Dendritic structures as non-viral vectors for gene delivery

Abstract

Disclosed herein are novel dendritic structures that can be used in combination with other lipid components to form nanoparticles bearing nucleic acids to facilitate intracellular delivery of the nucleic acids both in vitro and in vivo. Nanoparticle compositions comprising the compounds and methods of their use to treat or prevent diseases or conditions are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 170,119, filed April 2, 2021, and entitled "DENDRITIC ARCHITECTURES FOR NUCLEIC ACID DELIVERY," which is incorporated by reference herein as if fully set forth.

[0002] Field The present disclosure relates to dendritic structures as non-viral vectors or carriers for efficient delivery of nucleic acids to a subject for treating or preventing a disease and / or disorder, and nanoparticle compositions comprising the carrier and nucleic acid. The present disclosure also relates to methods of formulating the nanoparticle compositions, and methods of treating a disease and / or disorder in a subject using such nanoparticle compositions. [Background technology]

[0003] Nucleic acid vaccines and nucleic acid therapeutics have emerged as promising approaches to prevent and treat several diseases or conditions. Certain nucleic acids are stable only for a limited period in cells or plasma, and their highly charged nature further complicates delivery across cell membranes. Therefore, compositions that can stabilize and deliver such agents to cells are of particular interest (Mendes et al., 2017, Molecules 22(9), 1401; Jones et al., 2013, Mol.Pharmaceutics 10, 4082-4098; and Nishikawa and Huang, 2001 Hum.Gene Ther. 12, 861-870). Nucleic acid delivery vectors should ideally overcome different extracellular and intracellular barriers such as endonucleolytic degradation, cellular internalization, endosomal escape, payload release from the vector and access to the desired target, all with minimal toxicity (Jones et al., 2013, Mol. Pharmaceutics 10, 4082-4098; Nishikawa and Huang, 2001 Hum. Gene Ther. 12, 861-870; Gomes et al., 2014 MRS Bull. 39, 60-70; and Dufes et al., 2005, Adv. Drug Delivery Rev. 57, 2177-2202).

[0004] Lipid nanoparticle (LNP) systems are the most advanced delivery system for gene drugs due to their high nucleic acid encapsulation efficiency and strong transfection, etc., but mild to moderate local and systemic adverse events have been observed (Mol Ther Nucleic Acids. 2019 Apr 15;15:1-11; Ther Deliv. 2016;7(5):319-334. doi:10.4155 / tde-2016-0006).

[0005] So-called polyester dendrons have been reported, with basic units (building blocks) known to be biocompatible or degradable in vivo into natural metabolites (Carnahan and Grinstaff, 2001, J. Am. Chem. Soc. 123, 2905; Carnahan and Grinstaff, 2001, Macromolecules 34, 7648; and Carnahan and Grinstaff, 2006, Macromolecules 39, 609). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Mendes et al., 2017, Molecules 22(9), 1401 [Non-Patent Document 2] Jones et al., 2013, Mol.Pharmaceutics 10, 4082-4098 [Non-Patent Document 3] Nishikawa and Huang, 2001 Hum. Gene Ther. 12, 861-870 [Non-Patent Document 4] Gomes et al., 2014 MRS Bull. 39, 60-70 [Non-Patent Document 5] Dufes et al., 2005, Adv. Drug Delivery Rev. 57, 2177-2202 [Non-Patent Document 6] Mol Ther Nucleic Acids. 2019 / 04 / 15;15:1-11 [Non-Patent Document 7] Ther Deliv. 2016;7(5):319-334. doi:10.4155 / tde-2016-0006 [Non-Patent Document 8] Carnahan and Grinstaff, 2001, J. Am. Chem. Soc. 123, 2905 [Non-Patent Document 9] Carnahan and Grinstaff, 2001, Macromolecules 34, 7648 [Non-Patent Document 10] Carnahan and Grinstaff, 2006, Macromolecules 39, 609 Summary of the Invention [Means for solving the problem]

[0007] In one aspect, the invention relates to a nucleic acid carrier comprising a dendron or dendrimer of formula Ia, Ib, Ic, Id, Ie or If. [ka] In the above formula, PE is a polyester (PE) dendron comprising a core and a plurality of monomeric polyester units forming one or more generations, A is an amine, B is a hydrophobic unit, and z is the number of surface groups.

[0008] In one aspect, the present invention relates to a nanoparticle composition comprising any one of the nucleic acid carriers disclosed herein, a therapeutic or immunogenic nucleic acid agent encapsulated therein, and a conjugated lipid.In one aspect, the present invention relates to a nanoparticle composition comprising any one of the nucleic acid carriers disclosed herein, a therapeutic or immunogenic nucleic acid agent encapsulated therein, a conjugated lipid (e.g., PEG-lipid) disclosed herein, and a mixture of phospholipid and cholesterol or its derivatives for improving intracellular delivery and nanoparticle stability in vivo.

[0009] In one aspect, the present invention relates to a method of treating or preventing a disease or condition in a subject, the method comprising providing any one of the nanoparticle compositions disclosed herein and administering a therapeutically effective amount of these nanoparticle compositions to the subject. [Brief description of the drawings]

[0010] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, specific embodiments are shown in the drawings, it being understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

[0011] [Figure 1] FIG. 1 shows a dynamic light scattering (DLS) analysis illustrating the size distribution profile of a nanoparticle composition (PE-G2-hexadecyl-propylpyrrolidine as a nucleic acid carrier, formulated at pH 5.0). [Diagram 2] FIG. 2 shows dynamic light scattering (DLS) analysis showing the size distribution profile of a nanoparticle composition (PE-G2-hexadecyl-propylpyrrolidine as a nucleic acid carrier, formulated at pH 6.0). [Diagram 3] Figure 3 shows a photograph of an agarose gel showing the binding of PE-G2-hexadecyl-propylpyrrolidine to RNA. The gel was stained with ethidium bromide (EB) and the gel image was taken with a Syngene G Box Imaging System (Syngene, USA). [Figure 4] FIG. 4 shows in vivo expression of secreted alkaline phosphatase (SEAP) replicon RNA formulated in PE-G2-hexadecyl-propylpyrrolidine nanoparticles and harvested from mice 1, 4, and 6 days after administration. [Diagram 5] FIG. 5 shows dynamic light scattering (DLS) analysis demonstrating the size distribution profile of a nanoparticle composition (PE-G2-A1-ricinoleic acid as a nucleic acid carrier.PE-G2-linoleic acid, formulated at pH 5.0). [Figure 6] Figure 6 shows a photograph of an agarose gel showing the binding of PE-G2-A1-ricinoleic acid.PE-G2-linoleic acid to RNA. The gel was stained with ethidium bromide (EB) and the gel image was taken with a Syngene G Box Imaging System (Syngene, USA). [Figure 7]FIG. 7 shows in vitro SEAP expression by quantiblue assay for nanoparticles containing SEAP replicon RNA and PE-G2-A1-ricinoleic acid.PE-G2-linoleic acid. [Figure 8A] Figures 8A and 8B show dynamic light scattering (DLS) analyses showing the size distribution profiles of nanoparticle compositions containing PE-G1-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid or PE-G2-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid and SEAP replicon RNA as nucleic acid carriers. [Figure 8B] Figures 8A and 8B show dynamic light scattering (DLS) analyses showing the size distribution profiles of nanoparticle compositions containing PE-G1-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid or PE-G2-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid and SEAP replicon RNA as nucleic acid carriers. [Figure 9A] FIG. 9A shows in vitro SEAP expression by quantiblue assay for nanoparticles containing SEAP replicon RNA and PE-G1-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid or PE-G2-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid. [Figure 9B] FIG. 9B shows in vivo SEAP expression for nanoparticles containing SEAP replicon RNA and PE-G1-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid or PE-G2-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid harvested from mice 3 days after administration. [Figure 10] FIG. 10 shows endpoint dilution antibody titers of mouse serum IgG specific to SARS-CoV-2 spike protein following vaccination with SARS-CoV-2 spike replicon RNA formulated with PE-G1-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid or PE-G2-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid delivery materials. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Certain terminology is used in the following description for convenience only and is not intended to be limiting.

[0013] The term "substituted" refers to the ability to change one functional group or moiety contained therein to another functional group on a molecule, provided that the valences of all atoms are maintained on the parent structure. Substituted groups are referred to interchangeably herein as "substitutions" or "substituents." When multiple positions in any given structure are substituted with multiple substituents selected from a particular group, the substituents may be the same or different at all positions.

[0014] The term "amine" formally refers to a derivative of ammonia in which one or more hydrogen atoms have been replaced with a substituent such as an alkyl group.

[0015] The term "surface group" as used herein refers to the terminal group on the surface of a dendritic molecule. The surface of the nucleic acid carrier is modified with a hydrophobic tail (referred to herein as component "B") or an amine (referred to herein as component "A") to aid in the self-assembly properties and condense the nucleic acid.

[0016] The term "alkyl" as used herein means a straight or branched chain hydrocarbon containing 1 to 28 carbon atoms. The alkyl chain length may be useful for controlling the hydrophobicity and self-assembly properties of the nucleic acid carrier. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0017] The terms "a" and "one," as used in the claims and corresponding parts of the specification, are defined as including one or more of the referenced items, unless otherwise specified. This term includes the words specifically mentioned above, derivatives thereof, and words of similar import. The phrase "at least one" following a list of two or more items, such as "A, B, or C," means any individual one of A, B, or C, and any combination thereof.

[0018] Non-viral vectors have the potential to address many of the limitations associated with viral vectors, including carcinogenesis, immunogenicity, and broad tropism. "Nanoparticle composition" refers to a composition comprising a nucleic acid carrier and a nucleic acid payload. The nucleic acid payload is "carried" by the nucleic acid carrier and may be encapsulated by the nucleic acid carrier.

[0019] One embodiment includes polyester dendrons (a non-limiting example of which is 2,2-bismethylolpropionic acid (bis-MPA) monomer-based dendrons) as building blocks modified with amines and hydrophobic tails for collecting nucleic acids and for self-assembly into nanoparticle compositions that protect the nucleic acids from degradation, which in one embodiment may transport nucleic acids into cells.

[0020] One embodiment includes a nucleic acid carrier having formula Ia, Ib, Ic, Id, Ie, or If. [ka] In the above formula, PE is a polyester dendron comprising a core and monomeric polyester units layered around the core to form a dendritic structure, each layer referred to as a generation (G), A is an amine, B is a hydrophobic unit, and z is the number of surface groups.

[0021] The PE may have formula II. [ka] G is a layer or generation of the dendron, n is the generation number, the value of which ranges from 1 to 10, and the monomeric polyester unit can be, but is not limited to, 2,2-bis(hydroxymethyl)propionic acid or 2,2-bis(hydroxymethyl)butyric acid. Z has formula III. z=b n , formula III In the above formula, b is the branch point multiplicity, or the number of branches at each branch point, and n is the number of generations.

[0022] In a nucleic acid carrier containing 2,2-bis(hydroxymethyl)propionic acid or 2,2-bis(hydroxymethyl)butyric acid as a monomeric polyester unit, the branch point multiplicity or the number of branches at each branch point, b, is 2.

[0023] The structure of the core may affect the amine density and / or charge density, diameter, and flexibility of the resulting nucleic acid carrier. These attributes may affect the physicochemical properties of the nucleic acid carrier, its interaction with nucleic acids, and gene transfer activity.

[0024] In one embodiment, the core may be selected from the following scaffolds: [ka] In the above formula, A is an amine and B is a hydrophobic unit.

[0025] Amine A is a moiety that confers proton-accepting functionality to the nucleic acid carrier molecule by containing one or more nitrogen atoms with lone pairs of electrons. Thus, the amine can accept a free proton (H+) under acidic conditions. In a preferred embodiment, the nitrogen atom is present in the form of a secondary or tertiary amine. The amine may be selected from the following moieties: 1-(2-aminoethyl)piperazine, 1-piperidineethanamine, 1-dimethylamino-2-propylamine, 1-(3-aminopropyl)pyrrolidine, 1-(2-aminoethyl)pyrrolidine, 1-(2-aminoethyl)piperidine, 2-(1-piperazinyl)ethylamine, 2-(4-methyl-piperazin-1-yl)-ethylamine, 3-(dimethylamino)-1-propylamine, 3-(diethylamino)propylamine, (4-aminobutyl)dimethylamine, 4-(1-pyrrolidinyl)-1-butylamine, 4-morpholineethanamine, 4-morpholineproline. Panamine, 4-methyl-1-piperazineethanamine, 4-(diethylamino)butylamine, N,N-dimethylethylenediamine, N,N-dimethyldipropylenetriamine, N,N'-dimethyl-N,N'-bis(3-methylaminopropyl)trimethylenediamine, N1-(2-aminoethyl)-N1-methylpropane-1,3-diamine, N1-(3-aminopropyl)-N1-methylbutane-1,4-diamine, N1-(4-aminobutyl)-N1-methylbutane-1,4-diamine, N1-(3-aminopropyl)propane-1,3-diamine, N1-(3-aminopropyl)-N1-methylpropane-1,3-Diamine, 3-((3-aminopropyl)(methyl)amino)propan-1-ol, 1-dimethylamino-2-propanol, 1-[bis[3-(dimethylamino)propyl]amino]-2-propanol, 2-dimethylaminoethanol, 2-(diethylamino)ethanol, 2-[2-(dimethylamino)ethoxy]ethanol, 2-(2-aminoethoxy)ethanol, 2-{[2-(dimethylamino)ethyl]methylamino}ethanol, 3-dimethylamino-1-propanol, 3-diethylamino-1-propanol, 4-(dimethylamino)-1-butanol, 4-diethylamino-2-butyne-1- ol, N-methyldiethanolamine, 4-(diethylamino)butan-1-ol, 3-(azetidin-1-yl)propan-1-ol, 3-(pyrrolidin-1-yl)propan-1-ol, 3-(piperidin-1-yl)propan-1-ol, 4-(azetidin-1-yl)butan-1-ol, 4-(pyrrolidin-1-yl)butan-1-ol, 4-(piperidin-1-yl)butan-1-ol, 3-morpholinopropan-1-ol, 4-morpholinobutan-1-ol, 3-(4-methylpiperazin-1-yl)propan-1-ol, 4-(4-methylpiperazin-1-yl)butan-1-ol. These are as shown below. [ka]

[0026] The hydrophobic unit B of formula I may be introduced by contacting the PE dendron with a functional reagent such as a fatty acid or its derivative. 28The fatty acid may be a saturated or unsaturated fatty acid having a chain length, including, but not limited to, arachidonic acid, oleic acid, eicosapentaenoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, or linolenic acid. The fatty acid derivative may be, but is not limited to, 12-hydroxy-9-cis-octadecenoic acid, 12-methyltetradecanoic acid, 12-methyltridecanoic acid, 14-methylhexadecanoic acid, 14-methylhexadecanoic acid, 18-methylnonadecanoic acid, 19-methylarachidic acid, isopalmitic acid, isostearic acid, phytanic acid, (±)-2-hydroxyoctanoic acid, (±)-3-hydroxydecanoic acid, (±)-3-hydroxyoctanoic acid, 10-hydroxydecanoic acid, 12-hydroxyoctadecanoic acid, 15-hydroxypentadecanoic acid, 16-hydroxyhexadecanoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxytetradecanoic acid, 2-hydroxydodecanoic acid, DL-α-hydroxystearic acid, DL-β-hydroxylauric acid, DL-β-hydroxymyristic acid, or DL-β-hydroxypalmitic acid.

