Carriers for efficient nucleic acid delivery

Abstract

Nanoparticle compositions for the delivery of nucleic acids to a subject are described, comprising a carrier comprising a polyester (PE) dendrimer or dendron and a therapeutic or immunogenic nucleic acid agent encapsulated within the PE. Methods for treating or preventing a disease or disorder in a subject by administering the nanoparticle compositions that provide a synergistic therapeutic or prophylactic effect with an immune response are provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 005,853, filed April 6, 2020, entitled "CARRIERS FOR EFFICIENT NUCLEIC ACID DELIVERY," and U.S. Provisional Patent Application No. 63 / 145,086, filed February 3, 2021, entitled "CARRIERS FOR EFFICIENT NUCLEIC ACID DELIVERY," the entire contents of which are incorporated herein by reference.

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

[0003] In recent years, nucleic acid vaccines and nucleic acid therapeutics have emerged as promising approaches to prevent and treat several diseases or illnesses, including gene therapy applications. However, nucleic acids are large hydrophilic molecules that cannot penetrate cell membranes and are susceptible to enzymatic degradation in the bloodstream. (Mendes et al., 2017, Molecules 22(9), 1401; Jones et al., 2013, Mol. Pharmaceutics 10, 4082-4098; and Nishikawa and Huang, 2001, Hum. Gene Ther. 12, 861-870)

[0004] Therefore, most proposed nucleic acid strategies rely on delivery vectors that must overcome different extracellular and intracellular barriers to efficiently deliver nucleic acids into cells with as little toxicity as possible (Jones et al., 2013, Mol. Pharmaceutics 10, 4082-4098; Gomes et al., 2014 MRS Bull. 39, 60-70; and Nishikawa and Huang, 2001 Hum. Gene Ther. 12, 861-870).

[0005] Obstacles to successful nucleic acid therapy include nucleic acid degradation by endonucleases, cellular internalization, endosomal escape, payload release from the vector and access to the desired target, and intracellular and extracellular accumulation of the vector (Jones et al., 2013, Mol. Pharmaceutics 10, 4082-4098; Nishikawa and Huang, 2001 Hum. Gene Ther. 12, 861-870; Gomes et al., 2014 MRS Bull. 39, 60-70; and Dufes et al., 2005, Adv. Drug Delivery Rev. 57, 2177-2202).

[0006] In recent years, non-viral vectors such as lipids, polymers, and dendrimers have attracted much attention (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). Their common feature is cationic or ionized nature. Among non-viral vectors, dendrimer-based vectors have attracted great interest for over 20 years as potential nucleic acid delivery vehicles. However, access to biodegradable unimolecular carriers remains a challenge (Mintzer and Grinstaff, 2010, Chem. Soc. Rev. 40, 173-190; Ravina et al., 2010, Macromolecules 43, 6953-6961; Nouri et al., 2012, J. Mater. Sci. Mater. Med. 23, 2967-2980; Pandita et al., 2011, Biomacromolecules 12, 472-481; Rodrigues et al., 2011, New J. Chem. 35, 1938-1943; Santos et al., 2010, J. Controlled Release 144, 55-64; Santos et al., 2009, J. Controlled Release 134, 141-148; Santos et al., 2010, Mol. Pharmaceutics 7, 763-774; and Duncan and Izzo, 2005, Adv. Drug Delivery Rev. 57, 2215-2237).

