Nanoparticle Compositions Containing Sugar-Functionalized Nucleic Acid Carriers

Abstract

Nanoparticle compositions for delivering nucleic acids to a subject are described, the nanoparticle compositions comprising a carrier, the carrier comprising a sugar-functionalized nucleic acid carrier, and a therapeutic or immunogenic nucleic acid agent encapsulated within the delivery molecule. Methods are provided for treating or preventing a disease or condition in a subject by administering the nanoparticle compositions that provide an immune response and a synergistic therapeutic or prophylactic effect.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 233,505, filed August 16, 2021, and entitled "Nanoparticle Compositions Containing Sugar Functionalized Nucleic Acid Carriers," which is incorporated by reference herein as if fully set forth.

[0002] Field The present disclosure relates to a carrier for efficient delivery of a nucleic acid to a subject to treat or prevent a disease and / or disorder, and to a nanoparticle composition comprising the carrier and the nucleic acid. The present disclosure also relates to a method of formulating the nanoparticle composition, and a method of treating a disease and / or disorder in a subject with such a nanoparticle composition. [Background technology]

[0003] The industry continues to search for novel and safe nucleic acid carriers with multifunctional properties that can efficiently package and deliver genetic material to patient cells for ultimate therapeutic effect. A suitable non-viral gene delivery system may require a delicate balance of high cellular uptake, loading capacity, biocompatibility with low toxicity and high transfection efficiency (Jones et al., 2013, Mol.Pharmaceutics 10, 4082-4098; Nishikawa and Huang, 2001 Hum.Gene Ther. 12, 861-870; and Mintzer and Simanek, 2009, Chem.Rev. 109, 259-302). Carbohydrates are one of the most abundant natural compounds and important participants in many biological processes, with relevance across medical and industrial fields. Compared to synthetic polymers, carbohydrates are biocompatible and have inherent targeting properties, which allow them to interact with cell surface receptors (Hong et al., 2018, Carbohydr Polym. 181:1180-1193; Han et al., 2018, Polymers, 10(9), 1034) and participate in other biological processes to enhance uptake and eventual expression. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Jones et al., 2013, Mol.Pharmaceutics 10, 4082-4098 [Non-Patent Document 2] Nishikawa and Huang, 2001 Hum. Gene Ther. 12, 861-870 [Non-Patent Document 3] Mintzer and Simanek, 2009, Chem.Rev. 109, 259-302 [Non-Patent Document 4] Hong et al., 2018, Carbohydr Polym. 181:1180-1193 [Non-Patent Document 5] Han et al., 2018, Polymers, 10(9), 1034 Summary of the Invention [Problem to be solved by the invention]

[0005] In this invention, we have presented various classes of sugar-functionalized nucleic acid carriers for improved gene delivery, in which sugars are chemically conjugated to dendron or dendrimer systems for efficient and biocompatible delivery of nucleic acids. [Means for solving the problem]

[0006] In one aspect, the invention provides a nanoparticle composition comprising a nucleic acid carrier having the structure of one of formulas Ia, Ib, Ic, Id, Ie, If, Ig, Ih, Ii, Ij, IIa, or IIb, [ka] [ka] wherein A is an amine linker, B is a hydrophobic unit, n is 0-20, Y is a sugar moiety, and Z is a deoxy sugar moiety.

[0007] In one aspect, the present invention relates to a nanoparticle composition comprising at least one nucleic acid carrier as disclosed herein, optionally at least one therapeutic or immunogenic nucleic acid agent encapsulated in the nucleic acid carrier, and a conjugated lipid as disclosed herein (e.g., a PEG-lipid).

[0008] In one aspect, the present invention relates to a nanoparticle composition comprising at least one nucleic acid carrier as disclosed herein, at least one therapeutic or immunogenic nucleic acid agent encapsulated in the nucleic acid carrier, one conjugated lipid as disclosed herein (e.g., PEG-lipid), and a mixture of phospholipid and cholesterol or a derivative thereof for enhancing intracellular delivery and nanoparticle stability in vivo.

[0009] In one aspect, the invention relates to a method for treating or preventing a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a nanoparticle composition of the invention. [Brief description of the drawings]

[0010] The following detailed description of the preferred embodiment 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 should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

[0011] [Figure 1] FIG. 1 is a schematic diagram of a generation 2 modified polyester dendron with amine and ricinoleic acid tails as hydrophobic units, where the focal point of the dendron has been modified with various types of deoxy sugars. [Diagram 2] 1 shows the distribution of nanoparticle composition measured as intensity based on nanoparticle size (d). [Diagram 3] Figure 1 shows a photograph of an agarose gel showing the binding of PE Dendron_G2-2 deoxyglucose-A1-ricinoleic acid with 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] Quantification of SEAP expression in vitro and in vivo following administration of nanoparticle formulations containing deoxysugar-modified dendrons. SEAP mRNA formulated with PE dendron_G2-1 deoxyglucose-A1-ricinoleic acid, PE dendron_G2-2 deoxyglucose-A1-ricinoleic acid, and PE dendron_G2-6 deoxyglucose-A1-ricinoleic acid produced nanoparticles that led to SEAP protein production. [Diagram 5] Quantification of SEAP expression in vitro and in vivo following administration of deoxysugar-modified dendron-based nanoparticle formulations. SEAP-encoding mRNA was formulated with PE dendron_G2-2 deoxyglucose-A1-ricinoleic acid, PE dendron_G2-2 deoxyglucose-A5-ricinoleic acid, PE dendron_G2-1 deoxymannose-A5-ricinoleic acid, and PE dendron_G2-2 deoxygalactose-A5-ricinoleic acid to produce nanoparticles that were administered to cells in vitro or to mice. [Figure 6]Figure 1 shows endpoint dilution titers of mouse serum IgG specific for SARS-CoV-2 spike protein following vaccination with SARS-CoV-2 spike replicon RNA formulated with PE dendron_G2-2 deoxyglucose-A1-ricinoleic acid or PE dendron_G2-2 deoxyglucose-A5-ricinoleic acid delivery materials. An additional group of mice was vaccinated with the same RNA formulated with the amino lipid DLin-MC3-DMA (control). [Figure 7] FIG. 1 shows the distribution of nanoparticle composition measured as intensity based on size (d) of nanoparticles in which SEAP mRNA was formulated with PE Dendron_G2-Hexylmannose-A5-Ricinoleic acid (DMG PEG2000 as the PEG-lipid conjugate). [Figure 8] Quantification of SEAP expression in vivo following administration of nanoparticle formulations with sugar-modified dendrons. SEAP mRNA formulated with PE dendron_G2-hexylmannose A5-ricinoleic acid produced nanoparticles that led to SEAP protein production. [Figure 9] 1 shows the distribution of nanoparticle composition measured as intensity based on nanoparticle size (d). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In the following description, certain terminology is used for convenience only and is not limiting.

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

[0014] As used herein, the term "amine linker" refers to an amine-containing linker that links or connects a hydrophobic unit (conveniently described herein as component "B") to a terminal chemical group present on a dendrimer or dendron surface. The amine present in an amine linker is a functional group that contains a basic nitrogen atom bearing a lone pair of electrons. Amines are formally derivatives of ammonia in which one or more hydrogen atoms have been replaced with a substituent, e.g., an alkyl group.

[0015] As used herein, the term "alkyl" refers to a straight or branched chain hydrocarbon containing 1 to 28, preferably 1 to 20, carbon atoms, unless otherwise specified. The alkyl group may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbon atoms, or a number of carbon atoms in a range selected from any two of the above values. The length of the alkyl chain may be used to control the hydrophobicity and self-assembly properties of the nucleic acid carrier.

[0016] As used herein, the term "surface group" refers to a terminal group on the surface of the nucleic acid carrier. The surface of the nucleic acid carrier of the present invention is modified with hydrophobic units (referred to herein as component "B") to support self-assembly properties.

[0017] A numerical value or range preceded by "about" refers to the number explicitly recited and within the experimental error of the indication intended. An embodiment described with the modifier "about" may be modified to remove "about" to form further embodiments herein. Similarly, an embodiment described without the modifier "about" may be modified to add "about" to form further embodiments herein.

[0018] A range expressed as being between two numerical values, one with a low endpoint and the other with a high endpoint, includes the values ​​between those numerical values, as well as the low and high endpoints. Embodiments of the invention include subranges of the ranges herein, which include the low and high endpoints of that subrange selected from any increment within the range selected from each single increment of the least significant digit, provided that the high endpoint of the subrange is higher than the low endpoint of that subrange.

[0019] Further embodiments herein include replacing one or more of "including" or "comprising" in an embodiment with "consisting essentially of" or "consisting of." As used herein, "including" and "comprising" are open-ended and include the recited elements and do not exclude the addition of one or more other elements. "Consisting essentially of" means that the addition of one or more elements compared to those recited is within the scope, but the addition does not materially affect the basic and novel characteristics of the combination of elements expressly recited. "Consisting of" refers to the recited elements but excludes any unspecified elements, steps, or ingredients.

[0020] The words "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 indicated. This term includes the specifically mentioned words above, their derivatives and synonyms. The phrase "at least one" following a list of two or more items, such as "A, B, or C" or "A, B and C," means any one of A, B, or C, and any combination thereof.

[0021] One embodiment is a nanoparticle composition comprising a nucleic acid carrier having the structure of one of formulas Ia, Ib, Ic, Id, Ile, If, Ig, Ih, Ii, Ij, IIa, or IIb, [ka] [ka] wherein A is an amine linker, B is a hydrophobic unit, n is 0-20, Y is a sugar moiety, and Z is a deoxy sugar moiety.

[0022] The amine linker A is a moiety that contains one or more nitrogen atoms with lone pairs of electrons, thereby imparting proton-accepting functionality to the nucleic acid carrier molecule. Thus, the amine linker is capable of accepting 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 linker is selected from the group consisting of N1-(2-aminoethyl)ethane-1,2-diamine, N1-(2-aminoethyl)propane-1,3-diamine, N1-(3-aminopropyl)propane-1,3-diamine, N1,N1'-(ethane-1,2-diyl)bis(ethane-1,2-diamine), N1,N1'-(ethane-1,2-diyl)bis(N2-(2-aminoethyl)ethane-1,2-diamine), N1-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine, ... -1,2-diamine, N1-(2-aminoethyl)-N1-methylethane-1,2-diamine, N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine, N1-(3-aminopropyl)-N1-ethylpropane-1,3-diamine, 3-((3-aminopropyl)(methyl)amino)propan-1-ol, 3,3'-(methylazanediyl)bis(propan-1-ol), N1-(3-aminopropyl)-N1-methylbutane-1,4-diamine, 4-( (3-aminopropyl)(methyl)amino)butan-1-ol, 4-((3-hydroxypropyl)(methyl)amino)butan-1-ol, 4-((3-hydroxypropyl)(methyl)amino)butan-1-ol, N1-(4-aminobutyl)-N1-methylbutane-1,4-diamine, 4-((4-aminobutyl)(methyl)amino)butan-1-ol, 4,4'-(methylazanediyl)bis(butan-1-ol), 3-((3-aminopropyl)(ethyl) amino)propan-1-ol, 3,3'-(ethylazanediyl)bis(propan-1-ol), N1-(3-aminopropyl)-N1-ethylbutane-1,4-diamine, 4-((3-aminopropyl)(ethyl)amino)butan-1-ol, 4-(ethyl(3-hydroxypropyl)amino)butan-1-ol, N1-(2-aminoethyl)-N1-methylpropane-1,3-diamine, N1-(4-aminobutyl)-N1-ethylbutane-1,4-diamine, 4,4'-(ethylazanediyl)bis(butan-1-ol), 3-((3-aminopropyl)amino)propan-1-ol, N1-(3-aminopropyl)butane-1,4-diamine, 4-((3-hydroxypropyl)amino)butan-1-ol, N1-(4-aminobutyl)butane-1,4-diamine, 3,3'-azanediylbis(propan-1-ol), 4-((3-aminopropyl)amino) The linker may be derived from N1,N1'-(butane-1,4-diyl)bis(propane-1,3-diamine), 2-(bis(3-aminopropyl)amino)ethan-1-ol, 2-((4-aminobutyl)(3-aminopropyl)amino)ethan-1-ol, or 2-(bis(4-aminobutyl)amino)ethan-1-ol. For reference, the structures of amine linkers derived from the above amines are pictorially presented as the following structures: The pKa values ​​of the amines present in the linkers were calculated using the ACD / percepta pKa prediction tool. [ka] [ka]