[0027] In one embodiment, the nucleic acid carrier may include functional groups suitable for tracking the delivered material in vitro and in vivo. 13 C or 2 The nucleic acid carrier may have a fatty acid containing a stable isotope of carbon (C) or hydrogen (H), such as H (also referred to herein as deuterium, D or d). When the nucleic acid carrier is formulated into a nanoparticle with a nucleic acid, such as a replicon RNA, the nanoparticle may be tracked in vitro and in vivo after administration by techniques such as mass spectrometry or nuclear magnetic resonance imaging. The inclusion of a stable isotope may be beneficial for the identification of the delivery molecule, since these isotopes are abundant in tissues. 12 C and 1 H isotope. The tracking may be useful to determine the biodistribution, material clearance and molecular stability of nanoparticles after administration, as well as related items. The isotope-labeled fatty acid is octanoic acid-1- 13 C, Octanoic acid-8- 13C, Octanoic acid-8,8,8- 2 H3, Octanoic acid - 2 H15, Decanoic acid-1- 13 C, Decanoic acid-10- 13 C, Decanoic acid-10,10,10- 2 H3, decanoic acid - 2 H19, Undecanoic acid-1- 13 C, lauric acid-12,12,12- 2 H3, lauric acid - 2 H23, lauric acid-1- 13 C, lauric acid-1,12- 13 C2, Tridecanoic acid-2,2- 2 H2, myristic acid-14- 13 C, myristic acid-1- 13 C, Myristic acid-14,14,14- 2 H3, myristic acid-d27, palmitic acid-1- 13 C, Palmitic acid-16- 13 C, Palmitic acid-16- 13 C,16,16,16- 2 H3, palmitic acid - 2 H31, 1-stearic acid 13 C, stearic acid-18- 13 C, stearic acid-18,18,18- 2 H3, stearic acid 2 H35, oleic acid-1- 13 C, oleic acid - 2 H34, linolenic acid-1- 13 C, linoleic acid - 2 H32, Arachidonic acid-5,6,8,9,11,12,14,15- 2 H8, or eicosanoic acid - 2 It may be, but is not limited to, H39.

[0028] In one embodiment, a nanoparticle composition is provided comprising any one of the nucleic acid carriers described herein. The nanoparticle composition of the present disclosure may be useful for introducing a drug into a cell. The drug may be a nucleic acid. The nanoparticle composition of the present disclosure may be useful as a vaccine. The nanoparticle composition of the present disclosure may be useful as a therapeutic formulation. The nanoparticle composition of the present disclosure may be useful as a transfection agent. The nanoparticle composition of the present disclosure may be useful in a method for treating or preventing a disease.

[0029] In one embodiment, the nanoparticle composition may comprise a mixture of nucleic acid carriers. One or more of the nucleic acid carriers in the mixture may comprise at least one of different amines, different amine densities, or different side chains compared to other nucleic acid carriers in the mixture. These nucleic acid carriers may be in a fixed ratio in the mixture. For the example of a mixture with three dendritic molecules, the ratio of the first nucleic acid carrier to the second nucleic acid carrier and the third nucleic acid carrier may be i:j:k, where i, j, and k are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or a value between any two of the above numbers.

[0030] In one embodiment, the nanoparticle composition may comprise one or more therapeutic or immunogenic nucleic acid agents. As used herein, the term "nucleic acid" refers to any natural or synthetic DNA or RNA molecule, or hybrids thereof. Nucleic acids may comprise natural and / or synthetic nucleotides. Nucleic acid molecules may code for proteins with therapeutic benefits, such as antibodies, hormones, or receptors. Nucleic acid molecules may code for protein immunogens derived from diseases, such as viral or bacterial pathogens, or derived from cancerous cells. Nucleic acid molecules may not code for proteins, but instead exert a biological effect through cell signaling or modulation of protein expression, for example in the form of siRNA.

[0031] In one embodiment, the therapeutic or immunogenic nucleic acid agent may be an RNA molecule or a DNA molecule. The nucleic acid agent may be a mixture of one or more different RNA molecules, DNA molecules, or a combination of the two. The term "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. The DNA molecule may be a polynucleotide, an oligonucleotide, a DNA, or a cDNA. The DNA molecule may code for a wild-type or engineered protein, peptide, or polypeptide, such as an antigen. The term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of multiple ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The RNA molecule may be a replicon RNA (repRNA), a small interfering RNA (siRNA), a miRNA, a single-stranded guide RNA (sgRNA), a messenger RNA (mRNA), or a transfer RNA (tRNA). Replicon RNA (repRNA) refers to a replication-competent, progeny-defective RNA virus genome that is unable to produce infectious progeny virions. Viral genomes that are typically modified for use as repRNA include "positive-strand" RNA viruses. The modified viral genome serves as both an mRNA and a template for replication. Small interfering RNA (siRNA) refers to an RNA (or RNA analog) containing about 10-50 nucleotides (or nucleotide analogs) that can induce or mediate RNA interference. MicroRNA (miRNA) refers to a small (20-24 nt) regulatory non-coding RNA involved in post-transcriptional regulation of gene expression in eukaryotes by affecting either or both the stability and translation of coding mRNA. Messenger RNA (mRNA) is usually a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA. DNA or RNA molecules may be chemically modified in the nucleic acid backbone, the ribose sugar moiety, and the nucleic acid bases themselves.

[0032] The RNA molecule may be a monocistronic or polycistronic mRNA. Monocistronic mRNA refers to an mRNA that contains only one sequence that codes for a protein polypeptide or peptide. Polycistronic mRNA typically refers to two or more sequences that code for two or more proteins, polypeptides or peptides. The mRNA may code for a protein, polypeptide or peptide that acts as an antigen.

[0033] In one embodiment, the DNA molecule may be a polynucleotide, an oligonucleotide, DNA, or cDNA. The RNA molecule may be a replicon RNA (repRNA), a small interfering RNA (siRNA), a miRNA, a single-stranded guide RNA (sgRNA), a messenger RNA (mRNA), or a transfer RNA (tRNA). The therapeutic or immunogenic nucleic acid agent may be non-covalently or covalently bound to the nucleic acid carrier. The therapeutic or immunogenic nucleic acid agent may be electrostatically bound to the charged nucleic acid carrier via an ionic bond.

[0034] In one embodiment, the nanoparticle compositions described herein may include an immunogenic or therapeutic nucleic acid agent that encodes an antigen.

[0035] As used herein, "encapsulated" can refer to nanoparticles that provide an active agent or therapeutic agent, such as a nucleic acid (e.g., messenger RNA), fully encapsulated, partially encapsulated, or both. In a preferred embodiment, the nucleic acid is fully encapsulated in the nanoparticle. In the context of nucleic acid therapeutic agents, full encapsulation may be determined by the Ribogreen® assay. RiboGreen® is an ultrasensitive fluorescent nucleic acid stain for quantifying oligonucleotides and single-stranded DNA or RNA in solution (available from Thermo Fisher Scientific, USA).

[0036] An "antigen" as used herein is defined as a molecule that elicits an immune response. The immune response may include either antibody production or activation of specific immunologically active cells, or both. An antigen may refer to any molecule capable of stimulating an immune response, including macromolecules such as proteins, peptides, or polypeptides. An antigen may be a structural component of a pathogen or a cancer cell. An antigen may be synthesized in the host, recombinantly produced, or derived from a biological sample, including, but not limited to, a tissue sample, a cell, or a biological fluid.

[0037] The antigen may be, but is not limited to, a vaccine antigen, a parasitic antigen, a bacterial antigen, a tumor antigen, an environmental antigen, a therapeutic antigen, or an allergen. As used herein, a nucleotide vaccine is a DNA or RNA-based prophylactic or therapeutic composition that can stimulate adaptive immune response in a subject by delivering antigen(s). The immune response induced by vaccination typically results in the development of immunological memory and the ability of the organism to respond rapidly to subsequent encounters with antigens or infectious agents.

[0038] The use of "nucleic acid carrier" in the present disclosure as a carrier of nucleic acid is preferred, and for this reason the name "nucleic acid carrier" is applied. However, embodiments also include a combination of the nucleic acid carrier of the present disclosure with an agent containing a negative or partially negative charge. This agent may be a drug.

[0039] In one embodiment, the nanoparticle composition may be formulated to include a drug containing a negative or partial negative charge. The nanoparticle may be formulated via electrostatic association of the negative charge with the positive charge of the protonated amine group in the nucleic acid carrier. The negatively charged drug may be an ionizable drug. The term "ionizable drug" refers to an electrically asymmetric molecule that is water-soluble and ionizable in a solution of distilled water. The ionizable drug may contain a phosphate, phosphonate, or phosphinate functional group. The drug containing a phosphate group may be a phosphate-containing nucleotide analog, for example, a drug used for the treatment of cancer and viral chemotherapy. The phosphate-containing drug may be, but is not limited to, purine and pyrimidine nucleoside analogs, arabinosylcytosine (ara-C), ara-C monophosphate (ara-CMP), azidothymidine (AZT), AZT monophosphate (AZTMP), 2'3'-dideoxycytidine (ddCD), cyclic adenosine monophosphate (cAMP), tenofovir, or adefovir.

[0040] In one embodiment, the nanoparticle compositions described herein may include one or more "lipid conjugates". Lipid conjugates may be useful for preventing particle aggregation. Contemplated lipid conjugates include, but are not limited to, PEG-lipid conjugates. PEG-lipid conjugates may be, but are not limited to, PEG conjugated to lipids (e.g., DMG-PEG2000 or ALC 0159), PEG conjugated to phospholipids (e.g., phosphatidylethanolamine (PEG-PE), PEG conjugated to cholesterol or its derivatives, and mixtures thereof. In certain cases, PEG may be optionally substituted with alkyl, alkoxy, acyl, or aryl groups.

[0041] Phosphatidylethanolamines having various acyl chain groups of various chain lengths and degrees of saturation can be conjugated to PEG to form lipid conjugates. Phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques. Phosphatidylethanolamines are C 10 ~C 20 The phosphatidylethanolamine may comprise saturated or unsaturated fatty acids having carbon chain lengths ranging from 0 to 100 carbon atoms. The phosphatidylethanolamine may comprise monounsaturated or polyunsaturated fatty acids, as well as mixtures of saturated and unsaturated fatty acids. Contemplated phosphatidylethanolamines include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0042] The PEG-lipid may comprise PEG conjugated to cholesterol or a cholesterol derivative, examples of which include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0043] PEG is a linear water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEG is classified by its molecular weight. For example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. PEG is commercially available from Avanti Polar Lipids. The PEG portion of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons.

[0044] The size, relative amount and distribution of PEG-lipids included in the nanoparticle composition may affect the physical properties of the nanoparticle composition. Physical properties that may be controlled include, but are not limited to, the diameter of the nanoparticles, the tendency of the nanoparticles to aggregate, the number of nucleic acid molecules within each nanoparticle or the concentration of nanoparticles in the nanoparticle composition, the efficacy of intracellular delivery of therapeutic and immunogenic nucleic acid agents, and / or the efficacy of uptake of the nanoparticles by cells. See a) PCT / US19 / 67402 (Poulami Talukder, Jasdave S. Chahal, Justine S. McPartlan, Omar Khan, Karl Ruping. Nanoparticle Compositions for Efficient Nucleic Acid Delivery and Methods of Making and Using the Same; b) Mui, Barbara L, et al., "Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles," Molecular therapy. Nucleic acids Vol. 2, 12 e139. Dec. 17, 2013, doi:10.1038 / mtna.2013.66. These documents are incorporated by reference herein as if fully set forth.

[0045] The nanoparticle composition may comprise 10 mol% or less of PEG-lipid per nanoparticle composition. The nanoparticle composition may comprise about 10 mol%, about 9 mol%, about 8 mol%, about 7 mol%, about 6 mol%, about 5 mol%, about 4 mol%, about 3 mol%, about 2 mol%, or about 1 mol% of PEG-lipid per nanoparticle composition, or any amount between any two of the above integers. The nanoparticle composition comprising PEG-lipid may comprise nanoparticles having a smaller diameter than the nanoparticles of the composition lacking PEG-lipid. The nanoparticle composition may also comprise nanoparticles having a higher tendency to aggregate than the nanoparticles of the composition lacking PEG-lipid.