[0007] An ideal nucleic acid delivery vehicle must be biodegradable to prevent accumulation and subsequent cytotoxicity (Duncan and Izzo, 2005, Adv. Drug Delivery Rev. 57, 2215-2237). Polyester dendrimers, called "biodendrimers," have been reported, which contain building blocks known to be biocompatible or degradable into natural metabolites in vivo (Carnahan and Grinstaff, 2001, J. Am. Chem. Soc. 123, 2905; Carnahan and Grinstaff, 2001, Macromolecules 34, 7648; and Carnahan and Grinstaff, 2006, Macromolecules 39, 609). Polyester dendrimers have been identified as vehicles for small molecule delivery, but are not suitable for nucleic acid delivery. Among other requirements, to be efficient nucleic acid delivery vehicles, dendrimers must form complexes with nucleic acids and preferably self-assemble into nanoparticulate compositions that protect the nucleic acid from degradation while ensuring transport into cells. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Mendes et al., 2017, Molecules 22(9), 1401 [Non-patent document 2] Jones et al., 2013, Mol.Pharmaceutics 10, 4082-4098 [Non-patent document 3] Nishikawa and Huang, 2001 Hum. Gene Ther. 12, 861-870 [Non-patent document 4] Gomes et al., 2014 MRS Bull. 39, 60-70 [Non-Patent Document 5] Dufes et al., 2005, Adv. Drug Delivery Rev. 57, 2177-2202 [Non-patent document 6] Mintzer and Simanek, 2009, Chem. Rev. 109, 259-302 [Non-Patent Document 7] Mintzer and Grinstaff, 2010, Chem.Soc.Rev. 40, 173-190 [Non-patent document 8] Ravina et al., 2010, Macromolecules 43, 6953-6961 [Non-Patent Document 9] Nouri et al., 2012, J. Mater. Sci. Mater. Med. 23, 2967-2980 [Non-Patent Document 10] Pandita et al., 2011, Biomacromolecules 12, 472-481 [Non-Patent Document 11] Rodrigues et al., 2011, New J. Chem. 35, 1938-1943 [Non-Patent Document 12] Santos et al., 2010, J. Controlled Release 144, 55-64 [Non-Patent Document 13] Santos et al., 2009, J. Controlled Release 134, 141-148 [Non-Patent Document 14] Santos et al., 2010, Mol.Pharmaceutics 7, 763-774 [Non-Patent Document 15] Duncan and Izzo, 2005, Adv. Drug Delivery Rev. 57, 2215-2237 [Non-Patent Document 16] Carnahan and Grinstaff, 2001, J. Am. Chem. Soc. 123, 2905 [Non-Patent Document 17] Carnahan and Grinstaff, 2001, Macromolecules 34, 7648 [Non-Patent Document 18] Carnahan and Grinstaff, 2006, Macromolecules 39, 609 Summary of the Invention [Means for solving the problem]

[0009] In one aspect, the present invention provides a nucleic acid carrier represented by formula Ia or Ib, [ka] In the formula, PE is a polyester dendrimer or dendron comprising a core and a plurality of monomeric polyester units forming one or more generations, A is an amine linker, B is a hydrophobic unit, z is the number of surface groups, and P is a linker connecting two polyester dendrons, which relates to a nucleic acid carrier.

[0010] In one aspect, the invention relates to a nanoparticle composition comprising any of the nucleic acid carriers disclosed herein, a therapeutic or immunogenic nucleic acid agent encapsulated in the nucleic acid carrier, and a conjugated lipid.

[0011] In one aspect, the present invention relates to a nanoparticle composition comprising any of the nucleic acid carriers disclosed herein, a therapeutic or immunogenic nucleic acid agent encapsulated in the nucleic acid carrier, one of the conjugated lipids disclosed herein (e.g., PEG-lipid), and a mixture of phospholipids and cholesterol or a derivative thereof, for improving intracellular delivery and nanoparticle stability in vivo.

[0012] In one aspect, the present invention relates to a method of treating or preventing a disease or condition in a subject, the method comprising providing any of the nanoparticle compositions disclosed herein and administering a therapeutically effective amount of the nanoparticle composition to a subject. [Brief explanation of the drawings]

[0013] 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.