[0023] The hydrophobic unit B of formulae Ia, Ib, Ic, Id, Ie and If is a C1-C 28 Alkyl group or C2-C 28 It may be an alkenyl group. 28 Alkyl group or C2-C 28Each alkenyl group may be optionally substituted with 1 to 4 substituents selected from halogen, -CN, -NO2, -N3, C1-C6 alkyl, halo(C1-C6 alkyl), -OR, -NR2, -C2R, -OC(O)R, -CON(R)2, -OC(O)N(R)2, -NHC(O)N(R)2, -NHC(NH)N(R)2, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, aryl, heteroaryl, or heterocycle. Each R may be independently selected from hydrogen, C1-C6 alkyl, halo(C1-C6 alkyl), C3-C8 cycloalkyl, C3-C8 cycloalkenyl, aryl, heteroaryl, or heterocycle. Each cycloalkyl, cycloalkenyl, aryl, heteroaryl and heterocycle may be further optionally substituted with R', where R' may be independently selected from halogen, -CN, -NO2, -N3, C1-C6 alkyl and halo(C1-C6 alkyl). An embodiment of the invention includes a nucleic acid carrier in which one or more of the amine linkers are deprotonated. An embodiment of the invention includes a nucleic acid carrier in which one or more of the amine linkers are protonated. An embodiment of the invention includes a nucleic acid carrier in which all of the amine linkers are deprotonated. An embodiment of the invention includes a nucleic acid carrier in which all of the amine linkers are protonated.

[0024] The hydrophobic unit B of formula Ia, Ib, Ic, Id, Ie, If, Ig, Ih, Ii, Ij, IIa and IIb may be introduced by contacting the nucleic acid carrier with a functional reagent. In one embodiment, the functional reagent is a fatty acid. The fatty acid is a C4-C 28The fatty acid may be a saturated fatty acid having a chain, or may be an unsaturated fatty acid. The fatty acid may be caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid or linolenic acid, arachidonic acid, eicosapentanoic acid, 12-hydroxy-9-cis-octadecenoic acid (ricinoleic acid), 12-methyltetradecanoic acid, 12-methyltridecanoic acid, 14-methylhexadecanoic acid, 14-methylhexadecanoic acid, 18-methylnonadecanoic acid, 19-methylarachidonic acid, isopalmitic acid, isostearic acid, phytanic acid, (±)-2-hydroxyoctadecanoic acid, 12-methyltridecano ...2-methyltridecanoic acid, 14-methylhexadecanoic acid, 18-methylnonadecanoic acid, 19-methylarachidonic acid, 12-methyltridecanoic acid, 12-methyltridecanoic acid, 12-methyltridecanoic acid, 12-methyltridecanoic acid, 12-methyltridecanoic acid, 12-methyltridecanoic acid, 12-methyltridecanoic acid, 12-methyltridecanoic acid, 12-methyltridecanoic acid, 12-methyltridecanoic acid, 12-methyltridecanoic acid, 12-methyltridecanoic acid, 12-methyltridecanoic acid, 12-methyltridecanoic acid, 12-methyl The fatty acid may be, but is not limited to, 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. The fatty acid may be selected from conjugated fatty acids (e.g., conjugated isomers of linoleic acid); acetylenic fatty acids (e.g., crepenic acid); allenic fatty acids (e.g., lavalenic acid) or cyclopropenyl fatty acids (e.g., sterculic acid).

[0025] The hydrophobic unit B may be a methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, but-3-en-1-yl, oct-7-en-1-yl, 12-tridecenyl, 14-pentadecenyl, 17-octadecenyl, oleyl, linoleyl, arachidonyl or 16-hydroxyhexadecyl group, or a 12-hydroxy-9-cis-octadecenyl (ricinoleyl) group.

[0026] In one embodiment, the nucleic acid carrier may include a tracking moiety. The tracking moiety may be one or more functional groups suitable for tracking the delivery material in vitro and in vivo. The nucleic acid carrier may have a fatty acid containing stable isotopes of carbon (C) and / or hydrogen (H). In one embodiment, the stable isotopes of carbon (C) and / or hydrogen (H) are 13 C or 2 H (also referred to herein as deuterium, D or d). The tracking moiety in such a nucleic acid carrier would be a stable isotope of carbon or hydrogen. If the nucleic acid carrier is formulated into nanoparticles with nucleic acid, the nanoparticles may be tracked in vitro and in vivo after administration by techniques such as mass spectrometry or nuclear magnetic resonance imaging. In one embodiment, the nucleic acid in the nanoparticles to be tracked is a replicon RNA. These stable isotopes are abundant in tissues. 12 C and 1 The inclusion of a stable isotope may be beneficial in identifying the delivery molecule, since it is different from the H isotope. Tracking may be useful in identifying the biodistribution, material clearance and molecular stability of the nanoparticles after administration, and related issues. The isotope-labeled fatty acid is octanoic acid-1- 13 C, Octanoic acid-8- 13 C, 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- 13C, Palmitic acid-16- 13 C,16,16,16- 2 H3, palmitic acid - 2 H31, stearic acid-1- 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.

[0027] Y in formula Ia, formula Ib, formula Ie, formula If, formula Ii, formula Ij and formula IIa is a sugar. Non-limiting examples of sugars are furanose monosaccharides (e.g., xylofuranose, ribofuranose or arabinofuranose), pyranose monosaccharides (e.g., glucose, mannose, galactose), disaccharides (e.g., lactose, trehalose), polysaccharides (e.g., cyclodextrin), or sugar derivatives. In one embodiment, the sugar derivative is selected from nucleosides or nucleotides, etc. Z in formula Ic, formula Id, formula Ig or formula Ih or formula IIb is a deoxy sugar. Deoxy sugars are sugars in which a hydroxyl group is replaced with a hydrogen atom, and non-limiting examples of the sugar are furanose monosaccharides, pyranose monosaccharides, disaccharides, oligosaccharides, polysaccharides, or sugar derivatives. In one embodiment, the sugar derivative is selected from nucleosides or nucleotides. Non-limiting examples of deoxy sugars are 2-deoxy-D-ribose, 6-deoxy-L-tagatose, 5-deoxy-xylofuranose, 5-deoxy-ribofuranose and 5-deoxy-arabinofuranose, 1-deoxyglucose, 2-deoxyglucose, 6-deoxyglucose, 1-deoxymannose, 2-deoxygalactose, 6-deoxygalactose, 1-deoxylactose, 6-deoxy-trehalose, 6-deoxy-2,4-diacetamido-2,4,6-trideoxy-D-mannose and 6A-deoxy-β-cyclodextrin, which are building blocks of DNA.

[0028] Z of formula Ic or formula Id or formula IIb may be introduced by contacting the nucleic acid carrier with a functional reagent via click chemistry. In one embodiment, the functional reagent is selected from deoxysugar (monosaccharide, disaccharide, or polysaccharide) azides. The azide may be, but is not limited to, D-xylopyranosyl azide, 2,3,4-tri-O-acetyl-β-D-xylopyranosyl azide, 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl azide, 3,4,6-tri-O-acetyl-2-O-trifluoromethanesulfonyl-β-D-mannopyranosyl azide, 1,3,4,6-tetra-O-acetyl-2-azido-2-deoxy-D-galactopyranose, 1,2,3,4-tetra-O-acetyl-6-azido-6-deoxy-α-D-galactopyranose, 2,3,4,6-tetra-O-acetyl-β-D-galactopyranose, 2,3,4-tri-O-acetyl-6-azido-6-deoxy-α-D-galactopyranose, Oxy-β-D-glucopyranosylamine, 2,3,4-tri-O-acetyl-6-azido-6-deoxy-β-D-glucopyranosyl azide, 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide, 6-O-tosyl-β-D-glucopyranosyl azide, 1,3,4,6-tetra-O-acetyl-2-azido-2-deoxy-D-glucopyranose, 1,3,4-tri-O-acetyl-2-azido-2-deoxy-6-O-trityl-6-D-glucopyranose, 2,3,4-tri-O-acetyl-6-O-tosyl-β-D-glucopyranosyl azide, 3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-glucopyranosyl Trichloroacetimidate, 3,4,6-tri-O-acetyl-2-azido-2-deoxy-D-glucopyranosyl trichloroacetimidate, 1,2,4,6-tetra-O-acetyl-3-azido-3-deoxy-D-glucopyranose, 1,3,4-tri-O-acetyl-2-azido-2-deoxy-β-D-glucopyranuronic acid methyl ester, 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosyl azide, 1,3,4-tri-O-acetyl-2-azido-2-deoxy-α-L-fucopyranose, 1,2,3,4-tetra-O-acetyl-6-azido-L-fucopyranose, 1,2,3,4-Tetra-O-acetyl-6-azido-6-deoxy-D-galactopyranose, ethyl 3-azido-3-deoxy-N-methyl-β-D-glucopyranosiduronamide, ethyl 3-azido-3-deoxy-2,4-di-O-acetyl-β-D-glucopyranuronic acid benzyl ester, ethyl 3-azido-3-deoxy-β-D-glucopyranuronic acid methyl ester, β-L-fucopyranosyl azide, 2-fluoro-4-nitrophenyl 2-azido-2-deoxy-β-D-galactopyranoside, β-D-galactopyranosyl azide, β-D-maltosyl azidoheptaacetate, β-D-lactosyl azidoheptaacetate, methyl 4-Azido-4-deoxy-β-D-glucopyranoside, methyl 4-azido-2,3,6-tri-O-benzoyl-4-deoxy-β-D-glucopyranoside, methyl 2,3,4-tri-O-acetyl-6-azido-6-deoxy-α-D-glucopyranoside, methyl 2,3,4-tri-O-acetyl-β-D-glucopyranuronosyl azide, β-D-maltosyl azide, α-D-mannopyranosyl azide, phenyl 2-azido-2,6-dideoxy-1-seleno-α-D-galactopyranoside, phenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-seleno-α-D-galactopyranoside, phenyl 2-Azido-2-deoxy-1-seleno-α-D-galactopyranoside, phenyl 3,6-di-O-acetyl-2-azido-2-deoxy-1-seleno-α-D-galactopyranoside, 2,3,4-tri-O-acetyl-β-L-fucopyranosyl azide, 2-acetamido-2-deoxy-β-D-glucopyranosyl azide, 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl azide, β-D-glucopyranosyl azide , 6-azido-6-deoxy-β-D-glucopyranosylamine, 1-O-acetyl-4-azido-2,3,6-tri-O-benzoyl-4-deoxy-D-glucopyranose, 2-azido-2-deoxy-D-galactose, 3-azido-3-deoxy-D-galactose, 4-azido-4-deoxy-D-galactose, 3-azido-3-deoxy-4-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose, 6-azido-6-deoxy-2,3-O-isopropylidene-α-L-sorbofuranose, β-D-cellobiosyl azide, β-D-cellobiosyl azidoheptaacetate, 2-chloro-4-nitrophenyl 2-Azido-2-deoxy-β-D-galactopyranoside, 3,4-di-O-acetyl-1,6-anhydro-2-azido-2-deoxy-β-D-glucopyranose, 6,6'-diazido-6,6'-dideoxy-α,α-D-trehalose, 3,6-di-O-acetyl-2-azido-2-deoxy-α-D-glucopyranose, 2-deoxy-2-fluoro-β-D-glucopyranosyl azide, 1,6-di-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy -α-D-glucopyranose, 3,4-di-O-acetyl-2-azido-2-deoxy-D-glucopyranose, 6,6'-diazide-6,6'-dideoxy-α,α-D-trehalose hexaacetate, 3-azido-3-deoxy-1,2:5,6-di-O-isopropylidene-α-D-allofuranose, 3-azido-3-deoxy-1,2-O-isopropylidene-α-D-allofuranose, 4-O-(6-azido-6-deoxy-β-D-glucopyranosyl)-D-glucose , 2-azido-2-deoxy-L-fucopyranose, 6-azido-L-fucose, 6-azido-6-deoxy-D-galactose, 2-azido-2-deoxy-1-O-(t-hexyldimethylsilyl)-β-L-fucopyranose, 2-azido-2-deoxy-D-glucofuranurono-6,3-lactone, 6-azido-6-deoxy-1,2-O-isopropylidene-α-D-glucofuranose, 4-azido-4-deoxy-D-glucose, 6-azido-6-deoxy-D-glucoside Ranose, 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-β-D-glucopyranose, 1,6-anhydro-2-azido-4-O-benzyl-2-deoxy-β-D-glucopyranose, 1,6-anhydro-2-azido-3,4-di-O-benzyl-2-deoxy-β-D-glucopyranose, 1,6-anhydro-2-azido-2-deoxy-β-D-glucopyranose, 2-azido-2-deoxy-D-glucose, 6-O-acetyl-2-azido-3,Y of formula Ii or formula Ij may be introduced by contacting the nucleic acid carrier with a functional reagent by thiol chemistry. In one embodiment, the functional reagent is selected from sugar (monosaccharide, disaccharide, or polysaccharide) thiols. The sugar thiol may be, but is not limited to, β-D-GlcNAc-ethyl-thiol, β-LacNAc-PEG3-thiol, β-Lac-PEG3-thiol, α-Man-PEG3-thiol, β-Glc-PEG3-thiol, β-GlcNAc-PEG3-thiol, β-Gal-PEG3-thiol, α-GalNAc-PEG3-thiol, β-GalNAc-PEG3-thiol, β-Gal-PEG3-thiol, β-GlcNAc-PEG3-thiol, α-Man-PEG3-thiol, β-Glc-PEG3-thiol, β-Lac-PEG3-thiol, β-LacNAc-PEG3-thiol, and NeuAcα(2-6)LacNAc-PEG3-thiol.

