciferase messenger RNA (mRNA, Trilink Biotechnologies-L7602)-containing aNP formulations were prepared using the methods described in Example 2. mRNA-aNP formulations of the present invention and LNP-mRNA comparative formulation # were characterized for their particle size and particle size dispersity using dynamic light scattering (DLS) (FIG. 8A, left panel). The Ribogreen assay was used to assess mRNA capture efficiency (FIG. 8A, right panel).

[0260] FIG. 8B shows a cryo-transmission electron micrograph (scale bar 50 nm) of a representative mRNA-aNP (according to a particular embodiment of the present invention).

[0261] Human HEK293 cells were exposed to firefly mRNA-containing aNPs and comparative LNPs for 24 h. Reporter gene expression was determined by luminescence (FIG. 8C, left panel) and cell viability was determined by MTT assay (Promega-G3582) (FIG. 8C, right panel), which showed that mRNA-aNPs induced dose-dependent firefly luciferase expression without inducing toxicity in vitro.

[0262] Murine RAW264.7 macrophages were exposed to firefly mRNA-containing aNPs for 24 h. Gene expression was determined by luminescence, showing that mRNA-aNPs induced dose-dependent firefly luciferase expression in macrophage cell cultures (Figure 8D).

[0263] Primary mouse bone marrow-derived macrophages were exposed to firefly mRNA-containing aNPs for 24 h. Gene expression was determined by luminescence, showing that mRNA-aNPs induced dose-dependent firefly luciferase expression in primary cells (Figure 8E). # The LNP-mRNA comparative example consisted of Dlin-MC3-DMA, DSPC, cholesterol and PEG-DMG (50:38.5:10:1.5 mol %) containing mRNA.

[0264] Example 9. Synthesis of ionizable cationic lipids according to formulas (I)-(V) (shown in FIG. 9) Starting compounds, reagents, solvents, deuterated solvents and (purified) materials were purchased from commercial sources (e.g., Merck, ABCR, Cambridge Isotopes Laboratories, etc.). NMR analysis was performed on a Bruker 400 MHz spectrometer. MALDI-TOF-MS analysis was performed on a Bruker Autoflex spectrometer. HPLC-MS was performed on an LCQ Fleet (Thermo Scientific) equipped with an ESI ion trap MS detector as well as a PDA detector, applying a C18 reversed phase column (Kinetex 5 μm particles, 2.1 mm (id) × 50 mm, Phenomenex) with an eluent gradient from 5% acetonitrile and 95% water to 95% acetonitrile and 5% water (both containing 0.1% formic acid) at a flow rate of 0.2 mL / min.

[0265] Example of formula (II) (ICG-A type): 1,2-glycerol ionizable cationic lipid [ka] Scheme A: Synthetic route to ionizable cationic lipids according to formula (II) using ICG-A type. DPTS = 4-(dimethylamino)-pyridinium 4-toluenesulfonate; DIC = N,N'-diisopropylcarbodiimide; DCM = dichloromethane; RT = room temperature; DIPEA = diisopropylethylamine; Pd / C = palladium on carbon; THF = tetrahydrofuran; HAc = acetic acid; H2(g) = hydrogen gas.

[0266] Intermediate 1: (S)-4-((benzyloxy)methyl)-2,2-dimethyl-1,3-dioxolane This compound was obtained via benzyl protection of (S)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol according to literature procedures (Lee, Jong-Dae; et al, Organic Letters (2007), 9(2), 323-326). Yield: 16.8 g (88%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0267] Intermediate 2: (R)-3-((benzyloxy)propane-1,2-diol This compound was obtained by deprotection of the diol group of (S)-4-((benzyloxy)methyl)-2,2-dimethyl-1,3-dioxolane using acetic acid and water according to literature procedures (Lee, Jong-Dae; et al, Organic Letters (2007), 9(2), 323-326). Yield: 9.45 g (76%). 1 The 1 H-NMR spectrum was consistent with the desired structure. [ka]

[0268] Intermediate 3: (S)-3-(benzyloxy)propane-1,2-diyldidodecanoate This compound was obtained by coupling of (R)-3-((benzyloxy)propane-1,2-diol (3 g, 16.5 mmol) with dodecanoic acid (6.92 g, 34.6 mmol, 2.1 molar equivalents) in DCM (23 mL) using DPTS (0.4 g, 1.36 mmol, ca. 0.1 molar equivalents) and DIC (5.2 g, 41.3 mmol, 2.5 molar equivalents) as coupling reagents. The reaction mixture was stirred at room temperature for 24 h, after which the reaction mixture was filtered through a plug of Celite. The crude mixture was purified by silica column chromatography using 1 / 9 EtOAc / heptane as eluent. Yield: 8.21 g (91%). 1The 1 H-NMR spectrum was consistent with the desired structure.

[0269] 1 H NMR (400MHz, chloroform-d) δ7.43-7.27(m,5H,Ar-H),5.37-5.06(m,1H, chiral OCH2CHCH2O),4.67-4.43(m,2H,OCH2-Bn),4.43-4.08(m,2H,CHCH2OCO),3.67-3.31(m,2H,CHCH2OCH2),2.29(dt,J=16.9,7.5Hz, 4H,CH2CH2COO),1.59(dq,J=10.6,7.1Hz,4H,CH2CH2COO),1.26(d,J=4.2Hz,32H,CH3(CH2)8CH2),0.88(t,J=6.8Hz,6H,CH3CH2). [ka]

[0270] Intermediate 4: (S)-3-(benzyloxy)propane-1,2-diyldiundecanoate. The reaction between (R)-3-((benzyloxy)propane-1,2-diol and undecanoic acid was carried out in a similar manner as that carried out for intermediate 3. Yield: 478 mg (90%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0271] 1 H NMR (400MHz, chloroform-d) δ7.54-7.26(m,5H,Ar-H),5.33-5.12(m,1H, chiral OCH2CH CH2O),4.66-4.44(m,2H,OCH2-Bn),4.35-4.19(m,2H,CHCH2OCO),3.59(d,J=5.3 Hz,2H,CHCH2OCH2),2.30(dt,J=17.0,7.5Hz,4H,CH2CH2COO),1.74-1.49(m,4H, CH2CH2COO),1.50-1.08(m,28H,CH3(CH2)7CH2),0.88(t,J=6.8Hz,6H,CH3CH2). [ka]