[0046] The nanoparticle composition may include "amphiphilic lipids". As used herein, "amphiphilic lipids" refers to any material with a non-polar hydrophobic "tail" and a polar "head". Polar groups may include phosphate, carboxylate, sulfate (sulfato), amino, sulfhydryl, nitro, hydroxyl, or other similar groups. Non-polar groups may include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclic groups. Examples of amphiphilic lipids include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.

[0047] The nanoparticle composition may include amphipathic lipids in an amount ranging from 10 mol % to 15 mol % of amphipathic lipid per nanoparticle composition.

[0048] In one embodiment, the nanoparticle composition may include cholesterol or a cholesterol derivative, examples of which include cholestanol, 5,6-epoxycholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 24-ethylcholesterol, 24-methylcholesterol, cholenoic acid, 3-hydroxy-5-cholestenoic acid, cholesteryl palmitate, cholesteryl arachidonate, cholesteryl arachidate, cholesteryl myristate, cholesteryl palmitoleate, cholesteryl lignocerate, cholesteryl oleate, cholesteryl stearate, cholesteryl erucate, cholesterol α-linolenate, cholesteryl linoleate, homo-γ-linolenate, 4-hydroxycholesterol, 6-hydroxycholesterol, 7-hydroxycholesterol, 1 ... Examples of cholesterol derivatives include, but are not limited to, 9-hydroxycholesterol, 20-hydroxycholesterol, 22-hydroxycholesterol, 24-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 27-alkynylcholesterol, 7-ketocholesterol, 7-dehydrocholesterol, 8-dehydrocholesterol, 24-dehydrocholesterol, 5α-hydroxy-6-ketocholesterol, 20,22-dihydroxycholesterol, 7,25-dihydroxycholesterol, 7,27-dihydroxycholesterol, 7-keto-25-hydroxycholesterol, fucosterol, phytosterol, cholesteryl 11,14-eicosadienoate, dimethylhydroxyethylaminopropanecarbamoylcholesterol iodide, and mixtures thereof. Cholesterol derivatives may include sugar moieties such as mannose, galactose, etc. Cholesterol derivatives may include sugar moieties and / or amino acids such as serine, threonine, lysine, histidine, arginine, or derivatives thereof. The nanoparticle composition may include cholesterol or a cholesterol derivative in an amount ranging from 50 mol % to 75 mol % of cholesterol or a derivative thereof per nanoparticle composition.

[0049] In one embodiment, the nanoparticle composition may include a pharma- ceutically acceptable carrier. As used herein, the term "pharma-ceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle involved in carrying or transporting the subject compound from one organ or part of the body to another, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, or zinc stearate, or stearic acid), or solvent encapsulating material. Each pharma-ceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient.Some examples of materials that can serve as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, mannose, and / or sucrose; (2) starches, such as corn starch and / or potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and / or cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and / or talc; (8) excipients, such as cocoa butter and / or suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and / or soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, and and / or mannitol; (12) esters, such as glycerides, ethyl oleate and / or ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and / or aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) diluents, such as isotonic saline and / or PEG 400; (18) Ringer's solution; (19) C2-C12 alcohols, such as ethanol; (20) fatty acids; (21 ) pH buffer solutions; (22) bulking agents, such as polypeptides and / or amino acids; (23) serum components, such as serum albumin, HDL and LDL; (24) surfactants, such as polysorbates (Tween 80) and / or poloxamers; and / or (25) other non-toxic compatible substances employed in pharmaceutical formulations: for example, fillers, binders, wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and / or antioxidants. The terms "excipient", "carrier", "pharmaceutical acceptable carrier" and the like are used interchangeably herein.

[0050] One embodiment includes a method of treating or preventing a disease or condition in a subject. The method may include providing any one of the nanoparticle compositions described herein. The method may include administering a therapeutically effective amount of the nanoparticle composition to the subject.

[0051] As used herein, the term "therapeutically effective amount" refers to an amount of a nanoparticle composition effective to produce a desired therapeutic effect. The therapeutic effect may be observed in at least a subpopulation of cells in an animal. The therapeutic effect may be achieved with a reasonable benefit / risk ratio applicable to medical treatments. A "therapeutically effective amount" may refer to an amount sufficient to cause the appearance of antigen-specific antibodies in serum. A "therapeutically effective amount" may refer to an amount sufficient to cause a reduction in disease symptoms. A "therapeutically effective amount" may refer to an amount sufficient to cause the disappearance of disease symptoms. When treating a viral infection, the reduction in disease symptoms may be assessed by a reduction in virus in feces, body fluids, or secretory products. The nanoparticle composition may be administered in an amount and using a route of administration effective to produce an immune response. The nanoparticle composition may be administered in an amount and using a route of administration effective to modulate an immune response. The nanoparticle composition may be administered to induce immune tolerance, i.e., to reduce the subject's immune response to the encoded antigen. For example, one embodiment includes a method of treating an allergic disease or an autoimmune disease, which may include administering a nanoparticle composition to induce immune tolerance.

[0052] The therapeutic efficacy may depend on the effective amount of active agent and the administration time required to achieve the desired result. The administration of the nanoparticle composition may be a preventative measure. The administration of the nanoparticle composition may be a therapeutic measure to promote immunity against infectious agents and minimize complications associated with delayed immunity, especially in patients with weakened immune systems, the elderly, or infants.

[0053] The exact dosage may be selected by the clinician based on a variety of factors and taking into account the individual patient. Dosage and administration may be adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. For example, factors that may be considered may include the type and severity of the disease; age and sex of the patient; drug combinations; and individual response to treatment.

[0054] The therapeutic efficacy and toxicity of the active agents in the nanoparticle compositions may be determined by standard pharmaceutical procedures, for example, by determining the therapeutically effective dose in 50% of the population (ED50) and the lethal dose for 50% of the population (LD50) in cultured cells in vitro or in experimental animals. Nanoparticle compositions may be evaluated based on the toxic to therapeutic dose ratio (LD50 / ED50), called the therapeutic index, the larger of which may be used for evaluation. Data obtained from cell and animal studies may be used in formulating dosages for human use.

[0055] A therapeutically effective dose may be initially estimated from cell culture assays. A therapeutically effective dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the therapeutic agent that achieves half-maximal inhibition of symptoms) as determined in cell culture. Levels in plasma may be measured, for example, by high performance liquid chromatography. The effect of any particular dosage may be monitored by a suitable bioassay.

[0056] The amount of nanoparticle formulation administered will depend on the particular therapeutic agent (e.g., nucleic acid) used, the disease or disorder being treated, the age, weight, and condition of the patient, and the judgment of the clinician. A therapeutically effective dose may be from 0.001 ng to 50 mg of therapeutic or immunogenic nucleic acid per kilogram of subject body weight. A therapeutically effective dose may be from 0.001 ng, 0.002 ng, 0.003 ng, 0.004 ng, 0.005 ng, 0.006 ng, 0.007 ng, 0.008 ng, 0.009 ng, 0.01 ng, 0.02 ng, 0.03 ng, 0.04 ng, 0.05 ng, 0.06 ng, 0.07 ng, 0.08 ng, 0.09 ng, 0.1 ng, 0.2 ng, 0.001 ng, 0.0 ... 3ng, 0.4ng, 0.5ng, 0.6ng, 0.7ng, 0.8ng, 0.9ng, 0.001μg, 0.002μg, 0.003μg, 0.004μg, 0.005μg, 0.006μ g, 0.007μg, 0.008μg, 0.009μg, 0.01μg, 0.02μg, 0.03μg, 0.04μg, 0.05μg, 0.06μg, 0.07μg, 0.08μg, 0.09 μg, 0.1μg, 0.2μg, 0.3μg, 0.4μg, 0.5μg, 0.6μg, 0.7μg, 0.8μg, 0.9μg, 1mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 21mg, 22mg, 23mg, 24mg, 25m g, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, or 50 mg of the therapeutic or immunogenic nucleic acid, or a value within a range between any two of the above values. The therapeutic and immunogenic nucleic acids may be a combination of different nucleic acids used according to the therapeutic dose. The terms "subject" and "individual" are used interchangeably herein and refer to a human or an animal. Preferably, the animal is a vertebrate such as a primate, rodent, domestic or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys.The rodent may be selected from mouse, rat, guinea pig, woodchuck, ferret, rabbit and hamster. The livestock or game animal may be selected from cow, horse, pig, deer, bison, buffalo, feline species, such as domestic cat, canine species, such as dog, fox, wolf, avian species, such as chicken, emu, ostrich, and fish, such as trout, catfish, and salmon. The patient or subject may be selected from the above or a subset of the above. The patient or subject may be selected from all of the above, excluding one or more groups or species, such as humans, primates, or rodents. In one embodiment, the patient or subject may be a mammal, such as a primate, such as a human. The terms "patient" and "subject" are used interchangeably herein. The terms "patient" and "subject" are used interchangeably herein.

[0057] Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a pig, a horse, or a cow. The non-human mammal may be a subject that represents an animal model of a disease or disorder. In addition, the methods described herein may be directed to treating livestock and / or pets. The subject may be male or female.

[0058] As used herein, the terms "administer", "administering", "administration" and the like refer to the placement of a composition into a subject. Administration may be by a method or route that results in at least partial localization of the composition at a desired site so that a desired effect is produced. The nanoparticle compositions described herein may be administered by any suitable route known in the art, including, but not limited to, parenteral or oral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, or topical (including buccal and sublingual) administration.

[0059] Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In one embodiment, the composition may be administered by intravenous infusion or injection.

[0060] The nanoparticle composition may be used for delivery of therapeutic DNA or RNA to regulate gene expression. The therapeutic nucleic acid may be an antisense oligonucleotide (AON) or a double-stranded small interfering RNA (siRNA). Typically, the siRNA is 21-23 nucleotides in length. The siRNA may comprise a sequence complementary to a sequence contained in an mRNA transcript of a target gene when expressed in a host cell. The antisense oligonucleotide may be a morpholino antisense oligonucleotide. The antisense oligonucleotide may comprise a sequence complementary to a sequence contained in an mRNA transcript of a target gene. The therapeutic or immunogenic nucleic acid may be an interfering RNA (iRNA) directed to a specific target gene in a specific target organism. The iRNA may induce sequence-specific silencing of expression or translation of a target polynucleotide, thereby downregulating or preventing gene expression. The iRNA may completely inhibit expression of the target gene. The iRNA may reduce the expression level of the target gene compared to the expression level of an untreated control. The therapeutic or immunogenic nucleic acid may be a microRNA (miRNA). The miRNA may be a short RNA, such as a hairpin RNA (hpRNA). The miRNA may be cleaved into biologically active dsRNA in the target cell by the activity of endogenous cellular enzymes. The RNA may be double-stranded RNA (dsRNA). The dsRNA may be at least 25 nucleotides in length or may be longer. The dsRNA may contain a sequence complementary to the sequence of the target gene(s).

[0061] In one embodiment, the therapeutic or immunogenic nucleic acid may be or may encode an agent that reduces, inhibits, interferes with, or modulates, in whole or in part, the activity or synthesis of one or more genes encoding a target protein. The target gene may be any gene contained in the genome of the host organism. The sequence of the therapeutic or immunogenic nucleic acid may not be 100% complementary or identical to the nucleic acid sequence of the target gene.

[0062] In one embodiment, the nanoparticle composition may be used for targeted specific modification of genetic information in a subject. One embodiment includes targeted specific modification of genetic information sufficient to have a therapeutic or immunogenic effect in a subject, comprising administration of a nanoparticle composition of the present disclosure. As used herein, the term "modification" refers to any change in the genome in a cell of a subject. The modification may be an insertion or deletion of a nucleotide in the sequence of a target gene. An "insertion" refers to the addition of one or more nucleotides to the sequence of a target gene. The term "deletion" refers to the loss or removal of one or more nucleotides in the sequence of a target gene. The modification may be a modification of the sequence of a target gene. A "modification" refers to the modification of one or more nucleotides in the sequence of a target gene, for example by insertion, deletion or substitution, which may result in a more favorable expression of the gene, manifested by an improvement in the genotype and / or phenotype of the host organism.

[0063] The modification of genetic information may be achieved through genome editing techniques. As used herein, "genome editing" refers to the process of altering the nucleotide sequence in a genome in a precise or controlled manner.

[0064] An exemplary genome editing system is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, described, for example, in International Publication WO 2018 / 154387, published August 30, 2018, and incorporated by reference herein as if fully set forth. In general, a "CRISPR system" refers to the transcripts and other elements involved in the expression of CRISPR-associated (Cas) genes, such as sequences encoding Cas genes, tracr (transactivating CRISPR) sequences, tracr-mate sequences, guide sequences, or other sequences and transcripts from a CRISPR locus. One or more tracr-mate sequences may be operably linked to the guide sequence prior to processing by a nuclease, or to the crRNA after processing. The tracrRNA and crRNA may be linked, or in addition, the mature crRNA may be fused to a partial tracrRNA via a synthetic stem-loop to form a chimeric crRNA-tracrRNA hybrid that mimics the natural crRNA:tracrRNA duplex, as described in Cong et al., Science, 15:339(6121):819-823 (2013) and Jinek et al., Science, 337(6096):816-21 (2012), which are incorporated by reference herein as if set forth in their entirety. A single fused crRNA-tracrRNA construct is also referred to herein as a guide RNA or gRNA, or a single-stranded guide RNA (sgRNA). Within the sgRNA, the crRNA portion is identified as the "target sequence" and the tracrRNA is often referred to as the "scaffold." In one embodiment, the nanoparticle composition described herein may be used to deliver the sgRNA.