[0029] Z of formula Ig or formula Ih may be introduced by contacting the nucleic acid carrier with a functional reagent via disulfide chemistry. In one embodiment, the functional reagent is selected from deoxysugar (monosaccharide, disaccharide, or polysaccharide) thiols. The sugar thiols may be, but are not limited to, 1-thio-β-D-glucopyranose, 2-acetamido-2-deoxy-1-thio-β-D-glucopyranose, 1-thio-β-D-lactopyranose, C-glucosylpropylthiol, and C-mannosylthiol.

[0030] One embodiment includes a nanoparticle composition comprising any one or more of the nucleic acid carriers described herein. The nanoparticle composition may further comprise a drug, e.g., a nucleic acid. The nanoparticle composition of the invention may be useful for introducing a drug into a cell. The drug may be a nucleic acid. The nanoparticle composition of the invention may be useful as a transfection agent. The nanoparticle composition of the invention may be useful in a method for treating or preventing a disease.

[0031] In one embodiment, the nanoparticle composition may include a mixture of nucleic acid carriers, each containing a different amine and / or side chain and / or sugar. The nucleic acid carriers may be mixed in a fixed ratio. Also, as an example of a mixture of three nucleic acid carriers, the ratio of the first nucleic acid carrier to the second nucleic acid carrier to 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 any two values ​​between them.

[0032] In one embodiment, the nanoparticle composition may include one or more nucleic acid agents. The nucleic acid agent may be therapeutic or immunogenic. As used herein, the term "nucleic acid" refers to any natural or synthetic DNA or RNA molecule. The agent of the composition of the present invention, for example, a therapeutic or immunogenic nucleic acid agent, may be complexed with or encapsulated in the nucleic acid carrier of the nanoparticle composition.

[0033] In one embodiment, the 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 recombinant protein, peptide, or polypeptide. The encoded protein, peptide, or polypeptide may be an antigen. The term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides. This polymer has the following properties: 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000 , 20000, 21000, 22000, 23000, 24000, 25000, 26000, 27000, 28000, 29000, 30000, 31000, 32000, 33000, 34000, 35000, 36000, 37000, 38000, 39000, 40000, 41000, 42000, 43000, 44000, 45000, 46000, 47000, 48000, 49000, 50000 or more ribonucleotides.This polymer has the following properties: 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 21000, 220 In one embodiment, the nucleic acid sequence may have 00, 23000, 24000, 25000, 26000, 27000, 28000, 29000, 30000, 31000, 32000, 33000, 34000, 35000, 36000, 37000, 38000, 39000, 40000, 41000, 42000, 43000, 44000, 45000, 46000, 47000, 48000, 49000, or 50000 ribonucleotides, or a number of ribonucleotides within a range between any two of the aforementioned numbers. The RNA molecule may be a replicon RNA (repRNA), small interfering RNA (siRNA), miRNA, single-stranded guide RNA (sgRNA), messenger RNA (mRNA) or transfer RNA (tRNA). Replicon RNA (repRNA) refers to a genome-replicating but progeny-defective RNA virus genome that cannot produce infectious progeny virions. Typically, viral genomes 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 direct or mediate RNA interference. MicroRNA (miRNA) refers to a small (20-24 nt) regulatory non-coding RNA involved in the post-transcriptional regulation of eukaryotic gene expression by affecting either or both the stability and translation of the coding mRNA. Messenger RNA (mRNA) is typically a single-stranded RNA that defines the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA.The DNA or RNA molecules may be chemically modified in the nucleic acid backbone, the ribose sugar moiety and the nucleobases themselves.

[0034] 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 usually 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.

[0035] 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), 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 a nucleic acid agent electrostatically bound to the charged nucleic acid carrier via electrostatic interactions. The nucleic acid agent may be bound to the charged nucleic acid carrier via electrostatic interactions and hydrogen bonds.

[0036] In one embodiment, the nanoparticle compositions described herein may include an immunogenic or therapeutic nucleic acid agent that encodes an antigen.

[0037] As used herein, "encapsulation" can refer to nanoparticles that provide active or therapeutic agents with complete encapsulation, partial encapsulation, or both. In one embodiment, the therapeutic agent is a nucleic acid (messenger RNA, as a non-limiting example). In a preferred embodiment, the nucleic acid is fully encapsulated in the nanoparticle. For nucleic acid therapeutic agents, complete 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).

[0038] As used herein, an "antigen" is defined as a molecule that elicits an immune response. The immune response may be in terms of 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 a macromolecule. In one embodiment, the macromolecule is a protein, peptide, or polypeptide. An antigen may be a structural component of a pathogen or a cancer cell or its derivatives. An antigen may be synthesized, recombinantly produced in a host, or derived from a biological sample, including, but not limited to, a tissue sample, a cell, or a body fluid.

[0039] 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 the adaptive immune response of the subject's body by delivering an antigen.The immune response induced by vaccination usually leads to the development of immunological memory, which subsequently leads to the ability of the organism to respond quickly when it encounters an antigen or infectious agent.

[0040] In this specification, it is preferable to use "nucleic acid carrier" as a carrier for nucleic acids, and therefore the term "nucleic acid carrier" is given. However, non-nucleic acid agents may also be embodiments of the present invention.

[0041] In one embodiment, the nanoparticle composition described herein may include lipid complexes. In one embodiment, lipid complexes may be useful in preventing particle aggregation. Lipid complexes that may be present in the compositions herein include, but are not limited to, polyethylene glycol (PEG)-lipid complexes. Non-limiting examples of PEG-lipids include PEG bound to lipids (e.g., DMG-PEG 2000), PEG bound to phospholipids (e.g., phosphatidylethanolamine (PEG-PE)), PEG conjugated to cholesterol or its derivatives, and mixtures thereof. In some cases, PEG may be substituted with alkyl, alkoxy, acyl, or aryl groups as needed.

[0042] PEGs are classified by molecular weight, e.g., PEG 2000 has an average molecular weight of about 2000 daltons, and PEG 5000 has an average molecular weight of about 5000 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 550 daltons to 10,000 daltons.

[0043] Phosphatidylethanolamines with various acyl chain groups of different chain lengths and degrees of saturation may be conjugated to PEG to form lipid complexes. Phosphatidylethanolamines may be commercially available or may be isolated or synthesized using conventional techniques. Phosphatidylethanolamines include those with carbon chain lengths of C 10 -C 20The phosphatidylethanolamine may comprise saturated or unsaturated fatty acids ranging from 0 to 100%. The phosphatidylethanolamine may comprise mono- or polyunsaturated fatty acids and mixtures of saturated and unsaturated fatty acids. Phosphatidylethanolamines contemplated include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidylethanolamine (DSPE).

[0044] The PEG-lipid may comprise PEG conjugated to cholesterol or a derivative of cholesterol, examples of which include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0045] The size, relative amount and distribution of PEG-lipids in a nanoparticle composition may affect the physical properties of the nanoparticle composition and can be used to control particle properties. Controllable physical properties may be, but are not limited to, the diameter of the nanoparticles, the tendency of the nanoparticles to aggregate, the number of nucleic acid molecules in each nanoparticle, 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 International Application No. 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) and Reichmuth, A. M. et al., "mRNA vaccine delivery using lipid nanoparticles," Therapeutic Delivery 7,5 (2016):319-34. Both references are incorporated herein by reference as if fully set forth.

[0046] The nanoparticle composition may contain 10 mol% or less of PEG-lipid per nanoparticle composition. The nanoparticle composition may contain 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 containing PEG-lipid may contain nanoparticles with a smaller diameter than the nanoparticles of the composition lacking PEG-lipid.

[0047] The nanoparticle composition may contain "amphiphilic lipids". As used herein, "amphiphilic lipids" refers to any material having a non-polar hydrophobic unit or "tail" and a polar "head". Polar groups may include, but are not limited to, phosphate, carboxyl, sulfato, amino, sulfhydryl, nitro, and hydroxyl 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, and 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, palmitoyloleylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine. Representative examples of phosphatidylcholine include, but are not limited to, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and dilinoleoylphosphatidylcholine.Representative examples of phosphatidylethanolamine include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or DOPE.

[0048] The nanoparticle composition may contain amphiphilic lipids in an amount ranging from 10 mol % to 15 mol % per nanoparticle composition. The amphiphilic mol % may be 10 mol %, 11 mol %, 12 mol %, 13 mol %, 14 mol %, or 15 mol %, or a value within a range between any two of the aforementioned numbers.

[0049] In one embodiment, the nanoparticle composition may include cholesterol or a derivative of cholesterol. Examples of the derivative of cholesterol 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, Examples of the cholesterol derivatives include, but are not limited to, 19-hydroxycholesterol, 20-hydroxycholesterol, 22-hydroxycholesterol, 24-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 27-alkyne cholesterol, 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, dimethylhydroxyethylaminopropanecarbamoyl cholesterol iodide, and mixtures thereof. The cholesterol derivatives may include a sugar moiety and / or an amino acid. In one embodiment, the amino acid is selected from 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% per nanoparticle composition.The mole percent of cholesterol or a cholesterol derivative may be 50 mole%, 51 mole%, 52 mole%, 53 mole%, 54 mole%, 55 mole%, 56 mole%, 57 mole%, 58 mole%, 59 mole%, 60 mole%, 61 mole%, 62 mole%, 63 mole%, 64 mole%, 65 mole%, 66 mole%, 67 mole%, 68 mole%, 69 mole%, 70 mole%, 71 mole%, 72 mole%, 73 mole%, 74 mole%, or 75 mole%, or a value within a range between any two of the aforementioned numbers.