[0272] Intermediate 5: (S)-3-(benzyloxy)propane-1,2-diyldidecanoate The reaction between (R)-3-((benzyloxy)propane-1,2-diol and decanoic acid was carried out in a similar manner as that carried out for intermediate 3. Yield: 505 mg (93%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0273] 1 H NMR(400MHz,chloroform-d)δ7.42-7.27(m,5H,Ar-H),5.24(dtd,J=6.4,5.2,3.7Hz,1H, Chiral OCH2CHCH2O),4.65-4.44(m,2H,OCH2-Bn),4.35-4.19(m,2H,CHCH2OCO),3.59(dd ,J=5.2,1.2Hz,2H,CHCH2OCH2),2.30(dt,J=16.9,7.5Hz,4H,CH2CH2COO),1.74-1.5 1(m,4H,CH2CH2COO),1.51-1.08(m,24H,CH3(CH2)6CH2),1.08-0.67(m,6H,CH3CH2). Subexample 1: (R)-3-((4-(dimethylamino)butanoyl)oxy)propane-1,2-diyldidodecanoate

[0274] Step 1, Building Block 1: (S)-3-Hydroxypropane-1,2-diyldidodecanoate [ka]

[0275] This compound was obtained by debenzylation of (S)-3-(benzyloxy)propane-1,2-diyldidodecanoate (intermediate 3, 8.21 g, 15 mmol) in THF (50 mL) and acetic acid (0.5 mL) using a hydrogen balloon and Pd / C (250 mg, Degussa type) as catalyst. The reaction mixture was stirred at room temperature for 24 h, after which the reaction mixture was filtered through a plug of Celite and evaporated to dryness. The crude mixture was dissolved in chloroform, washed with demineralized water and then with a saturated NaCl solution (aqueous). An oil was obtained which slowly became solid. Yield: 7.1 g (100%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0276] 1 H NMR(400MHz,chloroform-d)δ5.09(p,J=5.0Hz,1H,chiral OCH2CHCH2O),4.48-4.19(m,2H,CHCH2OCO),3.73(t,J=4.0Hz,2H,CHCH2OH),2.33(dt,J =9.0,7.5Hz,4H,CH2CH2COO),1.61(h,J=5.2,3.1Hz,4H,CH2CH2COO),1.28(d,J=14.7Hz,32H,CH3(CH2)8CH2),1.04-0.66(m,6H,CH3CH2).

[0277] Step 2: (R)-3-((4-(dimethylamino)butanoyl)oxy)propane-1,2-diyldidodecanoate [ka]

[0278] This compound was obtained by coupling (S)-3-hydroxypropane-1,2-diyldidodecanoate (building block 1, 0.1 g, 0.22 mmol) to 4-(dimethylamino)butanoic acid hydrochloride (55 mg, 0.33 mmol, 1.5 molar equivalents) in DCM (1 mL) using DIPEA (74 mg, 0.58 mmol, 2.6 molar equivalents), DPTS (6.4 mg, 0.1 molar equivalents) and DIC (41.4 mg, 0.33 mmol, 1.5 molar equivalents) as reagents. The reaction mixture was stirred at room temperature for 24 h, after which the reaction mixture was filtered through a plug of Celite and the filtrate was diluted with DCM, followed by washing with 0.1 M HCl (aq), 0.1 M NaOH (aq) and saturated NaCl (aq). The organic layer was dried over Na2SO4. The crude mixture was stirred in acetonitrile and filtered. The filtrate was evaporated to dryness to give an oil that slowly became solid at 4 °C. Yield: 90mg (80%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0279] 1 H NMR(400MHz,chloroform-d)δ5.27(tt,J=6.0,4.3Hz,1H,OCH2CHCH2O),4.30-4.15(4H,CHCH2OCO),2.44-2.25(m,8H,CH2CH2COO and NCH2),2.21(s,6H, N(CH3)2),1.78(p,J=7.4Hz,2H,NCH2CH2CH2COO),1.70-1.51(m,4H,CH2CH2COO),1.40-1.08(m,32H,CH3(CH2)8CH2),1.08-0.69(m,6H,CH3CH2). MALDI-TOF-MS (CHCA matrix, positive reflector mode): m / z (M+H) + =570.48. Calculated value: C 33 H 63 NO6 (exact mass 569.47, molecular weight 569.87). Subexample 2: (R)-3-((4-(dimethylamino)butanoyl)oxy)propane-1,2-diyldiundecanoate

[0280] Step 1, Building Block 2: (S)-3-Hydroxypropane-1,2-diyldiundecanoate [ka]

[0281] The hydrogenation reaction of (S)-3-(benzyloxy)propane-1,2-diyldiundecanoate (intermediate 4) was carried out in a similar manner as that carried out for the preparation of building block 1. Yield: 405 mg (80%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0282] 1 H NMR(400MHz,chloroform-d)δ5.09(p,J=5.1Hz,1H,chiral OCH2CHCH2O),4.42-4.20(m,2H,CHCH2OCO),3.94-3.52(m,2H,CHCH2OH),2.33(dt, J=9.1,7.5Hz,4H,CH2CH2COO),1.76-1.53(m,4H,CH2CH2COO),1.28(d,J=14.9Hz,28H,CH3(CH2)7CH2),0.88(t,J=6.8Hz,6H,CH3CH2).

[0283] Step 2: (R)-3-((4-(dimethylamino)butanoyl)oxy)propane-1,2-diyldiundecanoate [ka]

[0284] The reaction between (S)-3-hydroxypropane-1,2-diyldiundecanoate (building block 2) and 4-(dimethylamino)butanoic acid hydrochloride was carried out in a manner similar to that carried out for the preparation of subexample 1 (step 2). Yield: 96 mg (87%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0285] 1H NMR(400MHz,chloroform-d)δ5.27(tt,J=6.0,4.3Hz,1H,OCH2CHCH2O),4.30-4.15(m,4H,CHCH2OCO),2.55-2.25(m,8H,(CH3)2NCH2CH2CH2COO,(CH3)2NCH2CH2CH2COO and C 10 Tail CH2CH2COO),2.21(s,6H,N(CH3)2),1.78(p,J=7.4Hz,2H,NCH2CH2CH2COO),1.61(td,J=7.3, 6.8,3.1Hz,4H,CH2CH2COO),1.28(d,J=14.1Hz,28H,CH3(CH2)7CH2),1.06-0.67(m,6H,CH3CH2).