[0014] [Figure 1] 1 is a schematic diagram of a modified polyester dendrimer of generation 1 with fatty acid side chains (B) used for modification, where the fatty acid side chains B can be selected from any of the C4-C28 fatty acids. [Figure 2] We demonstrate the preparation process of a nanoparticle composition designed for improved self-assembly, containing modified dendrimer (PE-stearin), 1,2 dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and mRNA. [Figure 3] 1 shows the distribution of nanoparticle composition measured as intensity based on nanoparticle size (d). [Figure 4] Figure 1 shows a photograph of an agarose gel demonstrating the binding of modified dendrimers to RNA. The gel was stained with ethidium bromide (EB) and the gel image was captured using a Syngene G Box imaging system (Syngene, USA). [Figure 5] 1 shows the stability of dendrimers at neutral pH and pH 5.0 used in the RNA formulation process. [Figure 6A] Figures 6A-6E show the stability of RNA-PE stearin nanoparticles in PBS as measured by DLS and agarose gel retention assays. Figure 6A shows the distribution of nanoparticle composition (immediately after dialysis), measured as intensity based on nanoparticle size (d). [Figure 6B] Figures 6A-6E show the stability of RNA-PE-stearin nanoparticles in PBS as measured by DLS and agarose gel retention assays, and Figure 6B shows the stability of PE-stearin PR8 HA mRNA as measured by DLS after 3 weeks of storage at 4°C. [Figure 6C]Figures 6A-6E show the stability of RNA-PE-stearin nanoparticles in PBS as measured by DLS and agarose gel retention assays, and Figure 6C shows the stability of PE-stearin PR8 HA mRNA as measured by DLS after 3 days of storage at room temperature (RT). [Figure 6D] Figures 6A-6E show the stability of RNA-PE-stearin nanoparticles in PBS as measured by DLS and agarose gel retention assays, and Figure 6D shows the stability of PE-stearin PR8 HA mRNA as measured by DLS after 2 hours of storage at 37°C. [Figure 6E] Figures 6A-6E show the stability of RNA-PE-stearin nanoparticles in PBS as measured by DLS and agarose gel retention assay. Figure 6E shows the stability results based on the gel retention assay: Lane 1—PR8 HA mRNA; Lane 2—PE-stearin PR8 HA mRNA, 4°C, 3 weeks; Lane 3—PE-stearin PR8 HA mRNA, rt, 3 days; Lane 4—PE-stearin PR8 HA mRNA, 37°C, 1 hour; Lane 5—PE-stearin PR8 HA mRNA, 37°C, 2 hours. [Figure 7A] Figure 7A shows luciferase expression in cell culture from luciferase mRNA delivered to mammalian cells by modified polyester dendrimer-based nanoparticles and measured by quantification of intracellular luciferase activity using a luminescence assay. Figure 7A shows cell culture expression of luciferase mRNA delivered by nanoparticles containing PE-linoleate, PE-stearate, and PE-palmitate compared to naked luciferase mRNA (negative control). [Figure 7B]Figure 7B shows luciferase expression in cell culture from luciferase mRNA delivered to mammalian cells by modified polyester dendrimer-based nanoparticles and measured by quantification of intracellular luciferase activity using a luminescence assay. Figure 7B shows cell culture expression of luciferase mRNA delivered by nanoparticles containing PE-heptadecane, PE-stearin, PE-olein, and PE-16-hydroxypalmitin compared to naked luciferase mRNA. [Figure 8] Figure 1 shows Western blot analysis of HA expression in cell culture following delivery of PR8 HA mRNA in modified polyester dendrimer-based nanoparticles. [Figure 9] Figure 1 shows HA-specific antibodies induced by intramuscular injection of PR8 HA mRNA-containing nanoparticles and assayed by hemagglutinin inhibition assay (HAI). [Figure 10] The distribution of DNA nanoparticle composition measured as intensity based on nanoparticle size (d) is shown. [Figure 11] Figure 1 shows in vitro SEAP expression via quantiblue assay for nanoparticles containing DNA and modified polyester dendrimers. [Figure 12] 1 shows the SEAP colorimetric signal of replicon RNA expressing SEAP formulated into modified polyester dendron nanoparticles at pH 4.0 or 5.0. [Figure 13] Western blot analysis of spike expression in cell culture following delivery of SARS-CoV-2 spike replicon RNA in polyester dendrimer and dendron-based nanoparticles. [Figure 14] Figure 1 shows endpoint diluted serum titers of mouse IgG specific to COVID-19 spike protein in response to vaccination with replicon spike RNA formulated with PE dendron-G2-ricinol delivery material. [Figure 15]Figure 1 shows relative luciferase expression, measured in relative light units (RLU), in the heart and spleen at 6, 16, and 42 h after treatment of mice injected with 7.2 μg of luciferase-encoding replicon RNA formulated with PE dendron G2-5A2-5 ricinol or 31.4 μg of the same RNA formulated as lipid nanoparticles (LNPs). [Figure 16] 1 shows the distribution of nanoparticle composition measured as intensity based on nanoparticle size (d). [Figure 17] Figure 1 shows the SEAP colorimetric signal for replicon RNA expressing SEAP formulated into PE dendron_G2-A1-ricinol and (PE dendron_G2-A1-ricinol)2 PEG 200 nanoparticles. DETAILED DESCRIPTION OF THE INVENTION

[0015] In the following description, certain terminology is used for convenience only and is not limiting.