[0050] In one embodiment, the nanoparticle composition, e.g., the pharmaceutical composition herein, may be sterilized by conventional, well-known sterilization techniques. Aqueous solutions may be packaged for use or lyophilized. Lyophilized formulations may be combined with sterile aqueous solutions prior to administration. 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, 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, involved in carrying or transporting a subject compound from one organ or part of the body to another. Each carrier is "pharma- ceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and that it is not injurious to the subject.Some examples of pharma- ceutically acceptable carrier materials include: (1) sugars, such as lactose, glucose, mannose, and / or sucrose; (2) starches, such as corn starch and / or potato starch; (3) celluloses, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and / or cellulose acetate, and derivatives thereof; (4) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and / or talc; (S) excipients, such as cocoa butter and / or suppository wax; and (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 / 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 PEG400, (18) Ringer's solution, (19) C2-C12 alcohols such as ethanol, (20) fatty acids, (21) pH buffers, (22) bulking agents such as polypeptides and / or amino acids, (23) serum components such as serum albumin, HDL, and LDL, (24) polysorbates (Tween and / or (25) other non-toxic compatible substances used in pharmaceutical formulations such as fillers, binders, wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and / or antioxidants. As used herein, the terms "excipient", "pharmaceutical acceptable carrier", and the like are used interchangeably.

[0051] One embodiment includes a method of treating or preventing a disease or condition in a subject. The method may include providing any of the nanoparticle compositions or pharmaceutical compositions described herein. The method may include administering a therapeutically effective amount of the nanoparticle composition to the subject.

[0052] As used herein, the term "therapeutically effective amount" refers to an amount of a nanoparticle composition effective to achieve a desired therapeutic effect. The therapeutic effect may be achieved at a reasonable benefit / risk ratio applicable to the treatment. 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 reduce symptoms of a disease. A "therapeutically effective amount" may refer to an amount sufficient to eliminate symptoms of a disease. When treating a viral infection, reduction in symptoms of a disease may be assessed by reduction of virus in feces, body fluids, or secretions. The nanoparticle composition may be administered at a dosage and route of administration effective to generate an immune response.

[0053] 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 slow immune development, especially in patients with weakened immune systems, the elderly, or infants.

[0054] The exact dosage may be selected by a physician in consideration of the individual patient, based on a variety of factors. Dosage and administration may be adjusted to provide sufficient levels of the active agent or agent or to maintain the desired effect. For example, factors that may be considered may include the type and severity of the disease, the age and sex of the patient, the drug combination, and the individual response to therapy.

[0055] The therapeutic efficacy and toxicity of the active pharmaceutical agent in the nanoparticle composition may be determined by standard pharmaceutical procedures, for example, by determining the therapeutically effective dose for 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 dose ratio between toxic and therapeutic effects (LD50 / ED50), referred to as the therapeutic index, and the larger value of the therapeutic index may be used for evaluation. Data obtained from cell and animal studies may be used to formulate dosages for humans.

[0056] The therapeutically effective dose may be initially estimated from cell culture assays. The therapeutically effective dose may be formulated in animal models to achieve a circulating plasma concentration range that contains the IC50 (i.e., the concentration of the therapeutic agent that achieves half-maximal inhibition of symptoms) determined in cell culture. Plasma levels may be measured, for example, by high performance liquid chromatography. The effect of any particular dosage may be monitored by a suitable bioassay.

[0057] The amount of particles 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 clinician's judgment. A therapeutically effective dose may be 0.001 ng to 50 mg of therapeutic or immunogenic nucleic acid per kilogram of subject body weight. A therapeutically effective dose may be 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, 0.3 ng, 0.4ng, 0.5ng, 0.6ng, 0.7ng, 0.8ng, 0.9ng, 0.001μg, 0.002μg, 0.003μg, 0.004μg, 0.005μg, 0.006μg , 0.007μg, 0.008μg, 0.009μg, 0.01μg, 0.02μg, 0.03μg, 0.04μg, 0.05μg, 0.06μg, 0.07μg, 0.08μg, 0.09μg , 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, 25mg, 26 The therapeutic or immunogenic nucleic acid may be at a dose of 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, or a value within a range between any two of the aforementioned numbers. The therapeutic and immunogenic nucleic acids may be a combination of different nucleic acids used per treatment dose. The term "subject" refers to a human or an animal. Preferably, the animal is a vertebrate. In one embodiment, the vertebrate is selected from a primate, a rodent, a domestic animal, or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques such as rhesus monkeys.The rodent may be selected from mice, rats, guinea pigs, woodchucks, ferrets, rabbits, and hamsters. The livestock or game animals may be selected from cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, avian species such as foxes, wolves, chickens, emus, ostriches, and fish such as trout, catfish, 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, but excluding one or more groups or species such as humans, primates, rodents, etc. In one embodiment, the patient or subject may be a mammal, such as a primate, a human. The terms "patient" and "subject" are used interchangeably herein. 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 horse, a cow, or a pig. 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 aimed at 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 performed in a manner or route that at least partially localizes the composition to a desired site. Placement at a desired site may lead to the achievement of a desired effect. The nanoparticle compositions described herein may be administered by any suitable route known in the art, including, but not limited to, oral or parenteral 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, drip, inhalation or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, intracerebral and intrasternal injection and infusion. In one embodiment, the composition may be administered by intravenous infusion or intravenous injection.

[0060] The nanoparticle composition may be used to deliver therapeutic or immunogenic nucleic acids for targeted gene targeting, gene silencing, or other methods of modulating gene expression. The therapeutic or immunogenic nucleic acid may be an antisense oligonucleotide (AON) or a double-stranded small interfering RNA (siRNA). Typically, siRNAs are 21-23 nucleotides in length. The siRNA may comprise a sequence that is complementary to a sequence contained in an mRNA transcript of the target gene when expressed in a host cell. The antisense oligonucleotide may be a morpholino antisense oligonucleotide. The antisense oligonucleotide may comprise a sequence that is complementary to a sequence contained in an mRNA transcript of the 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 downregulate or prevent expression of a gene by inducing sequence-specific silencing of expression or translation of a target polynucleotide. 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 a double-stranded RNA (dsRNA). The dsRNA may be at least 25 nucleotides in length or may be longer. The dsRNA may comprise a sequence complementary to the sequence of the target gene(s). One embodiment includes the use of the nanoparticle composition for targeted gene recombination in a subject. One embodiment includes a method of targeted gene recombination comprising administering to a subject a nanoparticle composition of the invention.

[0061] In one embodiment, the therapeutic or immunogenic nucleic acid may be or may encode an agent that completely or partially reduces, inhibits, prevents or modulates 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 to the nucleic acid sequence of the target gene.

[0062] In one embodiment, the nanoparticle composition may be used for targeted specific alteration of genetic information in a subject. One embodiment includes targeted specific alteration of genetic information in a subject, comprising administering a nanoparticle composition herein. As used herein, the term "alteration" refers to a genome modification in a cell of a subject. The alteration may be an insertion or deletion of a nucleotide in the sequence of a target gene. "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 alteration may be a modification of the sequence of a target gene. "Modification" refers to a change 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. One embodiment includes the use of the nanoparticle composition of the present invention for targeted specific alteration of genetic information in a subject. One embodiment includes a method of targeted specific alteration of genetic information in a subject, comprising administering to a subject a nanoparticle composition of the present invention. One embodiment includes the use of the nanoparticle compositions of the present invention to change genetic information in a subject's cells ex vivo (outside the body) by directly administering the nanoparticle composition to a solution in which the subject's cells are cultured or suspended.

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

[0064] An exemplary genome editing system is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, as 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, "CRISPR system" refers to transcripts and other elements involved in the expression and activity of CRISPR-associated (Cas) genes, including sequences encoding Cas proteins, tracr (transactivating CRISPR) sequences, tracr mate sequences, guide sequences, or other sequences and transcripts associated with gene editing or gene regulation by CRISPR-associated methods. One or more tracr mate sequences may be operably linked by a nuclease to the guide sequence before processing, or to the crRNA after processing. 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 as if fully set forth herein, the tracrRNA and crRNA may be linked, or 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. Herein, a single fused crRNA-tracrRNA construct is also referred to as a guide RNA, gRNA, or single 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 compositions described herein may be used for delivery of 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 the sequences of these gene editing tools, as well as any gene products, proteins, or other molecules associated with their function.

[0066] One embodiment includes the use of the nanoparticle composition of the present invention for genome editing in a subject. One embodiment includes a method of genome editing in a subject, comprising administering the nanoparticle composition of the present invention to a subject. The nucleic acid in these embodiments may be sgRNA and Cas protein-encoding RNA. The nucleic acid in these embodiments may be for genome editing via meganucleases, homing endonucleases, TALEN-based systems, or zinc finger nucleases.

[0067] In one embodiment, the nanoparticle compositions may be used for targeted gene modification or gene editing in a subject in vivo or ex vivo, for example, by isolating cells from the subject, editing the genes, and transplanting the edited cells back into the subject. One embodiment includes a method comprising administering a nanoparticle composition herein to cells isolated from a subject. The method may include targeted gene modification. The method may include transplanting the edited cells back into the subject (or into another subject).

[0068] One embodiment includes a method of introducing an agent into a cell. The method may include exposing the cell to a nanoparticle composition herein. The method may be a method of transfection when the agent is a nucleic acid. The agent may be introduced into the cell by mixing a solution of nanoparticles configured as described herein with a liquid medium in which the cells are cultured.

[0069] List of embodiments The following list of embodiments includes non-limiting embodiments. The present invention includes, but is not limited to, those in the list of embodiments.

[0070] 1. A nucleic acid carrier having, comprising, consisting essentially of, or consisting of a structure of formula Ia, Ib, Ic, Id, Ie, If, Ig, Ih, Ii, Ij, IIa, or IIb, [ka] [ka] wherein A is an amine linker, B is a hydrophobic unit, n ranges from 0 to 20, Y is a sugar moiety, and Z is a deoxy sugar moiety.

[0071] 2. A is N1-(2-aminoethyl)ethane-1,2-diamine, N1-(2-aminoethyl)propane-1,3-diamine, N1-(3-aminopropyl)propane-1,3-diamine, N1,N1'-(ethane-1,2-diyl)bis(ethane-1,2-diamine), N1,N1'-(ethane-1,2-diyl)bis(N2-(2-aminoethyl)ethane-1,2-diamine), N1-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine, N1-(2-aminoethyl)-N1-methylethane-1,2-diamine amine, N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine, N1-(3-aminopropyl)-N1-ethylpropane-1,3-diamine, 3-((3-aminopropyl)(methyl)amino)propan-1-ol, 3,3'-(methylazanediyl)bis(propan-1-ol), N1-(3-aminopropyl)-N1-methylbutane-1,4-diamine, 4-((3-aminopropyl)(methyl)amino)butan-1-ol, 4-((3-hydroxy ... N1-(4-aminobutyl)-N1-methylbutane-1,4-diamine, 4-((4-aminobutyl)(methyl)amino)butan-1-ol, 4,4'-(methylazanediyl)bis(butan-1-ol), 3-((3-aminopropyl)(ethyl)amino)propan-1-ol, 3,3'-(ethylazanediyl)bis(propan-1-ol), N1-(3-aminopropyl)-N1-ethylbutane-1,4-diamine, 4-((3-aminopropyl)(ethyl)amino)butan-1 -ol, 4-(ethyl(3-hydroxypropyl)amino)butan-1-ol, N1-(2-aminoethyl)-N1-methylpropane-1,3-diamine, N1-(4-aminobutyl)-N1-ethylbutane-1,4-diamine, 4,4'-(ethylazanediyl)bis(butan-1-ol), 3-((3-aminopropyl)amino)propan-1-ol, N1-(3-aminopropyl)butane-1,4-diamine, 4-((3-hydroxypropyl)amino)butan-1-ol, N1-(4-aminobutyl)butane-1,4-diamine, 3,The nucleic acid carrier according to embodiment 1, which is derived from the group consisting of 3'-azanediylbis(propan-1-ol), 4-((3-aminopropyl)amino)butan-1-ol, 4,4'-azanediylbis(butan-1-ol), N1,N1'-(butane-1,4-diyl)bis(propane-1,3-diamine), 2-(bis(3-aminopropyl)amino)ethan-1-ol, 2-((4-aminobutyl)(3-aminopropyl)amino)ethan-1-ol, or 2-(bis(4-aminobutyl)amino)ethan-1-ol.