[0286] MALDI-TOF-MS (CHCA matrix, positive reflector mode): m / z (M+H) + = 542.45, (M+Na) + =564.43. Calculated value: C 31 H 59 NO6 (exact mass 541.43, molecular weight 541.81). Subexample 3: (R)-3-((4-(dimethylamino)butanoyl)oxy)propane-1,2-diyldidecanoate

[0287] Step 1, Building Block 3: (S)-3-Hydroxypropane-1,2-diyldidecanoate [ka]

[0288] The hydrogenation reaction of (S)-3-(benzyloxy)propane-1,2-diyldidecanoate (intermediate 5) was carried out in a similar manner as that carried out for the preparation of building block 1. Yield: 412 mg (100%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0289] 1H NMR(400MHz,chloroform-d)δ5.09(p,J=5.0Hz,1H,chiral OCH2CHCH2O),4.46-4.20(m,2H,CHCH2OCO),3.74(t,J=5.6Hz,2H,CHCH2OH),2.33( dt,J=9.1,7.5Hz,4H,CH2CH2COO),1.78-1.54(m,4H,CH2CH2COO),1.28(d,J=8.8Hz,24H,CH3(CH2)6CH2),1.09-0.51(m,6H,CH3CH2).

[0290] Step 2: (R)-3-((4-(dimethylamino)butanoyl)oxy)propane-1,2-diyldidecanoate [ka]

[0291] The reaction between (S)-3-hydroxypropane-1,2-diyldidecanoate (building block 3) and 4-(dimethylamino)butanoic acid hydrochloride was carried out in a similar manner to that carried out for subexample 1 (step 2), but without DIPEA. Yield: 110 mg (80%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0292] 1 H NMR(400MHz,chloroform-d)δ5.27(tt,J=6.0,4.3Hz,1H,OCH2CHCH2O),4.30-4.15(m,4H,CHC H2OCO),2.48-2.23(m,8H,(CH3)2NCH2CH2CH2COO,(CH3)2NCH2CH2CH2COO and CH2CH2C of C9 tail OO),2.21(s,6H,N(CH3)2),1.78(p,J=7.4Hz,2H,NCH2CH2CH2COO),1.61(ddt,J=11.7,7. 8,4.7Hz,4H,CH2CH2COO),1.40-1.12(m,24H,CH3(CH2)6CH2),1.02-0.73(m,6H,CH3CH2).

[0293] MALDI-TOF-MS (CHCA matrix, positive reflector mode): m / z (M+H) + =514.43. Calculated value: C 29 H 55 NO6 (exact mass 513.40, molecular weight 513.76). Subexample 4: (R)-3-((3-(dimethylamino)propanoyl)oxy)propane-1,2-diyldidodecanoate [ka]

[0294] The reaction between (S)-3-hydroxypropane-1,2-diyldidodecanoate (building block 1) and 3-(dimethylamino)propanoic acid hydrochloride was carried out in a manner similar to that carried out for the preparation of subexample 1 (step 2). Yield: 83 mg (68%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0295] 1 H NMR(400MHz,chloroform-d)δ5.27(tt,J=6.0,4.3Hz,1H,OCH2CHCH2O),4.40-4.16(m,4H,CHCH2OCO),2.68 -2.55(m,2H,(CH3)2NCH2CH2COO),2.55-2.43(m,2H,(CH3)2NCH2CH2COO),2.31(td,J=7.5,3.7Hz,4H,C 11 Tail CH2CH2COO), 2.23(s,6H,N(CH3)2),1.62(qt,J=7.0,3.4Hz,4H,CH2CH2COO),1.27(d,J=9.9Hz,32H,CH3(CH2)8CH2),1.09-0.64(m,6H,CH3CH2).

[0296] MALDI-TOF-MS (CHCA matrix, positive reflector mode): m / z (M+H) + =556.48, (M+Na) + =578.44. Calculated value: C 32 H 61NO6 (exact mass 555.45, molecular weight 555.84). Subexample 5: (R)-3-((3-(dimethylamino)propanoyl)oxy)propane-1,2-diyldiundecanoate [ka]

[0297] The reaction between (S)-3-hydroxypropane-1,2-diyldiundecanoate (building block 2) and 3-(dimethylamino)propanoic acid hydrochloride was carried out in a manner similar to that carried out for the preparation of subexample 1 (step 2). Yield: 108 mg (82%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0298] 1 H NMR(400MHz,chloroform-d)δ5.27(tt,J=6.0,4.3Hz,1H,OCH2CHCH2O),4.31-4.04(m,4H,CHCH2OCO),2.74-2. 56(m,2H,(CH3)2NCH2CH2COO),2.56-2.42(m,2H,(CH3)2NCH2CH2COO),2.31(ddd,J=9.1,5.6,2.4Hz,4H,C 10 Tail CH2CH2COO), 2.25(d,J=1.2Hz,6H,N(CH3)2),1.62(tt,J=7.3,3.6Hz,4H,CH2CH2COO),1.51-1.07(m,28H,CH3(CH2)7CH2),1.07-0.66(m,6H,CH3CH2).

[0299] MALDI-TOF-MS (CHCA matrix, positive reflector mode): m / z (M+H) + =528.45. Calculated value: C 30 H 57 NO6 (exact mass 527.42, molecular weight 527.79). Subexample 6: (R)-3-((3-(dimethylamino)propanoyl)oxy)propane-1,2-diyldidecanoate [ka]

[0300] The reaction between (S)-3-hydroxypropane-1,2-diyldidecanoate (building block 3) and 3-(dimethylamino)propanoic acid hydrochloride was carried out in a manner similar to that carried out for the preparation of subexample 1, but without DIPEA (step 2). Yield: 131 mg (80%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0301] 1 H NMR(400MHz,chloroform-d)δ5.42-5.06(m,1H,OCH2CHCH2O),4.51-4.17(m,4H,CHCH2OCO),2.75-2.53(m,4H,(CH3)2NCH2CH2COO,(CH3)2NCH2CH2COO),2.48 -2.03(m,10H,N(CH3)2),C9 tail CH2CH2COO),1.61(td,J=7.3,3.5Hz,4H,CH2CH2COO),1.39-1.18(m,24H,CH3(CH2)6CH2),0.88(t,J=6.8Hz,6H,CH3CH2).