[0065] In one embodiment, the nanoparticle composition may be used to apply other exemplary genome editing systems, including meganucleases, homing endonucleases, TALEN-based systems, or zinc finger nucleases. The nanoparticle composition may be used to deliver nucleic acids (RNA and / or DNA) encoding sequences for these gene editing tools, as well as the actual gene products, proteins, or other molecules.

[0066] In one embodiment, the nanoparticle composition may be used for targeted gene targeting in a subject, for example, by isolating cells from a subject, editing the genes, and transplanting the edited cells back into the subject. One embodiment includes a method comprising administering a nanoparticle composition of the present disclosure to cells isolated from a subject. The method may include targeted gene targeting. The method may include transplanting the edited cells back into the subject.

[0067] One embodiment includes a method of introducing an agent into a cell. The method may include exposing the cell to a nanoparticle composition of the present disclosure. The agent may be a nucleic acid. The method may be a method of transfection where the agent is a nucleic acid.

[0068] LIST OF EMBODIMENTS - The following list is a non-limiting list of embodiments and does not limit the embodiments otherwise described or illustrated herein. The embodiments in this list may be supplemented with one or more elements from any other embodiment or example herein. An element in an embodiment in this list may be replaced by one or more elements from any other embodiment or example herein.

[0069] 1. A nucleic acid carrier comprising a dendron or dendrimer of formula Ia, Ib, Ic, Id, Ie or If, [ka] In the above formula, PE is a polyester dendron comprising a core and a plurality of monomeric polyester units forming one or more generations, A is an amine, B is a hydrophobic unit, and z is the number of surface groups.

[0070] 2. The PE has the formula II: [ka] In the above formula, G is a layer or generation of the dendron, and n is the number of generations, which is in the range of 1 to 10.

[0071] 3. The nucleic acid carrier described in embodiment 1 or 2, wherein the plurality of monomeric polyester units comprises 2,2-bis(hydroxymethyl)propionic acid or 2,2-bis(hydroxymethyl)butyric acid, and preferably, all of the plurality of monomeric polyester units are either 2,2-bis(hydroxymethyl)propionic acid or 2,2-bis(hydroxymethyl)butyric acid.

[0072] 4. z has formula III, z=b n ,III In the above formula, b is the branch point multiplicity, or the number of branches at each branch point, and n is the number of generations, which is in the range of 1 to 10. The nucleic acid carrier according to any one of the first to third embodiments.

[0073] 5. A is 1-(2-aminoethyl)piperazine, 1-piperidineethanamine, 1-dimethylamino-2-propylamine, 1-(3-aminopropyl)pyrrolidine, 1-(2-aminoethyl)pyrrolidine, 1-(2-aminoethyl)piperidine, 2-(1-piperazinyl)ethylamine, 2-(4-methyl-piperazin-1-yl)-ethylamine, 3-(dimethylamino)-1-propylamine, 3-(diethylamino)propylamine, (4-aminobutyl)dimethylamine, 4-(1 -pyrrolidinyl)-1-butylamine, 4-morpholineethanamine, 4-morpholinepropanamine, 4-methyl-1-piperazineethanamine, 4-(diethylamino)butylamine, N,N-dimethylethylenediamine, N,N-dimethyldipropylenetriamine, N,N'-dimethyl-N,N'-bis(3-methylaminopropyl)trimethylenediamine, N1-(3-aminopropyl)propane-1,3-diamine, N1-(3-aminopropyl)-N1-methylpropane-1,3-Diamine, 3-((3-aminopropyl)(methyl)amino)propan-1-ol, 1-dimethylamino-2-propanol, 1-[bis[3-(dimethylamino)propyl]amino]-2-propanol, 2-dimethylaminoethanol, 2-(diethylamino)ethanol, 2-[2-(dimethylamino)ethoxy]ethanol, 2-(2-aminoethoxy)ethanol, 2-{[2-(dimethylamino)ethyl]methylamino}ethanol, 3-dimethylamino-1-propanol, 3-diethylamino-1-propanol, 4-(dimethylamino)-1-butanol, 4-diethylamino-2-butyn-1-ol, N-methyldiethanol The nucleic acid carrier according to any one of the first to fourth embodiments, wherein the nucleic acid carrier is selected from the group consisting of amine, 4-(diethylamino)butan-1-ol, 3-(azetidin-1-yl)propan-1-ol, 3-(pyrrolidin-1-yl)propan-1-ol, 3-(piperidin-1-yl)propan-1-ol, 4-(azetidin-1-yl)butan-1-ol, 4-(pyrrolidin-1-yl)butan-1-ol, 4-(piperidin-1-yl)butan-1-ol, 3-morpholinopropan-1-ol, 4-morpholinobutan-1-ol, 3-(4-methylpiperazin-1-yl)propan-1-ol, and 4-(4-methylpiperazin-1-yl)butan-1-ol.

[0074] 6. A nucleic acid carrier according to any one of embodiments 1 to 5, wherein B is derived from a fatty acid (C4 to C28) or a derivative thereof.

[0075] 7. The nucleic acid carrier described in embodiment 6, wherein the fatty acid is selected from the group consisting of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid and eicosapentanoic acid.

[0076] 8. The nucleic acid carrier according to embodiment 6, wherein the fatty acid derivative is selected from the group consisting of 12-hydroxy-9-cis-octadecenoic acid, 12-methyltetradecanoic acid, 12-methyltridecanoic acid, 14-methylhexadecanoic acid, 14-methylhexadecanoic acid, 18-methylnonadecanoic acid, 19-methylarachidic acid, isopalmitic acid, isostearic acid, phytanic acid, (±)-2-hydroxyoctanoic acid, (±)-3-hydroxydecanoic acid, (±)-3-hydroxyoctanoic acid, 10-hydroxydecanoic acid, 12-hydroxyoctadecanoic acid, 15-hydroxypentadecanoic acid, 16-hydroxyhexadecanoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxytetradecanoic acid, 2-hydroxydodecanoic acid, DL-α-hydroxystearic acid, DL-β-hydroxylauric acid, DL-β-hydroxymyristic acid, and DL-β-hydroxypalmitic acid.

[0077] 9. A nucleic acid carrier according to any one of embodiments 6 to 8, wherein the fatty acid or derivative comprises one or more stable isotopes.

[0078] 10. The nucleic acid carrier described in embodiment 9, wherein the stable isotope is a stable isotope of carbon or hydrogen.

[0079] 11. Stable isotopes of carbon are 13 The nucleic acid carrier according to embodiment 10, which is C.

[0080] 12. What are the stable isotopes of hydrogen? 2 The nucleic acid carrier according to embodiment 10, wherein said nucleic acid carrier is H.

[0081] 13. The fatty acids containing the above stable isotopes are: octanoic acid-1-13C, octanoic acid-8-13C, octanoic acid-8,8,8-d3, octanoic acid-2H15, decanoic acid-1-13C, decanoic acid-10-13C, decanoic acid-10,10,10-d3, decanoic acid-d19, undecanoic acid-1-13C, lauric acid-12,12,12-2H3, lauric acid-2H23, lauric acid-1-13C, lauric acid-1,12-13C2, tridecanoic acid-2,2-2H2, myristic acid-14-13C, myristic acid-1-13C, myristic acid-14,14,14-2H3 ... 11. The nucleic acid carrier according to embodiment 10, wherein the nucleic acid carrier is selected from the group consisting of stearic acid-2H27, palmitic acid-1-13C, palmitic acid-16-13C, palmitic acid-16-13C,16,16,16-2H3, palmitic acid-2H31, stearic acid-1-13C, stearic acid-18-13C, stearic acid-18,18,18-2H3, stearic acid-2H35, oleic acid-1-13C, oleic acid-2H34, linolenic acid-1-13C, linoleic acid-2H32, arachidonic acid-5,6,8,9,11,12,14,15-2H8, and eicosanoic acid-2H39.

[0082] 14. A nanoparticle composition comprising a nucleic acid carrier according to any one of embodiments 1 to 13 and a therapeutic or immunogenic nucleic acid agent fully or partially encapsulated therein.

[0083] 15. The nanoparticle composition of embodiment 14, wherein the therapeutic or immunogenic nucleic acid agent is selected from the group consisting of polynucleotides, oligonucleotides, DNA, cDNA, RNA, repRNA, siRNA, miRNA, sgRNA and mRNA.

[0084] 16. The nanoparticle composition of embodiment 14, wherein the therapeutic or immunogenic nucleic acid agent encodes one or more antigens selected from the group consisting of infectious diseases (infectious diseases), pathogens, cancer, autoimmune diseases and allergenic diseases (allergy-induced diseases).

[0085] 17. The nanoparticle composition of embodiment 14, wherein the therapeutic or immunogenic nucleic acid agent comprises RNA or DNA capable of silencing, inhibiting or altering the activity of a gene.

[0086] 18. A nanoparticle composition described in any one of embodiments 14 to 17, further comprising a PEG-lipid.

[0087] 19. The nanoparticle composition of embodiment 18, wherein the PEG-lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0088] 20. The nanoparticle composition of embodiment 18 or embodiment 19, wherein the nanoparticle composition comprises the PEG-lipid in the range of 1 mol % to 10 mol % of the PEG-lipid per nanoparticle composition.

[0089] 21. The nanoparticle composition of any one of embodiments 18 to 20, further comprising a phospholipid and cholesterol or a derivative thereof.

[0090] 22. The nanoparticle composition of claim 21, wherein the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or distearoylphosphatidylcholine (DSPC).

[0091] 23. The nanoparticle composition of embodiment 21 or embodiment 22, wherein the nanoparticle composition comprises the phospholipid in the range of 10 mol % to 15 mol % of the phospholipid per nanoparticle composition.

[0092] 24. A nanoparticle composition described in any one of embodiments 21 to 23, wherein the nanoparticle composition contains cholesterol or a derivative thereof in the range of 50 mol % to 75 mol % of cholesterol or a derivative thereof per nanoparticle composition.

[0093] 25. A method for treating or preventing a disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of a nanoparticle composition described in any one of embodiments 1 to 24.

[0094] 26. The method of embodiment 25, wherein the therapeutically effective amount of the nanoparticle composition comprises the therapeutic or immunogenic nucleic acid agent in the range of 0.01 mg of nucleic acid to 10 mg of nucleic acid per kg of the subject's body weight.

[0095] 27. The method of embodiment 25 or embodiment 26, wherein the subject is a mammal.

[0096] 28. The method of any one of embodiments 25 to 27, wherein the mammal is selected from the group consisting of rodents, dogs, primates, horses, high-value agricultural animals, and humans. EXAMPLES

[0097] The following non-limiting examples are provided to illustrate certain embodiments. Throughout, the embodiments may be supplemented with one or more details from one or more of the following examples, and / or one or more elements from an embodiment may be substituted with one or more details from one or more of the following examples.

[0098] An ideal nucleic acid carrier would be biodegradable to prevent bioaccumulation and subsequent cytotoxicity. Higher generations have higher packaging and are therefore difficult to biodegrade. There is a need for low generation dendrons that can complex with nucleic acids and translocate across cell membranes while remaining biodegradable and avoiding cytotoxicity.

[0099] Example 1. Nucleic acid carrier of formula [I]a In one embodiment, the chemical bonds present on the monomer units are ester bonds, and the terminal layers of lower generation polyester dendrons are substituted with amines via amide bonds. This advances the understanding of structure-activity relationships for how surfactant-like dendrons with amine surface groups and lipophilic units at their focal points can self-assemble and subsequently bind to nucleic acids with high affinity.

[0100] Scheme 1 is a schematic diagram of a second generation modified polyester dendron and an amine (A) that can be used for the modification. In this scheme, the alkyl chain B is C 10 ~C 28 An example of the synthesis of PE-Dendron-G2-hexadecyl-propylpyrrolidine is as follows: [ka]

[0101] Compound 1: Hexadecyl azide (MW: 267.2, 106 mg, 0.39 mmol) was dissolved in 1 mL of THF in a 50 mL round-bottom flask (RBF), followed by the addition of the alkyne, PE-G2-acetylene-OH (80 mg, 0.2 mol) dissolved in THF (0.5 mL) along with CuSO4·5H2O (7.8 mg, 0.031 mmol, 10 mol %) and sodium ascorbate (12.3 mg, 0.062 mmol, 20 mol %) in degassed THF:H2O (2 mL, 1:1). The reaction mixture was stirred at 23 °C for 16 hours (h). The next day the solvent was then removed in vacuo and purified by flash chromatography on a silica column (24 g) using a gradient elution from 100% CH2Cl2 (mobile phase a) to 75:22:3 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 65% mobile phase b (R f =0.6) to give the desired product as a yellow oil in 120 mg (90%) yield.

[0102] Compound 2: Compound 1 (120 mg, 0.18 mmol) was dissolved in dry DCM or CHCl (3 mL) and pyridine (0.3 ml, 1.44 mmol) was added followed by p-nitrophenyl chloroformate (580 mg, 2.88 mmol, MW 201.6, 16 equiv) dissolved in dry DCM (5 mL). The next day, the reaction mixture was diluted with 1.33 M NaHSO and extracted with DCM. The organic layer was washed with brine, concentrated on a Rotavap and purified by flash chromatography using DCM / EtOAc. This compound eluted with 8.5% EtOAc / DCM (R in 9:1 DCM / EtOAc). f =0.7). The yield of the reaction was 175 mg (74%). 1H NMR (301 MHz, chloroform-d) δ ppm 0.83-0.89 (m, 3H) 1.20-1.35 (m, 40H) 1.79-1.90 (m, 2H) 2.02-2.04 (m, 3H) 4.06-4.15 (m, 2H) 4.26-4.45 (m, 13H) 4.45-4.49 (m, 1H) 5.25-5.30 (m, 2H) 7.33-7.41 (m, 8H) 7.57-7.61 (m, 1H) 8.18-8.30 (m, 8H).