[0072] 3. B is C1-C 28 Alkyl group or C2-C 28 The nucleic acid carrier according to embodiment 1 or embodiment 2, wherein the group is an alkenyl group.

[0073] 4. C1-C above 28 Alkyl group or C2-C 28 The nucleic acid carrier according to embodiment 3, wherein the alkenyl group is substituted with 1 to 4 substituents selected from the group consisting of halogen, -CN, -NO2, -N3, C1-C6 alkyl, halo(C1-C6 alkyl), -OR, -NR2, -C2R, -OC(O)R, -CON(R)2, -OC(O)N(R)2, -NHC(O)N(R)2, -NHC(NH)N(R)2, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, aryl, heteroaryl and heterocycle, and R is selected from the group consisting of hydrogen, C1-C6 alkyl, halo(C1-C6 alkyl), C3-C8 cycloalkyl, C3-C8 cycloalkenyl, aryl, heteroaryl and heterocycle.

[0074] 5. The nucleic acid carrier of embodiment 4, wherein each cycloalkyl, cycloalkenyl, aryl, heteroaryl and heterocycle is further substituted with R', wherein R' is independently selected from the group consisting of halogen, -CN, -NO2, -N3, C1-C6 alkyl and halo(C1-C6 alkyl).

[0075] 6. The nucleic acid carrier according to any one or more of the preceding claims, wherein B is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, but-3-en-1-yl, oct-7-en-1-yl, 12-tridecenyl, 14-pentadecenyl, 17-octadecenyl, oleyl, linoleyl, arachidonyl and ricinoleyl.

[0076] 7. A nucleic acid carrier according to any one of embodiments 1 to 3, wherein B is derived from a fatty acid.

[0077] 8. The above fatty acids are caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentanoic acid, 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-hydroxy-9-cis-octa ... The nucleic acid carrier according to embodiment 7, wherein the nucleic acid carrier is selected from the group consisting of thioctadecanoic 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, conjugated fatty acids (e.g., conjugated isomers of linoleic acid (e.g., 9,11-CLA)); acetylenic fatty acids (e.g., crepenic acid); allenic fatty acids (e.g., lavalenic acid) or cyclopropenyl fatty acids (e.g., sterculic acid).

[0078] 9. A nucleic acid carrier described in any one or more of embodiments 7 and 8, wherein the fatty acid comprises one or more stable isotopes.

[0079] 10. The nucleic acid carrier described in embodiment 9, wherein the stable isotope is a stable isotope of carbon or hydrogen.

[0080] 11. Stable isotopes of carbon are 13 11. The nucleic acid carrier according to embodiment 10, wherein said nucleic acid carrier is C.

[0081] 12. What are the stable isotopes of hydrogen? 2 11. The nucleic acid carrier according to embodiment 10, wherein said nucleic acid carrier is H.

[0082] 13. The fatty acid containing the above stable isotope is octanoic acid-1- 13 C, Octanoic acid-8- 13 C, Octanoic acid-8,8,8-d3, Octanoic acid- 2 H15, Decanoic acid-1- 13 C, Decanoic acid-10- 13 C, decanoic acid-10,10,10-d3, decanoic acid-d19, 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 - 2 H27, 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 acid2 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 and Eicosanoic Acid - 2 11. The nucleic acid carrier according to embodiment 10, selected from the group consisting of H39.

[0083] 14. A nucleic acid carrier according to any one or more of embodiments 1 to 13, wherein Y in formula Ia, formula Ib, formula Ie, formula If, formula Ii, formula Ij or formula IIa is a sugar.

[0084] 15. The nucleic acid carrier according to embodiment 14, wherein the sugar is selected from a furanose monosaccharide (e.g., xylofuranose, ribofuranose or arabinofuranose), a pyranose monosaccharide (e.g., glucose, mannose, galactose), a disaccharide (e.g., lactose, trehalose), a polysaccharide (e.g., cyclodextrin), or a sugar derivative. In one embodiment, the sugar derivative is a nucleoside or a nucleotide.

[0085] 16. The nucleic acid carrier described in embodiment 1, wherein Z in formula Ic, formula Id, formula Ig, formula Ih, or formula IIb is a deoxy sugar.

[0086] 17. The nucleic acid carrier according to embodiment 16, wherein the sugar in the deoxy sugar is a furanose monosaccharide, a pyranose monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide or a sugar derivative. In one embodiment, the sugar derivative is a nucleoside or a nucleotide.

[0087] 18. The nucleic acid carrier according to embodiment 16 or embodiment 17, wherein the deoxy sugar is 2-deoxy-D-ribose, 6-deoxy-L-tagatose, 5-deoxy-xylofuranose, 5-deoxy-ribofuranose or 5-deoxy-arabinofuranose, 1-deoxyglucose, 2-deoxyglucose, 6-deoxyglucose, 1-deoxymannose, 2-deoxygalactose, 6-deoxygalactose, 1-deoxylactose, 6-azido-trehalose, 6-azido-2,4-diacetamido-2,4,6-trideoxy-D-mannose or 6A-azido-6A-deoxy-β-cyclodextrin.

[0088] 19. A nanoparticle composition comprising a nucleic acid carrier according to any one or more of embodiments 1 to 18 and a drug encapsulated therein.

[0089] 20. The nanoparticle composition of embodiment 19, wherein the agent is a nucleic acid.

[0090] 21. The nanoparticle composition of embodiment 19 or embodiment 20, wherein the agent is therapeutic or immunogenic, preferably, the agent is a therapeutic or immunogenic nucleic acid.

[0091] 22. The nanoparticle composition of embodiment 21, 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.

[0092] 23. The nanoparticle composition of embodiment 21 or embodiment 22, 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.

[0093] 24. A nanoparticle composition described in any one or more of embodiments 21 to 23, wherein the therapeutic or immunogenic nucleic acid agent comprises RNA or DNA capable of silencing, inhibiting or modifying the activity of a gene.

[0094] 25. The nanoparticle composition of any one or more of embodiments 19 to 24, further comprising a PEG-lipid.

[0095] 26. The nanoparticle composition of embodiment 25, 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.

[0096] 27. The nanoparticle composition of embodiment 25 or embodiment 26, comprising the PEG-lipid in the range of 1 mol % to 10 mol % of the PEG-lipid per nanoparticle composition.

[0097] 28. A nanoparticle composition described in any one of embodiments 19 to 25, further comprising (1) a phospholipid and / or (2) cholesterol or a derivative thereof.

[0098] 29. The nanoparticle composition of embodiment 28, wherein the nanoparticle composition comprises the phospholipid, and the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or distearoylphosphatidylcholine (DSPC).

[0099] 30. The nanoparticle composition of embodiment 28 or embodiment 29, comprising the phospholipid in the range of 10 mol % to 15 mol % of the phospholipid per nanoparticle composition.

[0100] 31. The nanoparticle composition of any one or more of embodiments 28 to 30, comprising in the range of 50 mol % to 75 mol % of the cholesterol or derivative thereof per nanoparticle composition.

[0101] 32. A method for treating or preventing a disease or illness in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a nucleic acid carrier described in any one of embodiments 1 to 18 or a nanoparticle composition described in any one of embodiments 19 to 31.

[0102] 33. The method of embodiment 32, 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.

[0103] 34. The method of embodiment 33, wherein the subject is a mammal.

[0104] 35. The method of embodiment 34, wherein the mammal is selected from the group consisting of humans, non-human primates, mice, rats, dogs, cats, horses or cows, but is not necessarily limited to these examples. EXAMPLES

[0105] The following non-limiting examples are provided to illustrate certain embodiments.

[0106] Example 1. Nanoparticle Compositions Containing Dendrons Conjugated to Sugars or Deoxysugars Alkyne and azido sugars are known as versatile starting materials in the synthesis of several biologically active compounds, including amino sugars, nucleosides and many other glycosylated heterocycles. Furthermore, the development of cycloaddition reactions between acetylenic compounds and azides ("click chemistry") has opened new possibilities in the field of glycoconjugates. In view of the above facts, the present inventors have synthesized novel nucleic acid carriers bearing triazole moieties (formula Ia or Ib or Ic or Id) starting from sugar alkyne or deoxysugar azides and polyesteramine dendrons bearing azide or acetylene at the focal point, and evaluated them for their gene delivery capabilities in vitro and in vivo.

[0107] Example 1a. Nanoparticle Compositions Containing Deoxyglucose Dendrons An example of the synthesis of PE-G2-2-deoxyglucose-A1 ricinoleic acid is as follows. [ka] [ka]

[0108] Compound 2: 2-Azido-2-deoxy-D-glucose (MW: 205.17, 17 mg, 0.079 μmol) was taken in a 50 ml RBF (0.6 mL) and then compound 1, PE-G2-acetylene-Boc amine 1 (MW: 1489, 118 mg, 74 μmol; synthesized according to published procedure Barnard et al., 2011, which is incorporated by reference as if fully set forth) dissolved in THF (1.2 mL) was added along with CuSO4.5H2O (2 mg, 0.079 μmol, 10 mol%, MW 249.69) and sodium ascorbate (3.1 mg, 15.8 μmol, 20 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 reaction was found to be complete 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). The desired product eluted at 71% mobile phase b (R f =0.35) to give the desired product as a yellow oil (70 mg, 52%). MS(ESI) C 76 H 139 N 15 O 27 Calculated for [M+H]+ m / z 1695.0, found 1695.8; [M+2H]2+ m / z 848.1, found 848.0; [M+3H]3+ m / z 565.6, found 565.8. 1H NMR (301MHz, chloroform-d) δ ppm 1.11-1.21(m,7H) 1.26-1.29(m,3H) 1.37-1.44(m,1H) 1.42(s,40H) 1.54-1.72(m,18H) 2.12-2.20(m,12H) 2.28-2.43(m,17H) 3.03-3.21(m,19H) 3.41-3.49(m,10H) 3.92-4.38(m,15H) 5.11-5.46(m,4H) 5.98-6.36(m,2H).

[0109] Compound 3: 70 mg of compound 3 (0.041 mmol) was treated with 20 equivalents of AcCl (0.06 ml) 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, dissolved in 2 ml of DMF, and 0.11 ml of EtN (0.73 mmol, 20 equivalents) was added, followed by 97.0 mg of ricinoleic acid-NHS (synthesized according to published procedures Talukder et al., Publication No. WO 2020 / 132196, which is incorporated by reference herein as if fully set forth) dissolved in 2 ml of DMF. The reaction mixture was stirred at 23° C. for 24 hours, concentrated under reduced pressure in the Genevac, and the reaction mixture was purified by flash chromatography on a silica column (12 g) using a gradient elution from 100% CH2Cl2 (mobile phase a) to 75:22:3 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b). The desired product eluted at 55% mobile phase b (R f =0.3) to give the desired product as a yellow oil (30 mg, 30%). MS(ESI) C 76 H 139 N 15 O 27Calculated for [M+2H]2+ m / z 1209.0, found 1208.9; [M+3H]3+ m / z 805.9, found 806.2. 1H NMR (301 MHz, chloroform-d) δ ppm 0.83-0.89 (m, 13H) 1.11-1.32 (m, 81H) 1.39-1.47 (m, 11H) 1.54-1.74 (m, 23H) 1.92-2.23 (m, 38H) 2.35-2.55 (m, 15H) 3.04-3.31 (m, 17H) 3.52-3.65 (m, 5H) 3.93-4.37 (m, 13H). 5.30-5.60(m,8H) 6.18-6.44(m,2H) 6.82-7.01(m,2H).