[0302] MALDI-TOF-MS (CHCA matrix, positive reflector mode): m / z (M+H) + =500.42. Calculated value: C 28 H 53 NO6 (exact mass 499.39, molecular weight 499.73). Sub-example 7: (R)-3-((3-(dimethylamino)propanoyl)oxy)propane-1,2-diyl distearate [ka]

[0303] Building block 4, i.e. (S)-3-hydroxypropane-1,2-diyl distearate, was purchased from Merck (1,2-distearoyl-sn-glycerol, CAS 10567-21-2). The reaction between this building block 4 and 3-(dimethylamino)propanoic acid hydrochloride was carried out in a similar manner to that carried out for the preparation of sub-example 1, except without DIPEA (step 2). Also, the reaction was stirred at 40° C. instead of room temperature. 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0304] 1 H NMR(400MHz,chloroform-d)δ5.27(tt,J=6.0,4.2Hz,1H,OCH2CHCH2O),4.30-4.16(m,4H,CHCH2OCO),2.60(dd,J=7. 4,6.0Hz,2H,(CH3)2NCH2CH2COO),2.49(dd,J=7.5,6.2Hz,2H,(CH3)2NCH2CH2COO),2.31(td,J=7.6,3.7Hz,4H,C 18 CH2CH2COO in the tail), 2.23 (s, 6H, N(CH3)2), 1.70-1.51 (m, 4H, CH2CH2COO), 1.26 (s, 56H, CH3(CH2) 14 CH2),0.88(t,J=6.8Hz,6H,CH3CH2). Examples of formula (I) (ICG-A type): 1,2-glycerol ionizable cationic lipids Subexample 8: (R)-3-((4-(dimethylamino)butanoyl)oxy)propane-1,2-diyl dioleate [ka]

[0305] Building block 5, i.e. (S)-3-hydroxypropane-1,2-diyl dioleate, was purchased from ABCR. It is a racemic compound (CAS2442-61-7). The reaction between building block 5 and 4-(dimethylamino)butanoic acid hydrochloride was carried out in a manner similar to that carried out for the preparation of sub-example 1 (step 2). 1The 1 H-NMR spectrum was consistent with the desired structure.

[0306] 1 H NMR (400 MHz, chloroform-d) δ 5.35 (td, J = 7.2, 6.1, 4.2 Hz, 4H, CH=CH), 5.25 (ddd, J = 10.2, 5.8, 4.3 Hz, 1H, OCH2CHCH2O), 4.30-4.14 (m, 2H, CHCHOCO), 2.43-2.28 (m, 8H, (CH3)2NCH2CH2CH2COO, (CH3)2NCH2CH2CH2COO, CH2 of the oleic acid tail CH2COO),2.26(s,6H,N(CH3)2),2.11-1.92(m,8H,CH2CH=CHCH2),1.81(p,J=7.4Hz,2H,NCH2CH2CH2COO),1.62( q,J=7.2Hz,4H,CH2CH2COO),1.42-1.19(m,40H,CH3(CH2)6CH2CH=CH(CH2)4CH2COO),0.95-0.82(m,6H,CH3CH2). Subexample 9: (R)-3-((3-(dimethylamino)propanoyl)oxy)propane-1,2-diyl dioleate [ka]

[0307] Building block 5, i.e. (S)-3-hydroxypropane-1,2-diyl dioleate, was purchased from ABCR. It is a racemate (Cas[2442-61-7]). The reaction between building block 5 and 3-(dimethylamino)propanoic acid hydrochloride was carried out in a similar manner to that carried out for the preparation of subexample 1, except that DIPEA was not used (step 2). Yield: 120 mg (33%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0308] 1H NMR(400MHz,chloroform-d)δ5.46-5.19(m,5H,CH=CH,OCH2CHCH2O),4.31-4.15(m,4H,CHCH2OCO),2.62(td,J= 7.1,3.8Hz,2H,(CH3)2NCH2CH2COO),2.50(td,J=7.2,2.6Hz,2H,(CH3)2NCH2CH2COO),2.31(dd,J=9.1,6.1H z, 4H, CH2CH2COO of the oleic acid tail), 2.24 (s, 6H, N(CH3)2), 2.02 (dq, J = 12.8, 6.7Hz, 8H, CH2CH=CHCH2), 1.60 (q, J = 7.2Hz, 4H, CH2CH2COO), 1.52-1.18 (m, 40H, CH3(CH2)6CH2CH=CH(CH2)4CH2COO), 0.99-0.77 (m, 6H, CH3CH2).

[0309] HPLC-MS: (M+H+)=720. Calculated value: C 44 H 81 NO6 (exact mass, 719.61 molecular weight 720.13). Example of formula (III) (ICG-A type): 1,3-glycerol ionizable cationic lipid Subexample 10: 2-((3-(dimethylamino)propanoyl)oxy)propane-1,3-diyldidodecanoate

[0310] Step 1: 2-oxopropane-1,3-diyldidodecanoate [ka]

[0311] This compound was obtained by coupling 1,3-dihydroxypropan-2-one (0.5 grams, 5.6 mmol) with dodecanoic acid (2.28 grams, 11.4 mmol, 2.05 molar equivalents) in DCM (50 mL) using DIPEA (2.32 mL, 13.3 mmol, 2.4 molar equivalents), DMAP (67 mg, 0.56 mmol, 0.2 molar equivalents) and EDC.HCl (2.65 grams, 13.8 mmol, 2.4 molar equivalents) as coupling reagents. Extraction / washing steps followed by purification work by silica column chromatography. Yield: 2.27 g (90%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0312] 1 H NMR (400 MHz, chloroform-d) δ 4.75 (s, 4H, OCCHOCO), 2.42 (t, J = 7.5 Hz, 4H, CHCHCOO), 1.67 (q, J = 7.4 Hz, 4H, CHCHCOO), 1.50-1.07 (m, 32H, CH(CH)CHCH), 1.07-0.64 (m, 6H, CHCH).

[0313] Step 2: 2-Hydroxypropane-1,3-diyldidodecanoate [ka]

[0314] This compound was obtained by reduction of 2-oxopropane-1,3-diyldidodecanoate (1.54 grams, 3.37 mmol) with sodium borohydride (229 mg, 7.9 mmol, 2.4 molar equivalents) in THF (45 mL) and water (3 mL). Sodium borohydride was added to a cooled solution of ketone in THF / water (ice bath at 0° C.). The reaction mixture was stirred for 2 hours, after which the reaction was quenched by adding acetic acid (1 mL). The reaction mixture was diluted with chloroform (50 mL) and washed with saturated Na2CO3 solution and saturated NaCl solution. The organic layer was dried over Na2SO4. The crude product was then purified by silica column chromatography using 2% acetone in chloroform as eluent. A pure product fraction was obtained (440 mg, 28% yield) and also an impure fraction. 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0315] 1 H NMR(400MHz,chloroform-d)δ4.31-4.01(m,5H,OCH2CH(OH)CH2O),2.42(d,J=4.8Hz,1H,OH),2.35(t,J=7.6Hz,4H,CH 2CH2COO),1.74-1.58(m,4H,CH2CH2COO),1.27(d,J=12.5Hz,32H,CH3(CH2)8CH2),0.88(t,J=6.8Hz,6H,CH3CH2).