[0103] 13C NMR (76MHz, chloroform-d) δ ppm 14.09(s,1C) 14.17(s,1C) 17.58(s,1C) 17.62(s,1C) 21.03(s,1C) 22.65(s,1C) 26.45(s,1C) 28.95(s,1C) 29.31(s,1C) 29.36(s,1C) 29.49(s,1C) 29.56(s,1C) 29.61(s,1C) 29.64(s,1C) 30.22(s,1C) 31.88(s,1C) 46.53(s,1C) 46.67(s,1C) 50.44(s,1C) 58.42(s,1C) 60.37(s,1C) 65.70(s,1C) 69.08(s,1C) 77.20(s,1C) 121.75(s,1C) 123.91(s,1C) 125.26(s,1C) 125.28(s,1C) 125.30(s,1C) 141.61(s,1C) 145.54(s,1C) 152.07(s,1C) 155.17(s,1C) 170.97(s,1C) 172.01(s,1C).

[0104] Compound 3: A solution of compound 2 (90 mg, 0.07 mmol) dissolved in dry DCM (0.9 mL) was added to an excess of the amine 1-(3-aminopropyl)pyrrolidine (72 mg, 0.56 mmol) dissolved in dry DCM (1 mL). A solution of DMAP (17 mg, 0.28 mmol) and DIPEA (0.05 mmol, 0.56 mmol) in dry DCM (1 mL) was added and the reaction mixture was stirred at 23° C. under argon for 16 h. The next day, the solvent was then removed in vacuo and purified by flash chromatography on a silica column (24 g) using a gradient elution from 100% CHCl (mobile phase a) to 75:22:3 CHCl / MeOH / NHOHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 80% mobile phase b (R f = 0.2 (75:22:3 CH2Cl2 / MeOH / NH4OHaq) to give the desired product as a yellow oil (30 mg, 35%). MS(ESI) C 66 H 116 N 10 O 15Calculated for [M+H]+ m / z 1289.7, found 1289.7.

[0105] Example 2. Nucleic acid carrier of formula [I]b In this application, the chemical bonds present on the monomer units are ester bonds, and the terminal layers of low generation polyester dendrons with amines at their focal points were replaced with hydrophobic tails via ester bonds, making the entire structure susceptible to hydrolysis and therefore biodegradable. Such low generation dendrons with alkyl chains can non-covalently combine with nucleic acids to form nanoparticles through their dynamic equilibration nature. This advances the understanding of the structure-activity relationship of how surfactant-like dendrons with hydrophobic unit surface groups (B) and amines (A) at their focal points can self-assemble and subsequently bind to nucleic acids with high affinity. A representative structure of this example is shown below. [ka]

[0106] Example 3. Nucleic acid carrier of formula [I]c The structure contains two covalently linked dendritic segments, which may be of different generations, with a linear chain attached to one of the dendritic segments. The synthetic strategy exploited the chemoselectivity of the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Two separate dendrons with an azide and a primary acetylene at the focal point were efficiently coupled in THF. One dendron has amines at the periphery (periphery) and the other dendron has a hydrophobic tail at the periphery. A representative structure for this example is shown below. [ka]

[0107] [ka] [ka] Compound 4: The starting material PE dendron G2 acetylene-OH (300 mg, 0.62 mmol) was dissolved in dry DCM (6 mL) and pyridine (0.9 ml, 4.96 mmol) was added followed by p-nitrophenyl chloroformate (2.1 g, 10 mmol) dissolved in dry DCM (15 mL) and the reaction mixture was stirred at 0° C. to room temperature (rt) for 16 h. The next day, TLC showed the formation of the product. The reaction mixture was diluted with 1.33 M NaHSO4 and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4 and evaporated on a Rotavap. The crude product was then purified by flash chromatography using DCM / EtOAc. The compound started to elute at 45% EtOAc (R f =0.9) to give the desired product as a pale yellow oil (553 mg, 70%).

[0108] Compound 5: A solution of compound 4 (150 mg, 0.14 mmol) in dry DCM (3 mL) was added to excess NMe2EDA (0.08 ml, 0.7 mmol) in dry DCM (1 mL). A solution of DMAP (34 mg, 0.28 mmol) and DIPEA (0.1 ml, 0.56 mmol) in dry DCM (1 mL) was added and the reaction mixture was stirred at 23° C. under nitrogen for 16 h. The solvent was then removed in vacuo and loaded onto a 24 g column and eluted with 60% Ultra / DCM (silica gel TLC R in 75:22:3 CHCl / MeOH / NHOH aq.). f =0.25); yield 120 mg (90%). MS(ESI)C 38 H 68 N8O 14 Calculated value for [M]+ m / z 861.00, found value 861.4.

[0109] Compound 6: PE-G2-azide-OH (MW: 491.54, 54 mg, 114 μmol) was placed in a 50 mL RBF, followed by the addition of alkyne (MW: 860, 98 mg, 114 μmol) dissolved in THF (0.6 mL) along with CuSO4·5H2O (3 mg, 11.4 μmol, 10 mol%, MW 249.69), sodium ascorbate (4.5 mg, 22.8 μmol, 20 mol%, MW 198.11) and degassed THF:H2O (2 mL, 1:1) and the reaction mixture was stirred at 23 °C for 16 h. The next day the solvent was then removed in vacuo and purified by flash chromatography on a silica column (24 g) using a gradient elution from 100% CH2Cl2 (mobile phase a) to 75:22:3 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 70% mobile phase b (silica gel TLC R in mobile phase b). f =0.2); yield 120 mg (90%). MS(ESI)C 59 H 105 N 11 O 24 Calculated for [M+H]+ m / z 1352.73, found 1352.6.

[0110] Compound 7: Compound 6 (30 mg, 0.022 mmol) was dissolved in pyridine (2 ml). After flushing with argon for 10 min in an ice bath, linoleoyl chloride (0.04 ml, 0.11 mmol, 5.5 equiv, MW 298, SD 0.93) was added dropwise and stirred at 0° C. to 23° C. for 16 h. The next day, the solvent was then removed in vacuo and purified by flash chromatography on a silica column (24 g) using a gradient elution from 100% CHCl (mobile phase a) to 75:22:3 CHCl / MeOH / NHOHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 90% mobile phase b (silica gel TLC R in mobile phase b). f=0.45); yield 20 mg (36%). 1H NMR (301MHz, chloroform-d) δ ppm 0.82-0.90(m,12H) 1.12-1.16(m,6H) 1.19-1.39(m,79H) 1.50-1.67(m,9H) 1.97-2.07(m,16H) 2.16-2.24(m,24H) 2.24-2.31(m,9H) 2.37-2.45(m,6H) 2.71-2.79(m,7H) 3.18-3.28(m,6H) 4.01-4.29(m,24H) 4.32-4.39(m,1H) 5.24-5.40(m,16H) 5.59-5.70(m,2H) 7.71-7.76(m,1H). MS(ESI)C 131 H 225 N 11 O 28 Calculated for [M+2H]2+ m / z 1201.3, found 1201.8.

[0111] [ka] [ka] Compound 8: PE-G2-Azide-OH (250 mg, 0.51 mmol) was dissolved in dry DCM (6 mL) and pyridine (0.9 ml, 11.2 mmol) was added followed by p-nitrophenyl chloroformate (1.6 g, 8.16 mmol, MW 201.6, 16 equiv) dissolved in dry DCM (16 mL) and the reaction mixture was stirred at 0° C. to 23° C. for 16 h. The next day, TLC showed the formation of the product. The reaction mixture was diluted with 1.33 M NaHSO4 and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4 and evaporated on a Rotavap. The crude product was then purified by flash chromatography using DCM / EtOAc. The compound was eluted with 4% EtOAc / DCM (R f = 0.8 (19:1 DCM / EtOAc). Yield 135 mg (23%).

[0112] Compound 9: A solution of compound 8 (135 mg, 0.117 mmol) dissolved in dry DCM (1 mL) was added to excess NMe2EDA (0.077 ml, 0.7 mmol) dissolved in dry DCM (1 mL). A solution of DMAP (29 mg, 0.23 mmol) and DIPEA (0.08 ml, 0.46 mmol) in dry DCM (1 mL) was added and the reaction mixture was stirred at 23° C. under argon atmosphere for 16 h. The next day, the solvent was then removed in vacuo and purified by flash chromatography on a silica column (24 g) using a gradient elution from 100% CHCl (mobile phase a) to 75:22:3 CHCl / MeOH / NHOHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 60% mobile phase b (silica gel TLC R in mobile phase b). f =0.35). Yield: 70 mg (63%).

[0113] Compound 10: Compound 9 (70 mg, 0.074 mmol) was placed in a 50 ml RBF, the alkyne (29.9 mg, 0.074 mol) dissolved in 1 ml THF was added to the azide, CuSO4·5H2O (2 mg, 0.0074 mmol, 10 mol%) was added along with sodium ascorbate (3 mg, 0.015 mmol, 20 mol%) and degassed THF:H2O (2 mL, 1:1). The reaction mixture was stirred at 23 °C for 16 h. The next day, the solvent was then removed in vacuo and purified by flash chromatography on a silica column (24 g) using a gradient elution from 100% CH2Cl2 (mobile phase a) to 75:22:3 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 70% mobile phase b (silica gel TLC R in mobile phase b). f =0.25). Yield 50 mg (50%). MS(ESI)C 59 H 105 N 11 O 24 Calculated for [M+H]+ m / z 1352.73, found 1352.6.

[0114] Compound 11: Compound 10 (50 mg, 0.036 mmol) was dissolved in pyridine (2 ml). After flushing with argon for 10 min in an ice bath, linoleoyl chloride (0.065 ml, 0.203 mmol, 5.5 equiv, MW 298, SD 0.93) was added dropwise and stirred at 0° C. to 23° C. for 16 h. The next day, the solvent was then removed in vacuo and purified by flash chromatography on a silica column (40 g) using a gradient elution from 100% CHCl (mobile phase a) to 75:22:3 CHCl / MeOH / NHOHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 70% mobile phase b (silica gel TLC R in mobile phase b). f =0.85); yield 30 mg (34%). 1H NMR (301MHz, chloroform-d) δ ppm 0.82-0.91(m,12H) 1.14-1.41(m,84H) 1.50-1.68(m,10H) 1.96-2.07(m,15H) 2.17-2.25(m,26H) 2.26-2.31(m,6H) 2.36-2.45(m,7H) 2.70-2.79(m,7H) 3.17-3.27(m,6H) 4.07-4.24(m,22H) 4.27-4.38(m,3H) 5.22-5.41(m,16H) 5.57-5.66(m,2H) 7.65-7.69(m,1H). MS(ESI) C 131 H 225 N 11 O 28 Calculated for [M+2H]2+ m / z 1201.3, found 1201.8.

[0115] Example 4. Nucleic acid carrier of formula [I]d The structure contains two covalently linked dendritic segments, which may be of different generations, with a linear chain attached to one of the dendritic segments. This synthetic strategy also exploits the chemoselectivity of the CuAAC reaction. Two separate dendrons with azide and primary acetylene at the focal point were efficiently coupled in THF. This embodiment of the present example allows the introduction of different types of tails in the same structure. A representative structure of this example is shown below. [ka]

[0116] [ka] [ka] Compound 12: A solution of compound 4 (438 mg) in dry DCM (6 mL) was added to excess mono-Boc-DAPMA (0.45 ML) in dry DCM (6 mL). A solution of DMAP (100 mg) and DIPEA (0.29 ml) in dry DCM (1 mL) was added and the reaction mixture was stirred at 23° C. under argon for 16 h. TLC confirmed the reaction was complete. The crude product was concentrated under reduced pressure in a Rotavap and purified by flash chromatography on a silica column using a gradient elution from 100% CH2Cl2 (mobile phase a) to 75:22:3 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 45% mobile phase b (R at 1:1 mobile phase a / mobile phase b). f =0.4) to give the desired product as a pale yellow oil (404 mg, 66%).

[0117] Compound 13: PE-G2-Azide-OH (MW: 491.54, 58 mg, 114 μmol) was placed in a 50 mL RBF, followed by the addition of compound 12 (MW: 1475.82, 173 mg, 114 μmol) dissolved in THF (1 mL) along with CuSO4·5H2O (3 mg, 0.011 mmol, 10 mol%, MW 249.69) and sodium ascorbate (4.6 mg, 0.023 mmol, 20 mol%, MW 198.11) in degassed THF:H2O (2 mL, 1:1). The reaction mixture was stirred at 23 °C for 16 h. The next day, the reaction was complete as determined by TLC. The reaction mixture was purified by flash chromatography on a silica column using a gradient elution from 100% CH2Cl2 (mobile phase a) to 75:22:3 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 56% mobile phase b (Rf = 0.4 (75:22:3 CH2Cl2 / MeOH / NH4OHaq) to give the desired product as a yellow oil (140 mg, 60%). MS(ESI) C 91 H 165 N 15 O 32 Calculated for [M+4H]4+ m / z 795.5, found 794.9; [M+3H]3+ m / z 1059.96, found 1060.2.