[0110] Nanoparticle formulations Nanoparticles containing deoxysugar-modified polyesteramine dendrons (e.g., PE dendron_G2-2 deoxyglucose-A1-ricinoleic acid):DOPE:cholesterol:DMG-PEG2k in 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 to the final desired pH with DNase / RNase-free and endotoxin-free distilled water and sterile citrate buffer. For Benchtop formulations, the total flow rate was maintained at 8 mL per minute with a 3:1 ratio of aqueous to organic phase. 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 h. The dialyzed nanoparticles were sterile filtered through a 0.2 micron (0.2 μm) poly(ethersulfone) filter and characterized on a Zetasizer NanoZS machine (Malvern). The size distribution was characterized by a single peak with a low polydispersity index. Using the Ribogreen® assay (Geall et al., 10.1073 / pnas.1209367109, incorporated by reference herein as if fully set forth), the encapsulation efficiency was measured to be 95% for the nanoparticle composition containing PE dendron_G2-2 deoxyglucose-A1-ricinoleic acid and SEAP mRNA (formulated at pH 5).

[0111] Hydrodynamic size measurements Figure 2 shows the distribution of nanoparticle composition (Z-average) measured as intensity based on the size of the nanoparticles (d.nm, diameter in nm). The "Z-average" of nanoparticle composition containing PE dendron_G2-2 deoxyglucose-A1-ricinoleic acid and SEAP mRNA as a function of size was determined by dynamic light scattering (DLS) as shown in Figure 2. The strongest intensity was observed for nanoparticles with a size of 113.3 d.nm. The size distribution is characterized by a single peak with a low polydispersity index, indicating a relatively monodisperse size.

[0112] Gel retardation assay Agarose gel electrophoresis was performed to assess the binding of the sugar-modified dendrons to RNA according to published methods (Geall et al., 10.1073 / pnas.1209367109, incorporated herein by reference as if fully set forth). Figure 3 is a photograph of an agarose gel showing the binding of the sugar-modified dendrons to 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 Figure 3, lane 1 contained unformulated SEAP mRNA and lane 2 contained a formulation of PE dendron_G2-2 deoxyglucose-A1-ricinoleic acid and SEAP mRNA. Prior to loading, the samples were incubated with formaldehyde loading dye, denatured at 65°C for 10 min, and cooled to room temperature. The gel was run at 90V and gel images were taken with a Syngene G Box Imaging System (Syngene, USA). As shown in FIG. 4, the lower band corresponds to the small size of free RNA (lane 1), and the upper band indicates the large size of nanoparticles formed by binding the RNA to the PE dendron_G2-2 deoxyglucose-A1-ricinoleic acid carrier.

[0113] Results of in vitro SEAP production To test the ability of nanoparticles formulated with different deoxyglyco-based dendrons to express SEAP in vitro, BHK cells were treated with the nanoparticles. Each well of a BHK 12-well dish was treated with 20 μL (approximately 1 μg) of each formulation 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. Twenty-four hours after treatment or transfection, conditioned media was collected from each culture. The amount of SEAP was quantified using the Invitrogen NovaBright™ Phospha-Light™ EXP Assay kit for detection of SEAP according to the manufacturer's protocol. The amount of SEAP in the media samples was measured with a BioTek Synergy HTX microplate reader and is reported in arbitrary units (AU). Error bars are ± SEM. As shown in Figure 4a, the amount of SEAP was higher when PE dendron_G2-2 deoxyglucose-A1-ricinoleic acid was used than the other positional isomers PE dendron_G2-1 deoxyglucose-A1-ricinoleic acid and PE dendron_G2-6 deoxyglucose-A1-ricinoleic acid. As shown in Figure 5a, the amount of SEAP was higher when PE dendron_G2-2 deoxyglucose-A5-ricinoleic acid was used than the other positional isomers PE dendron_G2-1 deoxyglucose-A1-ricinoleic acid and PE dendron_G2-6 deoxyglucose-A1-ricinoleic acid. It was also found that PE dendron_G2-2 deoxygalactose-A5-ricinoleic acid nanoparticles expressed higher amounts of SEAP in BHK cells than other deoxysugar dendrons such as PE dendron_G2-2 deoxyglucose-A5-ricinoleic acid and PE dendron_G2-1 deoxymannose-A5-ricinoleic acid. For the no treatment negative control, 5 μL of medium from wells not treated with nanoparticles was used.

[0114] Results of in vivo SEAP production To test the formulation of different monosaccharide dendrons, a secreted placental alkaline phosphatase SEAP reporter system was used. For in vivo studies, mice were injected with nanoparticles at a dose of 5 μg SEAP mRNA and serum was collected from the mice 16 hours later. The amount 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 4b, the amount of SEAP was found to be higher with PE dendron_G2-1 deoxyglucose-A1-ricinoleic acid than with the other positional isomers PE dendron_G2-2 deoxyglucose-A1-ricinoleic acid and PE dendron_G2-6 deoxyglucose-A1-ricinoleic acid. As shown in Figure 5b, it was also observed that the SEAP amount was higher with PE dendron_G2-2 deoxyglucose-A5-ricinoleic acid than with PE dendron_G2-2 deoxyglucose-A1-ricinoleic acid, indicating that the pKa of the ionizable delivery molecule influences the ability of the nanoparticles to deliver functionally active RNA.

[0115] COVID-19 Spike Trimer Direct Serum ELISA Mice (BALB / c) were vaccinated with 2.5 μg of spike replicon RNA formulated in PE dendron_G2-2 deoxyglucose-A1-ricinoleic acid and PE dendron_G2-2 deoxyglucose-A5-ricinoleic acid (total volume of 100 μL PBS) by bilateral IM injection in leg muscles. Mice were bled 21 and 28 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 antibody titers by direct ELISA. Nunc MaxiSorp ELISA plates were coated overnight at 4° C. with bicarbonate coating buffer pH 9.5 containing recombinant spike trimer protein. Wells were blocked with PBS+1% BSA. For week 3 samples, serum was added to wells starting with a 1:50 dilution in PBS+1% BSA and using serial 1:2 solutions up to 1:6400, and for week 4 samples, serum was added to wells starting with a 1:100 dilution in PBS+1% BSA and using serial 1:2 solutions up to 1:12800. Samples were incubated for 1 h at RT and washed 3 times with PBST, after which goat anti-mouse IgG HRP was added at a dilution of 1:3000 in PBS+1% BSA and incubated for 1 h. Plates were washed again 5 times with PBST and developed using the chromogenic HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB). The reaction was stopped by the addition of H2SO4 and absorbance was measured at 450 nm and 570 nm. The end point titer was designated as the highest dilution that resulted in an absorbance value at 450-absorbance at 570 of ≥ 0.08. At weeks 3 and 4, the endpoint dilution titers in the groups immunized with 2.5 μg nanoparticle vaccine formulated with PE dendron_G2-2 deoxyglucose-A1-ricinoleic acid were higher than that of PE dendron_G2-2 deoxyglucose-A5-ricinoleic acid (Figure 6). As shown in Figure 6, both of our novel glyco-based dendrons were also found to be superior to Dlin-MC3-DMA in terms of immunogenicity upon IM administration.Dlin-MC3-DMA, also known as MC3, is one of the most widely used cationic lipids used to make lipid nanoparticles (LNPs) and is therefore used as a control in this assay.

[0116] Example 1b. Nanoparticle Compositions Containing Disaccharides To make interactions between receptors and carbohydrates biologically relevant, multiple copies of these interactions must be made, either sequentially or simultaneously (Mortell et al., Journal of American Chemical Society 1996, 118, 2297-2298).To study the effect of multiple monosaccharides on cellular uptake and nucleic acid delivery, in the present invention, the focus of the dendrons was modified with disaccharides.

[0117] An example of the synthesis of PE-G2-1-deoxylactose-A1-ricinoleic acid is as follows. [ka] Compound 4: 1-Azido-1-deoxy-β-D-lactopyranoside (MW: 367.31, 65 mg, 0.18 mmol) was taken in 50 ml RBF and dissolved in 1 ml THF. Then the alkyne, PE-G2-acetylene-Boc amine 1 (MW: 1489, 200 mg, 0.13 mmol) dissolved in THF (2 mL) was added along with CuSO4·5H2O (3.3 mg, 0.013 mmol, 10 mol%, MW 249.69) and sodium ascorbate (5.1 mg, 0.026 mmol, 20 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 reaction was complete as confirmed 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 75:24:6 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b). The desired product eluted with 100% mobile phase b (R f=0.6), compound 4 was obtained as a yellow oil (150 mg, 60%). MS(ESI) C 82 H 149 N 15 O 32 Calculated value for [M+2H] 2+ m / z 929.1, measured value 929.2. 1H NMR (301MHz, chloroform-d) δ ppm 1.08-1.28(m,8H) 1.30-1.45(m,41H) 1.54-1.77(m,15H) 2.22-2.38(m,12H) 2.40-2.61(m,15H) 2.96-3.23(m,16H) 3.41-3.47(m,8H) 3.60-4.55(m,37H) 4.60-4.79(m,2H) 5.11-5.38(m,1H) 5.55-5.85(m,4H) 6.19-6.41(m,3H) 8.10-8.30(m,1H).

[0118] Compound 5: 100 mg of compound 4 (0.054 mmol) was treated with 20 equivalents of AcCl (0.08 ml, 1.08 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, dissolved in 2 ml of DMF, and 0.15 ml of EtN (1.08 mmol, 20 equivalents) was added, followed by 128 mg of ricinoleic acid-NHS (synthesized according to published procedures Talukder et al., Publication No. WO 2020 / 132196, which is incorporated by reference herein as if fully set forth) dissolved in 2 ml of DMF. The reaction mixture was stirred at 23° C. for 24 hours, concentrated under reduced pressure in the Genevac, and the reaction mixture was purified by flash chromatography on a silica column (12 g) using a gradient elution from 98% CH2Cl2 (mobile phase a) to 75:24:6 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b). The desired product eluted at 100% mobile phase b (R f =0.6) to give the desired product as a yellow oil (28 mg, 17% over two steps). MS(ESI) C 134 H 245 N15 O 32 Calculated value for [M+2H] 2+ m / z 1289.5, actual value 1290.0. 1H NMR (301MHz, chloroform-d) δ ppm 0.85-0.90(m,12H) 1.21-1.33(m,76H) 1.40-1.48(m,10H) 1.51-1.72(m,25H) 1.95-2.07(m,10H) 2.07-2.25(m,32H) 2.31-2.49(m,17H) 3.05-3.29(m,17H) 3.52-3.66(m,6H) 3.73-4.34(m,21H) 5.29-5.55(m,8H) 6.24-6.37(m,2H) 6.89-7.09(m,2H) 8.05-8.24(m,1H).

[0119] Example 1c. Nanoparticle Compositions Containing Polysaccharides Such as Cyclodextrins Polysaccharides have various intrinsic biological functions, such as immune stimulating efficiency, anti-human immunodeficiency virus (anti-HIV), and antitumor activity (Hong et al., 2017). Among various polysaccharides, cyclodextrin has attracted special attention because it is water-soluble and has a hydrophobic cavity and a hydrophilic outer surface (Chem. Soc. Rev., 2011, 40, 1586-1608). To study the effect of the unique structural features of cyclodextrin on cellular uptake and nucleic acid delivery, in this invention, the focus of the dendron was modified with β-cyclodextrin. The amines present in the delivery molecule allow electrostatic interactions with negatively charged nucleic acids at low pH, while the presence of the hydrophilic outer surface of cyclodextrin should facilitate the complexation and cellular uptake of nucleic acid drugs.