[0316] Step 3: 2-((3-(dimethylamino)propanoyl)oxy)propane-1,3-diyldidodecanoate [ka]

[0317] The reaction between 2-hydroxypropane-1,3-diyldidodecanoate and 3-(dimethylamino)propanoic acid hydrochloride was carried out in a manner similar to that carried out for the preparation of subexample 1 (step 2). Yield: 165 mg (68%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0318] 1 H NMR(400MHz,chloroform-d)δ5.28(tt,J=5.8,4.4Hz,1H,OCH2CHCH2O),4.30-4.16(m,4H,CHCH2OCO),2.61(td,J=7. 1,1.1Hz,2H,(CH3)2NCH2CH2COO),2.50(ddd,J=7.9,6.9,1.1Hz,2H,(CH3)2NCH2CH2COO),2.31(t,J=7.6Hz,4H,C 11 Tail CH2CH2COO), 2.23(s,6H,N(CH3)2),1.62(q,J=7.1Hz,4H,CH2CH2COO),1.27(d,J=8.7Hz,32H,CH3(CH2)8CH2),0.99-0.71(m,6H,CH3CH2).

[0319] MALDI-TOF-MS (CHCA matrix, positive reflector mode): m / z (M+H) + =556.46. Calculated value: C 32 H 61 NO6 (exact mass 555.45, molecular weight 555.84). Example of formula (II) (ICG-B type): 1,2-glycerol ionizable cationic lipid Sub-example 11: (R)-3-((5-guanidinopentanoyl)oxy)propane-1,2-diyldidodecanoate

[0320] Step 1: (Z)-5-(2,3-bis(tert-butoxycarbonyl)guanidino)pentanoic acid [ka]

[0321] The reaction of tert-butyl (E)-(((tert-butoxycarbonyl)imino)(1H-pyrrol-1-yl)methyl)carbamate (0.5 grams, 4.25 mmol) with 5-aminopentanoic acid (1.45 grams, 4.68 mmol, 1.1 molar equivalents) was carried out in pyridine (10 mL) at room temperature for 48 hours. The reaction mixture suspension became a clear solution. The mixture was evaporated to dryness and the residue was dissolved in 1M NaOH (25 mL) and washed with EtOAc (50 mL). The aqueous layer was acidified to pH 3 using concentrated HCl solution. The aqueous layer was extracted twice with EtOAc and the collected organic layers were first washed with saturated NaCl solution and then dried over Na2SO4. The solvent was evaporated to give a white solid (1.22 grams). This crude product was further purified by stirring in a mixture of 1 / 3 EtOAc / heptane with dropwise addition of acetic acid (approximately 0.2 v / v%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0322] 1 H NMR(400MHz,chloroform-d)δ8.35(t,J=5.3Hz,1H),3.73-3.15(m,2H,NCH2CH2),2.40(t,J=7.0H z,2H,CH2CH2COOH),1.83-1.57(m,4H.NCH2CH2CH2CH2COOH),1.50(d,J=2.3Hz,18H,CH3Boc).

[0323] HPLC-MS: m / z (M+H) + =359.92. Calculated value: C 16 H 29 N3O6 (exact mass 359.21, molecular weight 359.42).

[0324] Step 2: (R,E)-6-((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,7-diazahexadec-5-ene-15,16-diyldidodecanoate [ka]

[0325] The reaction of (Z)-5-(2,3-bis(tert-butoxycarbonyl)guanidino)pentanoic acid (0.43 grams, 1.2 mmol, 1.1 molar equivalents) with (S)-3-hydroxypropane-1,2-diyldidodecanoate (building block 1, 0.5 grams, 1.1 mmol) was carried out in DCM (4 mL) using DIC (0.15 g, 1.2 mmol, 1.1 molar equivalents) and DPTS (32 mg, 0.11 mmol, 0.1 molar equivalents) as reagents. The mixture was stirred at room temperature for 72 hours. The mixture was diluted with DCM (25 mL) and then washed successively with 1 M NaOH (25 mL) and saturated NaCl (aq) solution. The solution was dried over Na2SO4. The crude product was purified by silica column chromatography using EtOAc / heptane (1 / 3) as eluent. Yield: 0.471g (54%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0326] 1 H NMR (400MHz, chloroform-d) δ11.50(s,1H,NH),8.32(t,J=5.2Hz,1H,NH),5.26(tt,J=6.0,4.3Hz,1H,OCH2CH CH2O),4.30-4.14(m,4H,CHCH2OCO),3.43(td,J=6.9,5.2Hz,2H,NCH2(CH2)2CH2COO),2.59-2.20(m,6H,C 11 Tail CH2CH2COO,NCH2(CH2)2CH2COO),1.84-1.56(m,8H,NCH2CH2CH2CH2COOH,CH2CH2COO),1. 50(d,J=3.9Hz,18H,CH3Boc),1.27(d,J=9.2Hz,32H,CH3(CH2)8CH2),1.11-0.64(m,8H,CH3).

[0327] Step 3: (R)-3-((5-guanidinopentanoyl)oxy)propane-1,2-diyldidodecanoate [ka]

[0328] (R,E)-6-((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,7-diazahexadec-5-ene-15,16-diyldidodecanoate (0.471 grams, 0.6 mmol) was deprotected with TFA in DCM at room temperature for 24 hours. The reaction mixture was evaporated and coevaporated with DCM several times to remove excess TFA. The product was diluted with chloroform and washed first with 0.05 M NaOH solution (25 mL) and then with saturated NaCl(aq) solution. The organic layer was dried over Na2SO4 and the solution was concentrated to give the product. Yield: 0.35 g (100%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0329] 1 H NMR(400MHz,chloroform-d)δ5.27(td,J=6.1,3.0Hz,1H,OCH2CHCH2O),4.49-4.00(m,4H,CHCH2OCO),3.21(q,J=6.8,6.1Hz,2H,NCH2(CH2)2CH2COO),2.57-2.26(m,6H,C 11 Tail CH2CH2COO,NCH2(CH2)2CH2COO),1.88-1.48(m,8H,NCH2CH2CH2CH2COO,CH2CH2COO),1.27(d,J=9.0Hz,32H,CH3(CH2)8CH2),0.88(t,J=6.7Hz,6H,CH3).