[0118] Compound 14: Compound 13 (137 mg, 0.069 mmol) was dissolved in pyridine (2 ml). After flushing with argon for 10 min in an ice bath, linoleoyl chloride (0.13 ml, 0.41 mmol, 6 eq, MW 298, SD 0.93) was added dropwise and stirred at 0° C. to 23° C. for 16 h. The reaction mixture was stirred at 23° C. for 16 h. The next day, TLC confirmed the reaction was complete. The reaction mixture was evaporated on a Rotavap and purified by flash chromatography on a silica column using a gradient elution from 100% CH2Cl2 (mobile phase a) to 75:22:3 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 50% mobile phase b (R f = 0.6 (75:22:3 CH2Cl2 / MeOH / NH4OHaq) to give the desired product as a yellow oil (100 mg, 48%). MS(ESI) C 163 H 285 N 15 O 36 Calculated value for [M+2H] 2+ m / z 1515.5, found 1516.1; [M+3H] 3+m / z 1010.3, measured value 1011.0; 1H NMR (301MHz, chloroform-d) δ ppm 0.84-0.90 (m, 12H) 1.11-1.36 (m, 91H) 1.40-1.43 (m, 40H) 1.52-1.70 (m, 26H) 1.98-2.07 (m, 15H) 2.16-2.29 (m, 20H) 2.34-2.45 (m, 15H) 2.70-2.78 (m, 6H) 3.07-3.25 (m, 17H) 4.01-4.25 (m, 26H) 4.32-4.40 (m, 1H) 5.27-5.43 (m, 19H) 5.93-6.20(m,1H) 7.71-7.77(m,1H).

[0119] Compound 15 (PE dendron-A1-ricinoleic acid.PE dendron-linoleic acid): 100 mg of compound 14 (0.033 mmol) was treated with 20 equivalents of AcCl (0.047 ml, 0.66 mmol) after dissolving the compound in 3 ml of MeOH, the reaction was stirred at 0° C. to 23° C. for 4 h, evaporated to dryness, half of the crude product was dissolved in 2 ml of DMF, 0.045 ml of Et3N (0.33 mmol) was added, followed by the addition of ricinoleic acid-NHS (39 mg, 0.099 mmol) dissolved in 1 ml of DMF. The next day, the reaction mixture was concentrated and purified by flash chromatography on a silica column using a gradient elution from 100% CHCl2 (mobile phase a) to 75:22:3 CHCl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product elutes at 45% mobile phase b (R f =0.85 (75:22:3 CH2Cl2 / MeOH / NH4OHaq) to give the desired product as a yellow oil (50 mg, 41%). 1H NMR(301MHz,chloroform-d) δ ppm 0.84-0.89(m,22H) 1.06-1.17(m,14H) 1.20-1.33(m,133H) 1.41-1.46(m,13H) 1.53-1.70(m,37H) 1.98-2.07(m,22H) 2.11-2.21(m,34H) 2.23-2.32(m,7H) 2.35-2.44(m,18H) 2.71-2.78(m,4H) 3.13-3.30(m,19H) 3.55-3.63(m,5H) 3.66-3.78(m,1H) 4.02-4.39(m,27H) 5.18-5.57(m,20H) 6.17-6.28(m,2H) 6.82-6.91(m,2H) 7.70-7.80(m,1H).

[0120] Example 5. Nucleic acid carrier of formula [I]e The structure comprises two covalently linked dendritic segments, which may be of different generations, and a linear chain attached to one of the dendritic segments. Two separate dendrons with azide and primary acetylene at the focal point are coupled to form a heterofunctional dendritic structure, one segment modified to incorporate an amine on the surface and the other segment modified to incorporate both an amine and a tail. The present invention allows the introduction of different types of amines on the same structure, where one amine may collect nucleic acids while the other amine may have a strong buffering capacity inside the endosome and thus may have an additional influx of hydrochloric acid and water, ultimately resulting in endosomal rupture of the payload. A representative structure of this example is shown below. [ka]

[0121] [ka] Compound 16: A solution of compound 8 (90 mg, MW 1151.95, 0.078 mmol) in dry DCM (0.5 mL) was added to excess 4-(2-aminoethyl)morpholine (80 mg, 0.992 g / ml, MW 130.19, 0.63 mmol, 8 eq) in dry DCM (1 mL). A solution of DMAP (19 mg, MW 122.17, 0.156 mmol, 2 eq) and DIPEA (0.312 mmol, MW 129.24, d0.742 g / ml, 4 eq) in dry DCM (1 mL) was added and the reaction mixture was stirred at 23° C. under argon for 16 h. TLC showed the reaction was complete. The crude product was concentrated under reduced pressure in a Rotavap and purified by flash chromatography on a 24 g silica column using a gradient elution from 100% CH2Cl2 (mobile phase a) to 75:22:3 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 24% mobile phase b (R at 1:1 mobile phase a / mobile phase b). f =0.7) to give the desired product as a pale yellow oil (79 mg, 91%).

[0122] Compound 17: Compound 16 (MW: 1116, 79 mg, 80 μmol) was placed in a 50 ml RBF, followed by the addition of the alkyne, PE-G2-acetylene-Boc amine 1 or compound 12 (MW: 1489, 105 mg, 70 μmol) dissolved in THF (0.6 mL) along with CuSO4·5H2O (2 mg, 0.07 μmol, 10 mol%, MW 249.69) and sodium ascorbate (3 mg, 14.8 μmol, 20 mol%, MW 198.11) in degassed THF:H2O (2 mL, 1:1). The reaction mixture was stirred at 23 °C for 16 h. The next day, the reaction was complete as determined by TLC. The reaction mixture was purified by flash chromatography on a 24 g silica column using a gradient elution from 100% CH2Cl2 (mobile phase a) to 75:22:3 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 65% mobile phase b (R f= 0.2 (2:1 mobile phase b / mobile phase a)), affording the desired product as a yellow oil (75 mg, 41%). MS (ESI) C 119 H 213 N 23 O 40 Calculated for [M+3H]3+ m / z 869.4, found 869.18.

[0123] Compound 18: 50 mg of compound 17 (0.019 mmol) was treated with 20 equivalents (0.024 ml, 0.38 mmol) after dissolving the compound in 3 ml of MeOH, the reaction was stirred at 0° C. to 23° C. for 4 hours, evaporated to dryness, the crude product was dissolved in 2 ml of DMF, Et3N (0.053 ml, 0.38 mmol, MW 101.19, d 0.726) was added, followed by ricinoleic acid-NHS (46 mg, 0.115 mmol, MW 395.27) dissolved in 1 ml of DMF. The next day, the reaction mixture was concentrated and purified by flash chromatography on a silica column using a gradient elution from 100% CHCl2 (mobile phase a) to 75:22:3 CHCl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 75% mobile phase b (R f =0.55 (75:22:3 CH2Cl2 / MeOH / NH4OHaq)) to give the desired product as a yellow oil (50 mg, 41%). MS(ESI) C 171 H 309 N 23 O 40 Calculated for [M+2H]2+ m / z 1664.6, found 1664.4.

[0124] Example 6. Nucleic acid carrier of formula [I]f The structure comprises two covalently linked dendritic segments, which may be of different generations, and a linear chain attached to one of the dendritic segments. Two separate dendrons, each with an azide and a primary acetylene at the focal point, are coupled to form a homofunctional dendritic structure. A representative structure for this example is shown below. [ka]

[0125] [ka] [ka] Compound 19: PE-G1-Azide-OH (250 mg, 0.96 mmol, MW 259.3) was dissolved in dry DCM (3 mL) and pyridine (0.6 ml, 7.68 mmol, 8 eq) was added followed by p-nitrophenyl chloroformate (525 mg, mmol, MW 201.6, 2.7 eq) dissolved in dry DCM (8 ml) and the reaction mixture was stirred at 0° C. to 23° C. for 16 h. The next day TLC showed the formation of the product. The reaction mixture was diluted with 1.33 M NaHSO4 and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4 and evaporated on a Rotavap. The crude product was then purified by flash chromatography using DCM / EtOAc. The compound was eluted with 2% EtOAc / DCM (R f = 0.8 (19:1 DCM / EtOAc). Yield 190 mg (33%).

[0126] Compound 20: A solution of compound 19 (190 mg, 0.32 mmol, 589.51) dissolved in dry DCM (0.5 mL) was added to an excess of Boc amine 1 (237 mg, 0.97 mmol, 3 equiv.) dissolved in dry DCM (1 mL). A solution of DMAP (79 mg, 0.64 mmol) and DIPEA (0.22 ml, 1.28 mmol, MW 129.24, d 0.742) in dry DCM (1 mL) was added and the reaction mixture was stirred at 23° C. under argon for 16 h. The next day, the solvent was then removed in vacuo and purified by flash chromatography on a silica column (24 g) using a gradient elution from 100% CHCl (mobile phase a) to 75:22:3 CHCl / MeOH / NHOHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 40% mobile phase b (silica gel TLC R in mobile phase a / mobile phase b f =0.55). Yield: 140 mg (54%).

[0127] Compound 21: PE-G1-Acetylene-OH (641 mg, 3.73 mmol) was dissolved in dry DCM (6 mL) and (5.4 mL, 30 mmol) was added followed by p-nitrophenyl chloroformate (2 g, 10 mmol, 2.7 equiv.) dissolved in dry DCM (15 mL) and the reaction mixture was stirred at 0° C. to 23° C. for 16 h. The next day, TLC showed the formation of the product. The reaction mixture was diluted with 1.33 M NaHSO4 and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4 and evaporated on a Rotavap. The crude product was then purified by flash chromatography using DCM / EtOAc. The compound was eluted with DCM (R f = 0.7 (19:1 DCM / EtOAc). Yield 765 mg (41%).

[0128] Compound 22: A solution of compound 21 (350 mg, 0.70 mmol, MW 502.4) dissolved in dry DCM (3 mL) was added to an excess of Boc amine 1 (513 mg, 2.09 mmol, MW 245.4) dissolved in dry DCM (6 mL). A solution of DMAP (170 mg, 1.39 mmol) and DIPEA (0.49 ml, 2.8 mmol) in dry DCM (1 mL) was added and the reaction mixture was stirred at 23 °C under argon atmosphere for 16 h. The next day, the solvent was then removed in vacuo and purified by flash chromatography on a silica column (40 g) using a gradient elution from 100% CHCl (mobile phase a) to 75:22:3 CHCl / MeOH / NHOHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 50% mobile phase b (silica gel TLC R in mobile phase a / mobile phase b). f =0.55). Yield: 300 mg (60%).

[0129] Compound 23: The azide compound 20 (MW: 802.03, 70 mg, 0.087 mol) was dissolved in 50 ml RBF, then the alkyne compound 22 (MW: 742.48, 65 mg, 0.087 mmol) dissolved in THF (0.6 mL) was added along with CuSO4·5H2O (7 mg, 30 mol%, MW 249.69) and sodium ascorbate (11 mg, 60 mol%, MW 198.11) and degassed THF:H2O (2 mL, 1:1). The reaction mixture was stirred at 23 °C for 16 h. The next day the solvent was then removed in vacuo and purified by flash chromatography on a silica column (24 g) using a gradient elution from 100% CH2Cl2 (mobile phase a) to 75:22:3 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 65% mobile phase b (silica gel TLC R in 1:1 mobile phase a / mobile phase b f =0.65). Yield 70 mg (50%). MS(ESI)C 71 H 133 N 15 O 20 Calculated for [M+2H]2+ m / z 759.0, found 758.6.

[0130] Compound 24: 70 mg of compound 23 (0.027 mmol) was treated with 20 eq. of AcCl (0.04 ml, 0.54 mmol) after dissolving the compound in 3 ml of MeOH, the reaction was stirred at 0° C. to 23° C. for 5 h, evaporated to dryness, the crude product was dissolved in 2 ml of DMF, Et3N (0.08 ml, 0.54 mmol) was added, followed by ricinoleic acid-NHS (64 mg, 0.16 mmol, 6 eq.) dissolved in 1 ml of DMF. The next day, the reaction mixture was concentrated and purified by flash chromatography on a silica column (24 g) using a gradient elution from 100% CHCl2 (mobile phase a) to 75:22:3 CHCl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 74% mobile phase b (R f =0.85 (2:1 mobile phase b / mobile phase a)), affording the desired product as a yellow oil (32 mg, 31%). MS(ESI) C123 H 229 N 15 O 20 Calculated for [M+2H]2+ m / z 1119.9, found 1119.3; [M+3H]3+ m / z 746.6, found 746.5.

[0131] [ka] [ka] Compound 25: The azide, PE-G1-azide-OH (230 mg, 0.89 mol, MW 259.3) was placed in a 25 mL RBF, followed by the alkyne, PE-G2-acetylene-OH (327 mg, 0.80 mmol, MW 404.4) dissolved in THF (0.6 mL) along with CuSO4·5H2O (22 mg, 10 mol%, MW 249.69, 35 mg used) and sodium ascorbate (35 mg, 20 mol%, MW 198.11: 50 mg actual added) and degassed THF:H2O (2 mL, 1:1) and the reaction mixture was stirred at 23 °C for 16 h. The next day the solvent was then removed in vacuo and purified by flash chromatography on a silica column (24 g) using a gradient elution from 100% CH2Cl2 (mobile phase a) to 75:22:3 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 65% mobile phase b (silica gel TLC R in 1:1 mobile phase a / mobile phase b). f =0.60). Yield 398 mg (74%, MW 663.32). MS(ESI) C 29 H 49 N3O 14 Calculated for [M+H]+ m / z 663.3, found 663.8.

[0132] Compound 26: Compound 25 (349 mg, 0.526 mmol) was dissolved in dry DCM (8.5 mL) and cooled in dry ice to which pyridine (0.34 ml, 4.21 mmol, 8 equiv.) was added, followed by p-nitrophenyl chloroformate (1.7 g, 8.42 mmol, 201.5 MW) dissolved in dry DCM (10 mL) and the reaction mixture was stirred at 0° C. to room temperature for 16 h. The next day, TLC showed the formation of the product. The reaction mixture was washed with 1.33 M NaHSO4 and brine and evaporated. The loaded compound was then purified by flash chromatography using DCM (A) / EtOAc (B). The product was eluted with 2% EtOAc (R f = 0.4 (19:1 DCM / EtOAc). Yield 573 mg (66%).