[0120] An example of the synthesis of PE-G2-6-deoxycyclodextrin-A1 ricinoleic acid is as follows. [ka] Compound 6: 6A-Azido-6A-deoxycyclodextrin (MW: 1160, 75 mg, 0.065 mmol) was taken in 50 ml RBF and dissolved in 1 ml THF. Then the alkyne, PE-G2-acetylene-Boc amine 1 (MW: 1489, 100 mg, 0.065 mmol) dissolved in THF (1 ml) was added along with CuSO4·5H2O (1.7 mg, 0.0065 mmol, 10 mol%, MW 249.69) and sodium ascorbate (2.6 mg, 0.013 mmol, 20 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 reaction was confirmed to be complete 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 75:24:6 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b). The desired product eluted with 100% mobile phase b (R f =0.35) to give the desired product as a yellow oil (58 mg, 52%). MS(ESI) C 112 H 197 N 15 O 56 Calculated value for [M+2H] 2+ m / z 1325.2, actual value 1325.2. 1H NMR (301MHz, solvent) δ ppm 1.15-1.30(m,9H) 1.44-1.49(m,36H) 1.61-1.83(m,16H) 2.29-2.42(m,12H) 2.47-2.65(m,16H) 3.01-3.19(m,17H) 3.28-3.33(m,5H) 3.37-3.63(m,15H) 3.69-3.97(m,22H) 4.05-4.33(m,11H) 4.92-5.00(m,11H) 5.03-5.17(m,3H) 5.22-5.32(m,2H) 8.06-8.12(m,1H).

[0121] Compound 7: 56 mg of compound 3 (0.021 mmol) was treated with 20 equivalents of AcCl (0.03 ml, 0.42 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, dissolved in 2 ml of DMF, and 0.06 ml of EtN (0.42 mmol, 20 equivalents) was added, followed by 50.2 mg of ricinoleic acid-NHS, 6 equivalents (synthesized according to published procedures Talukder et al., Publication No. WO 2020 / 132196, which is incorporated by reference herein as if fully set forth) dissolved in 2 ml of DMF. The reaction mixture was stirred at 23° C. for 24 hours, concentrated under reduced pressure in the Genevac, and the reaction mixture was purified by flash chromatography on a silica column (12 g) using a gradient elution from 100% CH2Cl2 (mobile phase a) to 75:24:6 CH2Cl2 / MeOH / NH4OHaq (by volume, mobile phase b). The desired product eluted with 100% mobile phase b (R f =0.3), affording the desired product as a yellow oil (20 mg, 30%). MS(ESI) C 164 H 293 N 15 O 56 Calculated value for [M+3H] 3+ m / z 1124.0, measured value 1124.5.

[0122] Example 1d. Nanoparticle Composition Containing a Dendron Having Formula Ib [ka] [ka] Compound 9: Mannose alkyne (MW: 218.21, 67 mg, 0.31 mmol; synthesized according to published procedure Percec et al., 2011 J. Am. Chem. Soc. 2013, 135, 9055-9077, which is incorporated by reference as if fully set forth herein) was taken in a 50 ml RBF (0.6 mL) and then dissolved in THF (1 mL), compound 8, PE-G2-azido-Boc amine 5 (MW: 1489, 118 mg, 74 μmol; synthesized from PE-G2-azido-OH according to published procedure Barnard et al., 2011, which is incorporated by reference as if fully set forth herein) was added to CuSO4·5H2O (7.6 mg, 10 mol%, MW 249.69) and sodium ascorbate (12 mg, 20 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, TLC confirmed the reaction was complete. 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). The desired product eluted at 70% mobile phase b (R at 2:1 mobile phase b / mobile phase a). f =0.2) to give the desired product as a yellow oil (136 mg, 92%). MS(ESI) C 86 H 159 N 15 O 28 Calculated for [M+2H]2+ m / z 926.0, found 925.9.

[0123] Compound 10: 136 mg of compound 9 (0.073 mmol) was treated with 20 equivalents of AcCl (0.105 ml) after dissolving the compound in 3 ml of MeOH, the reaction was stirred at 0° C. to 23° C. for 5 hours, evaporated to dryness, dissolved in 2 ml of DMF, and 0.11 ml of EtN (1.46 mmol, 20 equivalents) was added, followed by 174 mg of ricinoleic acid-NHS (synthesized according to published procedures Talukder et al., Publication No. WO 2020 / 132196, which is incorporated by reference herein as if fully set forth) dissolved in 2 ml of DMF. The reaction mixture was stirred at 23° C. for 24 hours, concentrated under reduced pressure in the Genevac, and the reaction mixture was 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). The desired product eluted at 80% mobile phase b (R f =0.65) to give the desired product as a yellow oil (56 mg, 30%). MS(ESI) C 139 H 256 N 14 O 28 Calculated for [M+2H]2+ m / z 1285.9, found 1285.0.

[0124] Nanoparticle formulations Nanoparticles containing sugar-modified polyesteramine dendron (e.g., PE dendron_G2-hexylmannose-A5-ricinoleic acid):DOPE:cholesterol:PEG in 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 to the final desired pH with DNase / RNase-free and endotoxin-free distilled water and sterile citrate buffer. For Benchtop formulation, the total flow rate was maintained at 8 mL per minute with a 3:1 ratio of aqueous to organic phase. 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 through a 0.2 micron (0.2 μm) poly(ethersulfone) filter and characterized on a Zetasizer NanoZS machine (Malvern). The size distribution was characterized by a single peak with a low polydispersity index. Using the Ribogreen® assay (Geall et al., 10.1073 / pnas.1209367109, incorporated by reference herein as if fully set forth), the encapsulation efficiency was determined to be 90% for the nanoparticle composition containing PE Dendron_G2-Hexylmannose-A5-Ricinoleic Acid and SEAP mRNA.

[0125] Hydrodynamic size measurements Figure 7 shows the distribution of nanoparticle composition (Z-average) measured as intensity based on the size of the nanoparticles (d.nm, diameter in nm). The "Z-average" of nanoparticle composition containing PE Dendron_G2-Hexylmannose-A5-Ricinoleic Acid and SEAP mRNA as a function of size was determined by dynamic light scattering (DLS) as shown in Figure 7. The strongest intensity was observed for nanoparticles with a size of 108.8 d.nm. The size distribution is characterized by a single peak with a low polydispersity index, indicating a relatively monodisperse size.

[0126] Results of in vivo SEAP production To test the formulations with PE Dendron_G2-Hexylmannose-A5-Ricinoleic acid using different classes of PEG in vivo, mice were injected with nanoparticles at a dose of 2 μg of SEAP mRNA, and serum was collected from the mice 3 days after administration. The amount 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 with a BioTek Synergy HTX microplate reader and is reported in arbitrary units (AU). Error bars are ± SEM. As shown in Figure 8, the amount of SEAP was found to be higher with PEG-lipid conjugates ALC 0159 and DMG PEG 2000 compared to formulations with 14:0 PEG 2000 and 184:0 PEG 2000.

[0127] Example 1e. Nanoparticle Composition Containing a Dendron Having Formula If [ka] [ka] Compound 12: Azido-PEG-β-D-glucose (MW: 381, 50 mg, 0.18 mmol) dissolved in 0.6 mL was taken in 25 mL RBF, then the alkyne, PE-G2-acetylene-Boc amine 5 (MW: 1546, 101 mg, 66 μmol; synthesized from PE-G2-acetylene-OH according to published procedure Barnard et al., 2011, which is incorporated herein by reference as if fully set forth) dissolved in THF (1 mL) was added along with CuSO4·5H2O (3.3 mg, 20 mol%, MW 249.69) and sodium ascorbate (5.2 mg, 40 mol%, MW 198.11) and degassed THF:H2O (2 mL, 1:1) was added to it. The reaction mixture was stirred at 23 °C for 2 days. The reaction mixture was purified by flash chromatography on a 12 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). The desired product eluted with 100% mobile phase b (R f =0.25) to give the desired product as a yellow oil (49 mg, 39%). MS(ESI) C 76 H 139 N 15 O 27 Calculated for [M+2H]2+ m / z 964.7, found 964.4; [M+3H]3+ m / z 643.4, found 643.5.

[0128] Compound 13: 49 mg of compound 12 (0.025 mmol) was treated with 20 equivalents of AcCl (0.04 ml, 0.51 mmol) after dissolving the compound in 3 ml of MeOH, the reaction was stirred at 0° C. to 23° C. for 5 hours, evaporated to dryness, dissolved in 2 ml of DMF, and 0.07 ml of EtN (0.51 mmol, 20 equivalents) was added, followed by 60.0 mg of ricinoleic acid-NHS (0.153 mmol; synthesized according to published procedures Talukder et al., Publication No. WO 2020 / 132196, which is incorporated by reference herein as if fully set forth) dissolved in 2 ml of DMF. The reaction mixture was stirred at 23° C. for 24 hours, concentrated under reduced pressure in the Genevac, and the reaction mixture was 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). The desired product eluted with 100% mobile phase b (R f =0.3) to give the desired product as a yellow oil (35 mg, 52%). MS(ESI) C 140 H 259 N 15 O 31 Calculated for [M+3H]3+ m / z 883.7, found 883.3.

[0129] Nanoparticle formulations Nanoparticles containing sugar-modified polyesteramine dendron (e.g., PE dendron_G2-PEG4-glucose-A5-ricinoleic acid):DOPE:cholesterol:PEG-lipid conjugates in 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 to the final desired pH with DNase / RNase-free and endotoxin-free distilled water and sterile citrate buffer. For Benchtop formulation, the total flow rate was maintained at 8 mL per minute with a 3:1 ratio of aqueous to organic phase. 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 through a 0.2 micron (0.2 μm) poly(ethersulfone) filter and characterized on a Zetasizer NanoZS machine (Malvern). The size distribution was characterized by a single peak with a low polydispersity index.

[0130] Hydrodynamic size measurements Figure 9 shows the distribution of nanoparticle composition (Z-average) measured as intensity based on the size of the nanoparticles (d.nm, diameter in nm). The "Z-average" of nanoparticle composition containing PE Dendron_G2-PEG4-Glucose-A5-Ricinoleic Acid and PR8 HA mRNA as a function of size was determined by dynamic light scattering (DLS) as shown in Figure 9. The strongest intensity was observed for nanoparticles with a size of 121.3 d.nm. The size distribution is characterized by a single peak with a low polydispersity index, indicating a relatively monodisperse size.

[0131] Example 2. Nanoparticle compositions containing amphiphilic dendrimers as nucleic acid carriers In this invention, we have synthesized sugar-modified amphiphilic dendrimer hybrids and deoxysugar-modified amphiphilic dendrimer hybrids. These amphiphilic molecules have hydrophobic units and therefore should be able to self-assemble in solution, while the hydrophilic sugar moieties should improve the biocompatibility and loading capacity of the nucleic acid carriers. [ka] where A is an amine linker, B is a hydrophobic unit, Y is a sugar moiety and Z is a deoxy sugar moiety.

[0132] References References cited throughout this application are incorporated for all purposes as if each reference were fully set forth herein and in the reference itself. For purposes of presentation, certain of these references are cited in particular locations herein. The citation of a reference in a particular location indicates the manner in which the teachings of the reference are incorporated. However, the citation of a reference in a particular location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.