[0330] MALDI-TOF-MS (CHCA matrix, positive reflector mode): m / z (M+H) + =598.46. Calculated value: C 33 H 63 N3O (exact mass 597.47, molecular weight 597.88). Examples of formula (IV) (ICG-A type): Ionizable cationic lipids of the serinol-derived type Subexample 12: 2-(4-(dimethylamino)butanamido)-2-methylpropane-1,3-diyldidodecanoate

[0331] Step 1: tert-Butyl (1,3-dihydroxy-2-methylpropan-2-yl)carbamate [ka]

[0332] 2-Amino-2-methylpropane-1,3-diol (5 grams, 48 ​​mmol) was reacted with BOC anhydride (8 grams, 96 mmol, 2 molar equivalents) in a mixture of methanol (120 mL) and THF (30 mL). The BOC anhydride solution was added dropwise to the reaction mixture cooled in an ice bath (0° C.). The reaction mixture was stirred at room temperature for 24 hours and then concentrated. The residue was dissolved in EtOAc, washed three times with demineralized water and dried over Na2SO4. The crude product was recrystallized from EtOAc. Yield: 3.5 g (36%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0333] 1 H NMR(400MHz,chloroform-d)δ4.98(s,1H,NH),3.78-3.62(m,4H,OCOCH2CCH2OCO),3.52(s,2H,OH),1.44(s,9H,CH3Boc),1.17(s,3H,NCCH3).

[0334] Step 2: 2-((tert-butoxy-carbonyl)amino)-2-methylpropane-1,3-diyldidodecanoate [ka]

[0335] tert-Butyl (1,3-dihydroxy-2-methylpropan-2-yl)carbamate (0.5 grams, 2.43 mmol) was coupled to dodecanoic acid (1.02 grams, 5.1 mmol, 2.1 molar equivalents) in DCM (4 mL) using DPTS (70 mg, 0.24 mmol, 0.1 molar equivalents) and DIC (0.77 grams, 41.2 mmol, 6.1 molar equivalents) as reagents. The reaction mixture was stirred at room temperature for 24 hours and then filtered through a plug of Celite. The filtrate was diluted with DCM (25 mL) and then washed with 0.1 M HCl (25 mL), 0.1 M NaOH (25 mL) and saturated NaCl (aq) solution (25 mL). The solution was dried over Na2SO4. Further purification was performed by silica column chromatography using EtOAc / Hept 1 / 20 as eluent. Yield: 1.13g (82%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0336] 1 H NMR (400 MHz, chloroform-d) δ 4.73 (s, 1H, NH), 4.42-3.96 (m, 4H, OCOCH2CCH2OCO), 2.33 (t, J = 7.5 Hz, 4H, C 11 Tail CH2CH2COO), 1.62(p,J=7.7Hz,4H,CH2CH2COO),1.52(s,9H,CH3Boc),1.36(s,3H,NCCH3),1.33-1.09(m,32H,CH3(CH2)8CH2),0.88(t,J=6.7Hz,6H,CH3).

[0337] Step 3: 2-Amino-2-methylpropane-1,3-diyldidodecanoate [ka]

[0338] (2-((tert-butoxycarbonyl)amino)-2-methylpropane-1,3-diyldidodecanoate (1.13 grams, 1.98 mmol) was stirred in TFA (2 mL) and DCM (4 mL) at room temperature for 24 hours. The solvent was evaporated and the crude product residue (white solid) was redissolved in chloroform, followed by washing the organic solution with 1.0 M NaOH (25 mL) and saturated NaCl(aq) solution. The product was isolated after drying in Na2SO4. Yield: 0.853 g (91%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0339] 1 H NMR (400 MHz, chloroform-d) δ 3.94 (q, J = 10.9 Hz, 4H, OCOCH2CCH2OCO), 2.33 (t, J = 7.6 Hz, 4H, C 11 tail CH2CH2COO), 1.63(q,J=7.2Hz,4H,CH2CH2COO),1.27(d,J=11.9Hz,32H,CH3(CH2)8CH2),1.12(s,3H,NCCH3),0.88(t,J=6.7Hz,6H,CH3).

[0340] MALDI-TOF-MS (CHCA matrix, positive reflector mode): m / z (M+H) + = 470.43, (M + Na) + =492.41. Calculated value: C 28 H 55 NO4 (exact mass 469.41, molecular weight 469.75).

[0341] Step 4: 2-(4-(dimethylamino)butanamido)-2-methylpropane-1,3-diyldidodecanoate [ka]

[0342] 2-Amino-2-methylpropane-1,3-diyldidodecanoate (0.32 grams, 0.68 mmol) was coupled to 4-(dimethylamino)butanoic acid hydrochloride (0.17 grams, 1.02 mmol, 1.5 molar equivalents) in DCM (2 mL) using DPTS (20 mg, 0.24 mmol, 0.07 molar equivalents) and DIC (0.127 grams, 1.01 mmol, 1.5 molar equivalents) as reagents. The reaction mixture was stirred at room temperature for 24 hours and then filtered through a plug of Celite. The filtrate was diluted with DCM (25 mL), washed with 0.1 M HCl (25 mL), 0.1 M NaOH (25 mL), saturated NaCl(aq) (25 mL), and finally dried over Na2SO4. The crude product was purified by silica column chromatography using an eluent gradient of 2% MeOH / chloroform to 10% MeOH / chloroform. Yield: 294 mg (75%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0343] 1 H NMR(400MHz,chloroform-d)δ6.86(s,1H,NH),4.50-4.09(m,4H,OCOCH2CCH2OCO),2.40(t,J=6.6Hz,2H.CH2N(CH3)2),2.36-2.16(m,10H,NCOCH2,OCOCH2 and CH2N(CH 3)2),1.79(q,J=6.8Hz,2H,CH2CH2N(CH3)2),1.75-1.54(m,4H,CH2CH2COO),1.3 9(s,3H,NCCH3),1.27(d,J=9.1Hz,32H,CH3(CH2)8CH2),1.12-0.52(m,6H,CH3).

[0344] MALDI-TOF-MS (CHCA matrix, positive reflector mode): m / z (M+H) + =583.47, (M+Na) + =605.46. Calculated value: C 34 H 66 N2O5 (exact mass 582.50, molecular weight 582.91).

[0345] Examples of formula (V) (ICG-A type): Cholesteryl ionizable cationic lipids [ka] Scheme B: Synthetic route to cholesteryl ionizable cationic lipids according to formula (V) using ICG-A type. DPTS = 4-(dimethylamino)-pyridinium 4-toluenesulfonate; DIC = N,N'-diisopropylcarbodiimide; DCM = dichloromethane. Subexample 13: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(dimethylamino)butanoate [ka]

[0346] Cholesterol (0.30 g, 0.77 mmol) was reacted with 4-(dimethylamino)butanoic acid hydrochloride (0.195 g, 1.16 mmol, 1.5 molar equivalents) in DCM (2 mL) using DPTS (21.7 mg, 0.077 mmol, 0.1 molar equivalents) and DIC (0.15 g, 1.16 mmol, 1.5 molar equivalents) as reagents. The reaction mixture was stirred at room temperature for 24 h and then filtered through a plug of Celite. The filtrate was diluted with DCM (25 mL), washed with 0.1 M NaOH (25 mL) and saturated NaCl(aq) (25 mL) and finally dried over Na2SO4. The crude mixture was precipitated from chloroform (1.5 mL) into acetonitrile (50 mL) at 0° C. The product precipitate was collected by filtration, washed with cold acetonitrile and dried at 40° C. Yield: 165 mg (42%).