[0133] Compound 27: A solution of compound 26 (250 mg, 0.15 mmol) dissolved in dry DCM (1 mL) was added to an excess of Boc amine 1 (223 mg, 0.91 mmol) dissolved in dry DCM (1 mL). A solution of DMAP (37 mg, 0.62 mmol) and DIPEA (0.11 ml, 1.26 mmol) in dry DCM (1 mL) was added and the reaction mixture was stirred at 23 °C under argon atmosphere for 16 h. The next day, the solvent was then removed in vacuo and purified by flash chromatography on a silica column (24 g) using a gradient elution from 100% CHCl (mobile phase a) to 75:22:3 CHCl / MeOH / NHOHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 53% mobile phase b (silica gel TLC R in 1:1 mobile phase a / mobile phase b). f =0.6). Yield: 260 mg (75%).

[0134] Compound 28 (PE dendron-G2-A1-ricinoleic acid.PE dendron-G1-A1-ricinoleic acid): 100 mg of compound 27 (0.044 mmol, MW 2290.46) was treated with 20 equivalents of AcCl (0.06 ml, 0.87 mmol) after dissolving the compound in 3 ml of MeOH, the reaction was stirred at 0° C. to 23° C. for 5 h, evaporated to dryness and the crude product was dissolved in 2 ml of DMF. 0.12 ml of Et3N (0.87 mmol) was added followed by the addition of ricinoleic acid-NHS (138 mg, 0.26 mmol) dissolved in 1 ml of DMF. The next day, the reaction mixture was concentrated and purified by flash chromatography on a silica column using a gradient elution from 100% CH2Cl2 (mobile phase a) to 75:22:3 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b) over 40 min. The desired product eluted at 60% mobile phase b (R at 1:1 mobile phase a / mobile phase b). f =0.7) to give the desired product as a yellow oil (98 mg, 45%). MS(ESI) C 185 H 343 N 21 O 32 Calculated for [M+3H]3+ m / z 1125.6, found 1125.2; [M+4H]4+ m / z 843.9, found 844.1.

[0135] Example 7. Nanoparticle formulations containing nucleic acid carriers of formula Ia Nanoparticles containing PE-G2-hexadecyl-propylpyrrolidine:DSPC:cholesterol:DMG-PEG2k in a molar ratio of 1:0.5:2.4:0.04 were formulated using a NanoAssemblr Benchtop (Precision NanoSystems Inc, Vancouver, BC, Canada). RNA was diluted with DNase / RNase-free, endotoxin-free distilled water and sterile citrate buffer to the final desired pH. The total flow rate was maintained at 12 mL / min with a 3:1 ratio of aqueous to organic phase for formulation on the Benchtop. Nanoparticles were dialyzed against sterile, endotoxin-free PBS using a 20,000 molecular weight cut-off dialysis using glassware that was depyrogenated by heating at 250°C for 24 hours. The dialyzed nanoparticles were sterile filtered using a 0.2 micron (0.2 μm) poly(ethersulfone) filter and characterized using a Zetasizer NanoZS instrument (Malvern). Encapsulation efficiency was determined to be 90% for a nanoparticle composition containing PE-G2-hexadecyl-propylpyrrolidine and SEAP replicon RNA (formulated at pH 5) using the Ribogreen® assay (Geall et al., 10.1073 / pnas.1209367109, incorporated by reference herein as if fully set forth).

[0136] Hydrodynamic size measurements The "Z-average" as a function of size of nanoparticle compositions containing PE-G2-hexadecyl-propylpyrrolidine and SEAP replicon RNA was determined by dynamic light scattering (DLS) as shown in Figures 1 and 2. The "Z-average" is the intensity-weighted average hydrodynamic size of an ensemble collection of particles measured by dynamic light scattering (DLS). As shown in Figure 1, the strongest intensity was observed for nanoparticles of size 192.5 d.nm (pH 5.0 formulation). The size distribution is characterized by a single peak with a low polydispersity index, indicating a relatively monodisperse size.

[0137] Gel retardation assay Agarose gel electrophoresis was performed to assess the binding of the dendron carrier to RNA according to known methods (Geall et al., 10.1073 / pnas.1209367109, which is incorporated by reference as if set forth in full). Figure 3 is a photograph of an agarose gel showing the binding of PE-G2-hexadecyl-propylpyrrolidine to replicon RNA. The gel was stained with ethidium bromide (EB) and the gel image was taken with a Syngene G Box Imaging System (Syngene, USA). As shown in FIG. 3, lane 1 contained the product of formulation of PE-G2-hexadecyl-propylpyrrolidine and SEAP replicon RNA (pH 5.0 formulation), lane 2 contained the product of formulation of PE-G2-hexadecyl-propylpyrrolidine and SEAP replicon RNA (pH 6.0 formulation), and lane 3 contained unformulated SEAP mRNA. Prior to loading, the samples were incubated with formaldehyde loading dye, denatured at 65° C. for 10 min, and cooled to 23° C. The gel was run at 90 V, and gel images were taken with a Syngene G Box Imaging System (Syngene, USA). For RNA detection, the gel was stained with ethidium bromide. As shown in FIG. 3, the lower band corresponds to small-sized free RNA (lane 3), and the upper band represents large-sized nanoparticles formed by binding of RNA to the nucleic acid carrier.

[0138] In vivo SEAP production results To test the formulation of the nucleic acid carrier of formula Ia, the secreted placental alkaline phosphatase SEAP reporter system was used. For in vivo testing, mice were injected with nanoparticles at a dose of 5 μg of SEAP replicon RNA, and serum was collected from the mice 1, 4, and 6 days after administration. SEAP was quantified using the Invitrogen NovaBright™ Phospha-Light™ EXP Assay kit for detection according to the manufacturer's protocol. The amount of SEAP in mouse serum samples was measured with a BioTek Synergy HTX microplate reader and reported in arbitrary units (AU). Error bars are ± SEM. As shown in Figure 4, it was observed that the amount of SEAP was higher in the pH 6.0 formulation compared to pH 5.0, because RNA was released faster from the pH 6.0 formulation due to weaker binding.

[0139] Example 8. Nanoparticle formulations containing nucleic acid carriers of formula Id Nanoparticles were formulated by direct mixing of 30 μl of ethanol phase containing the nucleic acid carrier of formula Id (e.g., PE-G2-A1-ricinoleic acid.PE-G2-linoleic acid) in combination with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-lipid, Avanti Polar Lipids) with 90 μl of SEAP replicon RNA diluted in ultrapure, DNase / RNase-free, endotoxin-free distilled water (Invitrogen) and sterile 100 mM (pH 5.0) QB citrate buffer (Teknova) to a final citrate concentration of 10 mM. The resulting nanoparticles contained a mass ratio of nucleic acid carrier to PEG-lipid to replicon RNA of 16:2.3:6. The formulations were diluted 1000-fold for analysis of particle size distribution, Z-average, using a Zetasizer Nano ZS (Malvern Panalytical).

[0140] Hydrodynamic size measurements Figure 5 shows the distribution of nanoparticle composition measured as intensity based on nanoparticle size (d.nm; diameter in nm). As shown in Figure 5, "Z-average" is the intensity-weighted average hydrodynamic size of an ensemble population of particles measured by dynamic light scattering (DLS). As shown in Figure 5, the strongest intensity was observed for nanoparticles of size 234.5 d.nm.

[0141] Gel retardation assay Agarose gel electrophoresis was performed to assess the binding of PE-G2-A1-ricinoleic acid.PE-G2-linoleic acid to RNA according to known methods (Geall et al., 10.1073 / pnas.1209367109, which is incorporated by reference as if set forth in its entirety). FIG. 6 is a photograph of an agarose gel showing the binding of PE-G2-A1-ricinoleic acid.PE-G2-linoleic acid to SEAP replicon RNA. The gel was stained with ethidium bromide (EB) and gel images were taken with a Syngene G Box Imaging System (Syngene, USA). As shown in FIG. 6, lane 1 contained unformulated SEAP RNA replicon and lane 2 contained the product of formulation of PE-G2-A1-ricinoleic acid.PE-G2-linoleic acid and SEAP replicon RNA. Prior to loading, the samples were incubated with formaldehyde loading dye, denatured at 65°C for 10 min, and cooled to 23°C. The gels were run at 90V, and gel images were taken with a Syngene G Box Imaging System (Syngene, USA). For RNA detection, the gels were stained with ethidium bromide. As shown in Figure 6, the lower band corresponds to the small size of free RNA (lane 1), and the upper band represents the large size of nanoparticles formed by the binding of RNA to the nucleic acid carrier (lane 2).

[0142] In vitro SEAP production results To test the ability of the nanoparticle composition to express SEAP in vitro, BHK cells were treated with nanoparticles. Each well of a BHK 12-well dish was treated with 10 μL (approximately 1 μg) of the formulation product diluted with a 1:1 Optimem:PBS mixture to a final volume of 500 μL. Non-treated wells had 500 μL of 50 / 50 PBS / OptiMEM. 24 hours after treatment or transfection, conditioned medium was collected from each culture. The medium was analyzed using the QuantiBlue assay (InvivoGen). 150 μL of QuantiBlue reagent was mixed with 50 μL of medium. Readings were taken after 10 minutes by measuring absorbance at 650 nm as instructed by the manufacturer's protocol. For untreated samples or negative controls, 50 μL of medium from wells not treated with nanoparticles was added to 150 μL of QuantiBlue reagent. FIG. 7 shows in vitro SEAP expression of nanoparticle formulations containing PE-G2-A1-ricinoleic acid.PE-G2-linoleic acid as nucleic acid carriers.

[0143] Example 9. Nanoparticle formulations containing a nucleic acid carrier of formula If Nanoparticles containing PE-G1-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid or PE-G2-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid:DOPE:cholesterol:DMG-PEG2k at a molar ratio of 1:0.6:2.88:0.1 were formulated using a NanoAssemblr Benchtop (Precision NanoSystems Inc, Vancouver, BC, Canada). RNA was diluted with DNase / RNase-free, endotoxin-free distilled water and sterile citrate buffer to the final desired pH. The total flow rate was maintained at 10 mL / min with a 3:1 ratio of aqueous phase to organic phase for formulation on the Benchtop. Nanoparticles were dialyzed against sterile, endotoxin-free PBS using a 20,000 molecular weight cut-off dialysis using glassware that was depyrogenated by heating at 250° C. for 24 hours. The dialyzed nanoparticles were sterile filtered using a 0.2 micron (0.2 μm) poly(ethersulfone) filter and characterized using a Zetasizer NanoZS instrument (Malvern). Encapsulation efficiency was determined to be 93% and 90% for nanoparticle compositions containing PE-G1-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid or PE-G2-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid and SEAP replicon RNA (formulated at pH 6) using the Ribogreen® assay (Geall et al., 10.1073 / pnas.1209367109, incorporated herein by reference as if fully set forth).

[0144] Hydrodynamic size measurements Figures 8A and 8B show the distribution of nanoparticle composition measured as intensity based on nanoparticle size (d.nm; diameter in nm). As shown in Figures 8A and 8B, the strongest intensity was observed for nanoparticles of size 86.90 d.nm and 102.6 d.nm, respectively. These size distributions are characterized by a single peak with a low polydispersity index, indicating relatively monodisperse sizes.

[0145] In vitro SEAP production results To test the ability of the nanoparticle composition to express SEAP in vitro, BHK cells were treated with nanoparticles. Each well of a BHK 12-well dish was treated with 10 μL (approximately 1 μg) of the formulation product diluted with a 1:1 Optimem:PBS mixture to a final volume of 500 μL. Non-treated wells had 500 μL of 50 / 50 PBS / OptiMEM. 24 hours after treatment or transfection, conditioned medium was collected from each culture. The medium was analyzed using the QuantiBlue assay (InvivoGen). 150 μL of QuantiBlue reagent was mixed with 50 μL of medium. Readings were taken after 10 minutes by measuring absorbance at 650 nm as instructed by the manufacturer's protocol. For untreated samples or negative controls, 50 μL of medium from wells not treated with nanoparticles was added to 150 μL of QuantiBlue reagent. FIG. 9A shows in vitro SEAP expression of nanoparticle formulations containing PE-G1-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid or PE-G2-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid as nucleic acid carriers.

[0146] In vivo SEAP production results To test formulations containing PE-G1-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid or PE-G2-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid in vivo, mice were injected with nanoparticles with a dose of 2 μg of SEAP replicon RNA and serum was collected from the mice 3 days after administration. SEAP was quantified using the Invitrogen NovaBright™ Phospha-Light™ EXP Assay kit for SEAP detection according to the manufacturer's protocol. The amount of SEAP in mouse serum samples was measured on a BioTek Synergy HTX microplate reader and is reported in arbitrary units (AU). Error bars are ± SEM. As shown in Figure 9B, the amount of SEAP was observed to be higher with PE-G1-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid compared to PE-G2-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid.