[0133] (1)Jones, CH, Chen, C.-K., Ravikrishnan, A., Rane, S., and Pfeifer, BA (2013) Overcoming nonviral gene delivery barriers: perspective and future. Mol.Pharmaceutics 10, 4082-4098. (2) Nishikawa, M., and Huang, L. (2001) Nonviral vectors in the new millennium: delivery barriers in gene transfer. Hum.Gene Ther. 12, 861-870 (3) Mintzer, MA, and Simanek, EE (2009) Nonviral Vectors for Gene Delivery. Chem.Rev. 109, 259-302. (4)a. Hong SJ, Ahn MH, Sangshetti J, Choung PH, Arote RB. Sugar-based gene delivery systems: Current knowledge and new perspectives. Carbohydr Polym. 2018 Feb 1;181:1180-1193. doi:10.1016 / j.carbpol.2017.11.105. Published online 28 Nov 2017. PMID:29253947; b. Han,S., Ganbold,T., Bao,Q., Yoshida,T., and Baigude,H. (2018). Sugar Functionalized Synergistic Dendrimers for Biocompatible Delivery of Nucleic Acid Therapeutics. Polymers, 10(9), 1034. (5)Geall AJ, Verma A, Otten GR, Shaw CA, Hekele A, Banerjee K, Cu Y, Beard CW, Brito LA, Krucker T, O'Hagan DT, Singh M, Mason PW, Valiante NM, Dormitzer PR, Barnett SW, Rappuoli R, Ulmer JB, Mandl CW. Nonviral delivery of self-amplifying RNA vaccines. Proc Natl Acad Sci USA. 2012 September 4;109(36):14604-9. doi:10.1073 / pnas.1209367109. Published electronically August 20, 2012. PMID:22908294;PMCID:PMC3437863. (6) Mortell, K.H.; Weatherman, R.V.; Kiessling, L.L. Recognition specificity of neoglycopolymers prepared by ring-opening metathesis polymerization. Journal of the American Chemical Society 1996, 118, 2297 - 2298. (7) Carmen Ortiz Mellet, Jose M. Garcia Fernandezb and Juan M. Benito. Chem.Soc.Rev., 2011, 40, 1586 - 1608 (8) Barnard A, Posocco P, Pricl S, Calderon M, Haag R, Hwang ME, Shum VW, Pack DW, Smith DK. Degradable self-assembling dendrons for gene delivery: experimental and theoretical insights into the barriers to cellular uptake. J Am Chem Soc. December 21, 2011; 133(50): 20288 - 300. (9) 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. PCT / US19 / 67402. (10) Percec V, Leowanawat P, Sun HJ, Kulikov O, Nusbaum CD, Tran TM, Bertin A, Wilson DA, Peterca M, Zhang S, Kamat NP, Vargo K, Moock D, Johnston ED, Hammer DA, Pochan DJ, Chen Y, Chabre YM, Shiao TC, Bergeron-Brlek M, Andre S, Roy R, Gabius HJ, Heiney PA. Modular synthesis of amphiphilic Janus glycodendrimers and their self-assembly into glycodendrimersomes and other complex architectures with bioactivity to biomedically relevant lectins. J Am Chem Soc. 2013 June 19;135(24):9055-77. doi:10.1021 / ja403323y. Published electronically on June 6, 2013. PMID:23692629.

[0134] It is understood therefore that the invention is not limited to the particular embodiments disclosed, but is intended to encompass all modifications that fall within the spirit and scope of the invention as defined by the appended claims, the above description, and / or the accompanying drawings as shown.

Claims

**Claim 1** A nucleic acid carrier comprising a structure of Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ie, Formula If, Formula Ig, Formula Ih, Formula Ii, Formula Ij, Formula IIa or Formula IIb, 【Chemical Formula 1】 【Chemical 2】 wherein A is an amine linker, B is a hydrophobic unit, n takes a value of 0 to 20, Y is a sugar moiety, and Z is a deoxy sugar moiety Nucleic acid carrier. **Claim 2** A is N1-(2-aminoethyl)ethane-1,2-diamine, N1-(2-aminoethyl)propane-1,3-diamine, N1-(3-aminopropyl)propane-1,3-diamine, N1,N1'-(ethane-1,2-diyl)bis(ethane-1,2-diamine), N1,N1'-(ethane-1,2-diyl)bis(N2-(2-aminoethyl)ethane-1,2-diamine), N1-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine, N1-(2-aminoethyl)-N1-methylethane-1,2-diamine, N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine, N1-(3-aminopropyl)-N1-ethylpropane-1,3-diamine, 3-((3-aminopropyl)(methyl)amino)propan-1-ol, 3,3'-(methylazanediyl)bis(propan-1-ol), N1-(3-aminopropyl)-N1-methylbutane-1,4-diamine, 4-((3-aminopropyl)(methyl)amino)butan-1-ol, 4-((3-hydroxypropyl)(methyl)amino)butan-1-ol, 4-((3-hydroxypropyl)(methyl)amino)butan-1-ol, N1-(4-aminobutyl)-N1-methylbutane-1,4-diamine, 4-((4-aminobutyl)(methyl)amino)butan-1-ol, 4,4'-(methylazanediyl)bis(butan-1-ol), 3-((3-aminopropyl)(ethyl)amino)propan-1-ol, 3,3'-(ethylazanediyl)bis(propan-1-ol), N1-(3-aminopropyl)-N1-ethylbutane-1,4-diamine, 4-((3-aminopropyl)(ethyl)amino)butan-1-ol, 4-(ethyl(3-hydroxypropyl)amino)butan-1-ol, N1-(2-aminoethyl)-N1-methylpropane-1,3-diamine, N1-(4-aminobutyl)-N1-ethylbutane-1,4-diamine, 4,4'-(ethylazanediyl)bis(butan-1-ol), 3-((3-aminopropyl)amino)propan-1-ol, N1-(3-aminopropyl)butane-1,4-diamine, 4-((3-hydroxypropyl)amino)butan-1-ol, N1-(4-aminobutyl)butane-1,4-diamine, 3,The nucleic acid carrier according to claim 1, which is derived from the group consisting of 3'-azanediylbis(propan-1-ol), 4-((3-aminopropyl)amino)butan-1-ol, 4,4'-azanediylbis(butan-1-ol), and N1,N1'-(butane-1,4-diyl)bis(propan-1,3-diamine). **Claim 3** B is C １ -C ２８ an alkyl group or C ２ -C ２８ an alkenyl group, and the C1-C28 alkyl group or C2-C28 alkenyl group is substituted with 1 to 4 substituents selected from the group consisting of halogen, -CN, -NO2, -N3, C1-C6 alkyl, halo(C1-C6 alkyl), -OR, -NR2, -CO2R, -OC(O)R, -CON(R)2, -OC(O)N(R)2, -NHC(O)N(R)2, -NHC(NH)N(R)2, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, aryl, heteroaryl, and heterocyclic ring, and R is selected from the group consisting of hydrogen, C1-C6 alkyl, halo(C1-C6 alkyl), C3-C8 cycloalkyl, C3-C8 cycloalkenyl, aryl, heteroaryl, and heterocyclic ring. The nucleic acid carrier according to claim 1. **Claim 4** Each cycloalkyl, cycloalkenyl, aryl, heteroaryl and heterocycle is further substituted with R', where R' is independently halogen, -CN, -NO ２ , -N ３ , C １ -C ６ alkyl and halo(C １ -C ６ alkyl), the nucleic acid carrier according to claim 3, selected from the group consisting of. **Claim 5** The nucleic acid carrier according to claim 3, wherein B is selected from the group consisting of a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a but-3-en-1-yl group, an oct-7-en-1-yl group, a 12-tridecenyl group, a 14-pentadecenyl group, a 17-octadecenyl group, an oleyl group, a linoleyl group, an arachidonyl group, and a ricinoleyl group. **Claim 6** The nucleic acid carrier according to claim 3, wherein B is derived from a fatty acid or a derivative thereof, and 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, eicosapentaenoic acid, 2-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, DL-β-hydroxypalmitic acid, conjugated fatty acids, conjugated isomers of linoleic acid, 9,11-CLA, acetylenic fatty acids, crepenynic acid, allene fatty acids, laballenic acid, cyclopropenyl fatty acids, and sterculic acid. **Claim 7** The nucleic acid carrier according to claim 6, wherein the fatty acid contains one or more stable isotopes, and the stable isotopes are stable isotopes of carbon or hydrogen.

8. The stable isotope of carbon is 13C, and the stable isotope of hydrogen is 2H, or The fatty acid containing the stable isotope is octanoic acid-1- １３ C, octanoic acid-8- １３ C, octanoic acid-8,8,8-d3, octanoic acid- ２ H15, decanoic acid-1- １３ C, decanoic acid-10- １３ C, decanoic acid-10,10,10-d3, decanoic acid-d19, 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 C ２ , 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- ２ H27, palmitic acid-1- １３ C, palmitic acid-16- １３ C, palmitic acid-16- １３ C, 16,16,16- ２ H3, palmitic acid- ２ H31, stearic acid-1- 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 and eicosanoic acid- ２ The nucleic acid carrier according to claim 7, which is selected from the group consisting of H39.

9. Y in Formula Ia, Formula Ib, Formula Ie, Formula If, Formula Ii, Formula Ij or Formula IIa is a sugar, and the sugar is a furanose monosaccharide (for example, xylofuranose, ribofuranose or arabinofuranose), a pyranose monosaccharide (for example, glucose, mannose, galactose), a disaccharide (for example, lactose, trehalose), or a polysaccharide (for example, cyclodextrin) The nucleic acid carrier according to claim 1.

10. Z in Formula Ic, Formula Id, Formula Ig or Formula Ih or Formula IIb is a deoxy sugar, The deoxy sugar is a furanose monosaccharide, a pyranose monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide, or The deoxy sugar is 2-deoxy-D-ribose, 6-deoxy-L-tagatose, 5-deoxy-xylofuranose, 5-deoxy-ribofuranose or 5-deoxy-arabinofuranose, 1-deoxyglucose, 2-deoxyglucose, 6-deoxyglucose, 1-deoxymannose, 2-deoxygalactose, 6-deoxygalactose, 1-deoxylactose, 6-azido-trehalose, 6-azido-2,4-diacetamido-2,4,6-trideoxy-D-mannose or 6A-azido-6A-deoxy-β-cyclodextrin The nucleic acid carrier according to claim 1.

11. A nanoparticle composition comprising the nucleic acid carrier according to claim 1 and a drug encapsulated therein.

12. The nanoparticle composition according to claim 11, wherein the drug is a nucleic acid.

13. The drug is a therapeutic or immunogenic nucleic acid, 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 The nanoparticle composition according to claim 11 or claim 12.

14. The nanoparticle composition according to claim 13, 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 allergic diseases.

15. The nanoparticle composition according to claim 13, wherein the therapeutic or immunogenic nucleic acid agent comprises RNA or DNA capable of silencing, inhibiting, or modifying the activity of a gene.

16. further comprising a PEG-lipid, 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, The nanoparticle composition according to claim 11, wherein the nanoparticle composition comprises the PEG-lipid in the range of 1 mol% to 10 mol% of the PEG-lipid per nanoparticle composition.

17. The nanoparticle composition according to claim 16, further comprising (1) a phospholipid and / or (2) cholesterol or a derivative thereof.

18. The nanoparticle composition comprises the phospholipid in the range of 10 mol% to 15 mol% of the phospholipid per nanoparticle composition, wherein the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or distearoyl phosphatidylcholine (DSPC), and / or The nanoparticle composition according to claim 17, wherein the nanoparticle composition comprises the cholesterol or a derivative thereof in the range of 50 mol% to 75 mol% of the cholesterol or a derivative thereof per nanoparticle composition.

19. The nanoparticle composition according to claim 11, for use as a medicament for treating a disease or disorder of a subject.

20. The therapeutically effective amount of the nanoparticle composition comprises a therapeutic or immunogenic nucleic acid agent in the range of 0.001 ng nucleic acid to 10 mg nucleic acid per kg of body weight of the subject, and the subject is a mammal selected from the group consisting of humans, non-human primates, mice, rats, dogs, cats, horses, or cows.

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