[0347] 1H NMR(400MHz,chloroform-d)δ5.37(d,J=5.0Hz,1H,C=CHCH2),4.71-4.49(m,1H,CH2CHCOO),2.37-2.2 5(m,6H,CH2N(CH3)2,CH=CCH2CHCOO,CH2COO),2.22(s,6H,CH2N(CH3)2),2.08-1.91(m,2H),1.91- 1.71(m,5H),1.57-1.41(m,6H),1.34(d,J=8.2Hz,3H),1.26(d,J=10.9Hz,1H),1.22-1.05(m,7H) ,1.02(s,5H),0.99-0.94(m,2H),0.91(d,J=6.5Hz,3H),0.86(dd,J=6.6,1.8Hz,6H),0.68(s,3H). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0348] HPLC-MS: m / z (M+H) + =500.42. Calculated value: C 33 H 57 NO2 (exact mass 499.44, molecular weight 499.82). Subexample 14: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(dimethylamino)propanoate [ka]

[0349] Cholesterol (0.097 grams, 0.25 mmol) was coupled to 3-(dimethylamino)propanoic acid hydrochloride (0.058 grams, 0.375 mmol, 1.5 molar equivalents) using a procedure similar to that described in Example 13. Yield: 76 mg (63%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0350] 1H NMR (400MHz, chloroform-d) δ5.37(d,J=4.9Hz,1H),4.62(t,J=5.9Hz,1H),2.67-2.56(m,2H) ,2.51-2.41(m,2H),2.32(d,J=7.5Hz,2H),2.24(s,6H),2.05-1.91(m,2H),1.84(tt,J=9. 5,4.2Hz,3H),1.66-1.42(m,9H),1.42-1.23(m,4H),1.23-1.04(m,7H),1.02(s,4H),0.96 (dd,J=11.3,5.3Hz,2H),0.91(d,J=6.5Hz,3H),0.86(dd,J=6.6,1.8Hz,6H),0.68(s,3H).

[0351] HPLC-MS: m / z (M+H) + =486.33. Calculated value: C 32 H 55 NO2 (exact mass 485.42, molecular weight 485.80). Subexample 15: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl dimethyl glycinate [ka]

[0352] Cholesterol (0.193 grams, 0.5 mmol) was coupled to dimethyl-glycine hydrochloride (0.077 grams, 0.75 mmol, 1.5 molar equivalents) using a procedure similar to that described in Example 13. Yield: 147 mg (63%). 1 The 1 H-NMR spectrum was consistent with the desired structure.

[0353] 1H NMR(400MHz,chloroform-d)δ5.38(d,J=5.1Hz,1H),4.78-4.61(m,1H),3.14(s,2H),2.35(s,8H),2.08-1.92(m,2H),1.92-1.75(m,3H),1.72-1.41(m,10) H),1.30(dd,J=31.8,9.6Hz,4H),1.23-1.04(m,7H),1.02(s,4H),0.99-0. 94(m,2H),0.91(d,J=6.5Hz,3H),0.86(dd,J=6.6,1.8Hz,6H),0.68(s,3H).

[0354] HPLC-MS: m / z (M+H) + =472.17. Calculated value: C 31 H 53 NO2 (exact mass 471.41, molecular weight 471.77).

[0355] Example 10. Apolipoprotein lipid nanoparticles (aNPs) containing siRNA (siRNA-aNPs) can be prepared using a variety of ionizable cationic materials to yield stable formulations (Figure 10). siRNA-aNP formulations containing phospholipids, cholesterol, selected ionizable cationic materials as shown in Figure 9, triglyceride, apolipoprotein A1 and siRNA. The siRNA-aNPs were prepared using the procedure described in Example 2. One day after formulation, the individual siRNA-aNP formulations of the library and the LNP-siRNA comparative formulation # The particles were analyzed for (A) particle size and (B) particle size dispersity using dynamic light scattering (DLS), and (C) siRNA retention using the Ribogreen assay. # The LNP-siRNA comparative example consisted of Dlin-MC3-DMA, DSPC, cholesterol, and PEG-DMG (50:38.5:10:1.5 mol %) containing siRNA.

[0356] In addition, non-optimized siRNA-aNP formulations of ionizable cationic materials 17 and 19 were tested and showed moderate (~50%) silencing capacity in murine RAW264.7 macrophages transfected with pmirGLO plasmid (Promega) for stable dual reporter luciferase expression (firefly and Renilla luciferase).

Claims

1. A nanoparticle comprising a core surrounded by a surface layer, wherein the core comprises a nucleic acid and a cationic or ionizable cationic lipid, and the surface layer comprises a phospholipid, a sterol, and an apolipoprotein or apolipoprotein mimetic or a combination thereof is a nanoparticle.

2. The nanoparticle according to claim 1, wherein the apolipoprotein, apolipoprotein mimetic, or a combination thereof is located on the outer surface of the surface layer.

3. The nanoparticle according to claim 1, wherein the nanoparticle core further comprises a filler, preferably a filler selected from triacylglycerol and cholesteryl ester or a combination thereof.

4. The nanoparticle according to claim 3, wherein the triacylglycerol is tricaprylin and / or the cholesteryl ester is cholesteryl caprylate and / or cholesteryl oleate.

5. The nanoparticle according to claim 1, wherein the nucleic acid is RNA, DNA, or a nucleic acid analog.

6. The nanoparticle according to claim 5, wherein the RNA is microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), 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 a combination and / or modification thereof.

7. The nanoparticle according to claim 5, wherein the DNA is single-stranded or double-stranded DNA.

8. The nanoparticle according to claim 1, wherein 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 modification of the phosphodiester backbone or 2'-ribose.

9. The nanoparticle according to claim 8, wherein the nucleotide or nucleoside analog is selected from locked nucleic acid (LNA), bridged nucleic acid (BNA), morpholino, or peptide nucleic acid (PNA).

10. The nanoparticle according to claim 1, wherein the apolipoprotein is selected from ApoA1, ApoA1-Milano, ApoA2, ApoA4, ApoA5, ApoB48, ApoB100, ApoC-I, ApoC-II, ApoC-III, ApoC-IV, ApoD, ApoE, ApoF, ApoH, ApoL, ApoM, and combinations thereof.