[0147] COVID-19 Wuhan Spike RBD Direct Serum ELISA Mice (BALB / c) were vaccinated with 2 μg of spike repllicon RNA formulated with PE-G1-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid and PE-G2-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid (in a total volume of 100 μL PBS) by IM injection into both leg muscles. Mice were housed for 14 days after injection and serum was isolated from whole blood by centrifugation of clotted samples at 10000 RCF for 1.5 minutes. The serum was assayed for anti-spike RBD antibody titers by direct ELISA. Greiner Bio High Binding Microlon ELISA plates were coated overnight at 4° C. with PBS containing recombinant Wuhan spike RBD protein. Wells were blocked with PBS+0.5% BSA for 1 hour at room temperature. Serum samples were added to wells in serial 1:2 dilutions starting at 1:50 dilution and up to 1:6400 in PBS+0.5% BSA. Samples were incubated for 1 hour at room temperature, washed 3 times with PBST, then goat anti-mouse IgG HRP was added at 1:20,000 dilution in PBS+0.5% BSA and incubated for 1 hour. Plates were washed again 5 times with PBST and developed with the chromogenic HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB). The reaction was stopped by the addition of 0.5M H2SO4 and absorbance was measured at 450 nm and 570 nm. The endpoint antibody titer was designated as the highest dilution where the absorbance at 450-absorbance at 570 value was ≧0.08. At week 2, groups immunized with 2 μg of nanoparticle vaccines formulated with PE-G1-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid and PE-G2-A1-ricinoleic acid.PE-G1-A1-ricinoleic acid had endpoint dilution titers ( FIG. 10 ) ranging from 200 to 1600. This is in contrast to the non-immunized seronegative group, whose titers remained at baseline.

[0148] References: References cited throughout this application are incorporated for all purposes apparent in the specification and in the references themselves as if each reference were set forth in full. For purposes of presentation, certain of these references are cited at specific locations in this specification. The citation of a reference at a specific location indicates the manner in which the teachings of the reference are incorporated. However, the citation of a reference at a specific location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.

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Claims

1. A nucleic acid carrier comprising a dendron or dendrimer of formula Ia, Ib, Ic, Id, Ie or If, 【Chemistry 1】 In the above formula, PE is a polyester dendron comprising a core and a plurality of monomeric polyester units forming one or more generations, A is an amine, B is a hydrophobic unit, and z is the number of surface groups.

2. PE has formula II: 【Chemistry 2】 2. The nucleic acid carrier according to claim 1, wherein, in the formula, G is a layer or generation of the dendron, and n is the number of generations, which is in the range of 1 to 10.

3. 2. The nucleic acid carrier according to claim 1, wherein the plurality of monomeric polyester units are 2,2-bis(hydroxymethyl)propionic acid or 2,2-bis(hydroxymethyl)butyric acid.

4. z has the formula II: z=b ｎ 、III 2. The nucleic acid carrier according to claim 1, wherein in the formula, b is the branch point multiplicity or the number of branches at each branch point, and n is the number of generations, which is in the range of 1 to 10.

5. A is 1-(2-aminoethyl)piperazine, 1-piperidineethanamine, 1-dimethylamino-2-propylamine, 1-(3-aminopropyl)pyrrolidine, 1-(2-aminoethyl)pyrrolidine, 1-(2-aminoethyl)piperidine, 2-(1-piperazinyl)ethylamine, 2-(4-methyl-piperazin-1-yl)-ethylamine, 3-(dimethylamino)-1-propylamine, 3-(diethylamino)propylamine, (4-aminobutyl)dimethylamine, 4-(1 -pyrrolidinyl)-1-butylamine, 4-morpholineethanamine, 4-morpholinepropanamine, 4-methyl-1-piperazineethanamine, 4-(diethylamino)butylamine, N,N-dimethylethylenediamine, N,N-dimethyldipropylenetriamine, N,N'-dimethyl-N,N'-bis(3-methylaminopropyl)trimethylenediamine, N1-(3-aminopropyl)propane-1,3-diamine, N1-(3-aminopropyl)-N1-methylpropane-1,3-Diamine, 3-((3-aminopropyl)(methyl)amino)propan-1-ol, 1-dimethylamino-2-propanol, 1-[bis[3-(dimethylamino)propyl]amino]-2-propanol, 2-dimethylaminoethanol, 2-(diethylamino)ethanol, 2-[2-(dimethylamino)ethoxy]ethanol, 2-(2-aminoethoxy)ethanol, 2-{[2-(dimethylamino)ethyl]methylamino}ethanol, 3-dimethylamino-1-propanol, 3-diethylamino-1-propanol, 4-(dimethylamino)-1-butanol, 4-diethylamino-2-butyn-1-ol, N-methyl The nucleic acid carrier according to claim 1, which is diethanolamine, 4-(diethylamino)butan-1-ol, 3-(azetidin-1-yl)propan-1-ol, 3-(pyrrolidin-1-yl)propan-1-ol, 3-(piperidin-1-yl)propan-1-ol, 4-(azetidin-1-yl)butan-1-ol, 4-(pyrrolidin-1-yl)butan-1-ol, 4-(piperidin-1-yl)butan-1-ol, 3-morpholinopropan-1-ol, 4-morpholinobutan-1-ol, 3-(4-methylpiperazin-1-yl)propan-1-ol, or 4-(4-methylpiperazin-1-yl)butan-1-ol.

6. The nucleic acid carrier according to claim 1, wherein B is derived from a fatty acid (C4 to C28) or a derivative thereof.

7. 7. The nucleic acid carrier of claim 6, wherein the fatty acid is selected from the group consisting of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, and eicosapentanoic acid.

8. The fatty acid derivatives include 12-hydroxy-9-cis-octadecenoic acid, 12-methyltetradecanoic acid, 12-methyltridecanoic acid, 14-methylhexadecanoic acid, 14-methylhexadecanoic acid, 18-methylnonadecanoic acid, 19-methylarachidic acid, isopalmitic acid, isostearic acid, phytanic acid, (±)-2-hydroxyoctanoic acid, (±)-3-hydroxydecanoic acid, (±)-3-hydroxyoctanoic acid, 10-hydroxyoctanoic acid, ...hydroxy-9-cis-octadecenoic acid, 12-hydroxy-9-cis-octadecanoic acid, 12-hydroxy-9-cis-octadecanoic acid, 12-hydroxy-9-cis-octadecanoic acid, 12-hydroxy-9-cis-octadecanoic acid, 12-hydroxy-9-cis-octadecanoic acid, 12-hydroxy-9-cis-octadecanoic acid, 12-hydroxy-9-cis-octadecanoic acid, 12-hydroxy-9-cis-octadecanoic acid, 12-hydroxy-9-cis-octadecanoic acid, 12-hydroxy-9-cis-octa The nucleic acid carrier according to claim 6, wherein the hydroxyl group is selected from the group consisting of DL-α-hydroxystearic acid, DL-β-hydroxylauric acid, DL-β-hydroxymyristic acid, and DL-β-hydroxypalmitic acid.

9. The nucleic acid carrier according to claim 6 , wherein the fatty acid or derivative contains one or more stable isotopes.

10. The nucleic acid carrier according to claim 9, wherein the stable isotope is a stable isotope of carbon or hydrogen.

11. The stable isotope of carbon is １３ The nucleic acid carrier according to claim 10, which is C.

12. The stable isotope of hydrogen is ２ The nucleic acid carrier according to claim 10, which is H.

13. The fatty acid containing the stable isotope is selected from the group consisting of octanoic acid-1-13C, octanoic acid-8-13C, octanoic acid-8,8,8-d3, octanoic acid-2H15, decanoic acid-1-13C, decanoic acid-10-13C, decanoic acid-10,10,10-d3, decanoic acid-d19, undecanoic acid-1-13C, lauric acid-12,12,12-2H3, lauric acid-2H23, lauric acid-1-13C, lauric acid-1,12-13C2, tridecanoic acid-2,2-2H2, myristic acid-14-13C, myristic acid-1-13C, myristic acid-14,14,14-2H3, myri The nucleic acid carrier according to claim 10, which is selected from the group consisting of stearic acid-2H27, palmitic acid-1-13C, palmitic acid-16-13C, palmitic acid-16-13C,16,16,16-2H3, palmitic acid-2H31, stearic acid-1-13C, stearic acid-18-13C, stearic acid-18,18,18-2H3, stearic acid-2H35, oleic acid-1-13C, oleic acid-2H34, linolenic acid-1-13C, linoleic acid-2H32, arachidonic acid-5,6,8,9,11,12,14,15-2H8, and eicosanoic acid-2H39.

14. 14. A nanoparticle composition comprising a nucleic acid carrier according to any one of claims 1 to 13 and a therapeutic or immunogenic nucleic acid agent fully or partially encapsulated therein.

15. The nanoparticle composition of claim 14, wherein the therapeutic or immunogenic nucleic acid agent is selected from the group consisting of polynucleotides, oligonucleotides, DNA, cDNA, RNA, repRNA, siRNA, miRNA, sgRNA and mRNA.

16. The nanoparticle composition of claim 14, wherein the therapeutic or immunogenic nucleic acid agent encodes one or more antigens selected from the group consisting of infectious diseases, pathogens, cancer, autoimmune diseases and allergenic diseases.

17. 15. The nanoparticle composition of claim 14, wherein the therapeutic or immunogenic nucleic acid agent comprises RNA or DNA capable of silencing, inhibiting or altering the activity of a gene.

18. The nanoparticle composition of claim 14, further comprising a PEG-lipid.

19. 20. The nanoparticle composition of claim 18, wherein the therapeutic or immunogenic nucleic acid agent is selected from the group consisting of polynucleotides, oligonucleotides, DNA, cDNA, RNA, repRNA, siRNA, miRNA, sgRNA and mRNA.

20. The nanoparticle composition of claim 18, wherein the therapeutic or immunogenic nucleic acid agent encodes one or more antigens selected from the group consisting of infectious diseases, pathogens, cancer, autoimmune diseases and allergenic diseases.

21. 20. The nanoparticle composition of claim 18, wherein the therapeutic or immunogenic nucleic acid agent comprises RNA or DNA capable of silencing, inhibiting or altering the activity of a gene.

22. The nanoparticle composition of claim 18, wherein the PEG-lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

23. 20. The nanoparticle composition of claim 18, wherein the nanoparticle composition comprises the PEG-lipid in the range of 1 mol % to 10 mol % of the PEG-lipid per nanoparticle composition.

24. 20. The nanoparticle composition of claim 18, further comprising a phospholipid and cholesterol or a derivative thereof.

25. The nanoparticle composition of claim 24, wherein the PEG-lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

26. 25. The nanoparticle composition of claim 24, wherein the nanoparticle composition comprises the PEG-lipid in the range of 1 mol % to 10 mol % of the PEG-lipid per nanoparticle composition.

27. 25. The nanoparticle composition of claim 24, wherein the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or distearoylphosphatidylcholine (DSPC).

28. 25. The nanoparticle composition of claim 24, wherein the nanoparticle composition comprises the phospholipid in the range of 10 mol % to 15 mol % of the phospholipid per nanoparticle composition.

29. 25. The nanoparticle composition of claim 24, wherein the nanoparticle composition comprises cholesterol or a derivative thereof in the range of 50 mol % to 75 mol % cholesterol or a derivative thereof per nanoparticle composition.

30. 15. Use of the nanoparticle composition of claim 14 for the manufacture of a medicament for treating or preventing a disease or condition in a subject.

31. 31. The use of claim 30, wherein the therapeutic or immunogenic nucleic acid agent is selected from the group consisting of polynucleotides, oligonucleotides, DNA, cDNA, RNA, repRNA, siRNA, miRNA, sgRNA and mRNA.

32. The use of claim 30, wherein the therapeutic or immunogenic nucleic acid agent encodes one or more antigens selected from the group consisting of infectious diseases, pathogens, cancer, autoimmune diseases and allergenic diseases.

33. 31. The use of claim 30, wherein the therapeutic or immunogenic nucleic acid agent comprises RNA or DNA capable of silencing, inhibiting or altering the activity of a gene.

34. The use of claim 30, wherein the nanoparticle composition further comprises a PEG-lipid.

35. 35. The use of claim 34, wherein the therapeutic or immunogenic nucleic acid agent is selected from the group consisting of polynucleotides, oligonucleotides, DNA, cDNA, RNA, repRNA, siRNA, miRNA, sgRNA and mRNA.

36. The use of claim 34, wherein the therapeutic or immunogenic nucleic acid agent encodes one or more antigens selected from the group consisting of infectious diseases, pathogens, cancer, autoimmune diseases and allergenic diseases.

37. 35. The use of claim 34, wherein the therapeutic or immunogenic nucleic acid agent comprises RNA or DNA capable of silencing, inhibiting or altering the activity of a gene.

38. 35. The use according to claim 34, wherein the PEG-lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

39. 35. The use of claim 34, wherein the nanoparticle composition comprises the PEG-lipid in the range of 1 mol % to 10 mol % of the PEG-lipid per nanoparticle composition.

40. 40. The use of claim 39, wherein the nanoparticle composition further comprises a phospholipid and cholesterol or a derivative thereof.

41. 41. The use according to claim 40, wherein the PEG-lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

42. 41. The use of claim 40, wherein the nanoparticle composition comprises the PEG-lipid in the range of 1 mol % to 10 mol % PEG-lipid per nanoparticle composition.

43. The use according to claim 40, wherein the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or distearoylphosphatidylcholine (DSPC).

44. 41. The use of claim 40, wherein the nanoparticle composition comprises the phospholipid in the range of 10 mol % to 15 mol % of the phospholipid per nanoparticle composition.

45. The use according to claim 40, wherein the nanoparticle composition comprises cholesterol or a derivative thereof in the range of 50 mol % to 75 mol % cholesterol or a derivative thereof per nanoparticle composition.

46. The use described in claim 30, wherein the therapeutically effective amount of the nanoparticle composition comprises the therapeutic or immunogenic nucleic acid agent in the range of 0.01 mg nucleic acid to 10 mg nucleic acid per kg body weight of the subject.

47. 47. The use of claim 46, wherein the subject is a mammal.

48. 48. The use of claim 47, wherein the mammal is selected from the group consisting of rodents, dogs, primates, horses, high value agricultural animals, and humans.

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