11. The nanoparticle according to claim 10, wherein the apolipoprotein is selected from ApoA1, ApoA2, ApoA4, ApoA5, ApoB100, ApoC-I, ApoC-II, ApoC-III, ApoC-IV, ApoE, and combinations thereof.

12. The nanoparticle according to claim 10, wherein the apolipoprotein is selected from ApoA1, ApoA4, ApoA5, ApoB100, ApoC-III, ApoE, and combinations thereof.

13. The nanoparticle according to claim 10, wherein the apolipoprotein is selected from ApoA1, ApoB100, ApoE, and combinations thereof.

14. The apolipoprotein in the nanoparticle is - to prevent aggregation during preparation and storage, - to improve in vivo stability, - to provide natural stealth properties, and / or - to promote interaction with immune cells for use in the nanoparticle according to claim 1.

15. The cationic or ionizable cationic lipid of the nanoparticle is selected from ionizable cationic esters of long-chain alcohols, ionizable cationic esters of diglycerides, or ionizable cationic esters of sterols, or combinations thereof, of the nanoparticle according to claim 1.

16. The ionizable cationic lipid is a molecule described in any one of formulas (I), (II), (III), (IV), or (V), 【Chemical 35】 wherein ICG is 【Chemical 36】 and the wavy line indicates the point of attachment to the compound of formulas (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, Each R １ 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 said alkyl or alkenyl group optionally contains up to 5 heteroatoms independently selected from O and N, R ２ is selected from the group consisting of hydrogen, methyl, ethyl and -CH ２ -O-C(O)-R １ａ and is selected from the group consisting of; R ３ is selected from the group consisting of hydrogen, aryl, arylene-alkyl, alkylene-aryl and straight-chain C1-C6 alkyl groups, R １ａ 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 said alkyl or alkenyl group optionally contains up to 5 heteroatoms independently selected from O and N, Each R ｘ is independently selected from the group consisting of methyl, ethyl, propyl and -CH ２ -CH ２ -OH, Each R ｙ group is independently selected from the group consisting of hydrogen, linear or branched C1-C18 alkyl, aryl, arylene-alkyl or alkylene-aryl groups, and said alkyl group optionally contains up to 5 heteroatoms independently selected from O and N, molecule, or a rotational isomer, tautomer, stereoisomer, or positional isomer thereof, of the nanoparticle according to claim 1.

17. The nanoparticle according to claim 1, wherein the sterol is selected from cholesterol, desmosterol, stigmasterol, β-sitosterol, ergosterol, hopanoid, hydroxysteroid, phytosterol, steroid, hydrogenated cholesterol, campesterol, animal sterol, or a combination thereof.

18. The nanoparticle according to claim 1, wherein the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine, and phosphatidylglycerol, or a combination thereof.

19. The nanoparticle according to claim 18, wherein at least one, more preferably both, of the acyl groups in the phospholipid are long-chain fatty acids.

20. The nanoparticle according to claim 19, wherein the long-chain fatty acid is selected from myristoleic acid, palmitoleic acid, and oleic acid, or a combination thereof.

21. The nanoparticle according to claim 1, wherein the phospholipid is selected from the group consisting of dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dilauroyl phosphatidylglycerol (DLPG), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylglycerol (DSPG), dioleoyl phosphatidylglycerol (DOPG), dilauroyl phosphatidylethanolamine (DLPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE), dilauroyl phosphatidylserine (DLPS), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylserine (DSPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or a combination thereof.

22. The amount of the apolipoprotein is in the range of 0.08 to 2.0 mol%, for example, 0.10 to 2.0 mol%, and / or The amount of the phospholipid is in the range of 5 to 90 mol%, for example, 15 to 90 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%, and / or The amount of the cationic or ionizable cationic lipid is in the range of 5.0 to 80 mol%, for example, 8.0 to 80 mol%, and the mole percentage is based only on the total amount of the apolipoprotein, the phospholipid, the sterol, and the cationic or ionizable cationic lipid in the nanoparticles. The nanoparticles according to claim 1

23. The amount of the apolipoprotein and / or apolipoprotein mimetic is in the range of 0.1 to 90% by weight, The amount of the nucleic acid is in the range of 0.01 to 90% by weight, The amount of the phospholipid is in the range of 0.1 to 95% by weight, The amount of the sterol is in the range of 0.1 to 95% by weight, and / or The amount of the cationic and / or ionizable cationic lipid is in the range of 0.1 to 95% by weight, These weight percentages are based on the total amount of the apolipoprotein and / or apolipoprotein mimetic, the nucleic acid, the phospholipid, the sterol, and the cationic and / or ionizable cationic lipid. The nanoparticles according to claim 1

24. The ratio of the apolipoprotein to the phospholipid based on the 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. The nanoparticles according to claim 1

25. The ratio of the apolipoprotein to the phospholipid based on the weight is 2:1 to 1:10, more preferably 1:1 to 1:5, and even more preferably 1:1.5 to 1:

4. The nanoparticles according to claim 1

26. The average size is 10 to 100 nm, for example, 30 to 100 nm. The nanoparticles according to claim 1

27. A composition comprising the nanoparticles according to claim 1 and a physiologically acceptable carrier

28. The composition according to claim 27, wherein the composition is a pharmaceutical composition

29. The nanoparticles according to claim 1, or the composition according to claim 27, for use in the treatment of diseases by stimulating or inhibiting the innate immune response

30. The nanoparticle or composition for use according to claim 29, wherein the disease is cancer, cardiovascular disease, autoimmune disorder or xenograft rejection.

31. A method for preparing a nanoparticle, comprising: a) a step of rapidly mixing a lipid component in an organic solvent and a nucleic acid in an aqueous buffer to prepare lipid nanoparticles, wherein the lipid component comprises a phospholipid, a sterol, a cationic lipid or an ionizable cationic lipid, and the pH of the aqueous buffer is 5.0 or less, a step of preparing lipid nanoparticles; b) a step of rapidly mixing the lipid nanoparticles with an apolipoprotein, an apolipoprotein mimetic, or a combination thereof to prepare the nanoparticles at a pH of 6.0 to 8.

0.

32. The nanoparticle according to claim 1, wherein the nanoparticle can be obtained or is obtained by the method according to claim 31.

33. An in vitro or ex vivo method for introducing a nucleic acid into a cell, comprising contacting the cell with the nanoparticle according to claim 1 or the composition according to claim 27.

34. The nanoparticle according to claim 1, or the composition according to claim 27, for use in the in vivo delivery of a nucleic acid to a subject, optionally to a bone marrow compartment or spleen of the subject.

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