[0016] "Nanoparticle composition" refers to a composition comprising a modified dendrimer and a nucleic acid payload molecule encapsulated in the modified dendrimer.

[0017] The term "substituted" refers to the ability to change one functional group or moiety contained therein to another functional group or moiety contained therein, provided that the valences of all atoms on the parent structure are maintained. Substituted groups are referred to interchangeably herein as "substituted" or "substituents." 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.

[0018] As used herein, the term "amine linker" refers to an amine-containing linker that links or connects a hydrophobic tail (referred to herein for convenience 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 with a lone pair of electrons. Amines are formally derivatives of ammonia, in which one or more hydrogen atoms have been replaced with a substituent, such as an alkyl group.

[0019] As used herein, the term "alkyl" refers to a straight-chain or branched-chain hydrocarbon containing 1 to 28, preferably 1 to 20, carbon atoms, unless otherwise specified. The length of the alkyl chain may be used to control the hydrophobicity and self-assembly properties of the nucleic acid carrier. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. When an "alkyl group" is a linking group between two other moieties, it may be straight-chain or branched. Examples of alkyl groups include, but are not limited to, -CH2-, -CH2CH2-, -CHCHCHC(CH) and CHCH(CHCH)CH-.

[0020] As used herein, the term "surface group" refers to a terminal group on the surface of a nucleic acid carrier. The surface of the nucleic acid carrier herein is modified with a hydrophobic tail (referred to herein as component "B") to support self-assembly properties.

[0021] The words "a" and "one," as used in the claims and corresponding parts of the specification, are defined to include one or more of the referenced items, unless otherwise specified. This term includes the specifically mentioned words above, their derivatives, and words of similar meaning. 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.

[0022] In one embodiment, there is provided a nucleic acid carrier represented by formula Ia or Ib. [ka] where PE is a polyester dendrimer or dendron comprising a core and monomeric polyester units layered around the core to form a tree-like structure, where each layer is called a generation (G), A is an amine linker, B is a hydrophobic unit, z is the number of surface groups, and P is a linker connecting two polyester dendrons. The amine linker may contain an amine group that is protonated and charged at physiological pH.

[0023] In one embodiment, the PE may have formula II: [(core) c -Gn-O] Formula II where c is the core multiplicity or number of wedges from the core, and the value of c ranges from 1 to 6. The dendron may consist of hyperbranched wedges arising from a single chemically addressable focal point, also referred to herein as the core. Thus, for a dendron having a unidirectional core, c is equal to 1. G is the layer or generation of the dendrimer or dendron, n is the generation number, the value of which ranges from 1 to 10, the monomer polyester unit may be 2,2-bis(hydroxymethyl)propionic acid or 2,2-bis(hydroxymethyl)butyric acid, and z has formula III: z=cb n Formula III In the formula, b is the branch point multiplicity or the number of branches at each branch point, c is in the range of 1 to 6, and n is the number of generations.

[0024] In a nucleic acid carrier containing 2,2-bis(hydroxymethyl)propionic acid or 2,2-bis(hydroxymethyl)butyric acid as the monomer polyester unit, the branch point multiplicity or the number of branches at each branch point, b, is 2.

[0025] The structure of the core may affect the number of functional groups, amine and / or charge density, diameter and flexibility of the surface of the resulting nucleic acid carrier, which may modulate the physicochemical properties, interaction with nucleic acids and gene transfer activity of the carrier.

[0026] In one embodiment, the core may be unidirectional, where in Formula II, c is 1. The unidirectional core may be a carboxylic acid or a derivative thereof.

[0027] The unidirectional core may be selected from the following scaffolds: [ka] wherein Y is selected from methyl, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, neopentyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, azide (N), halogen (Cl, Br, or I), acetylene (C2H2), hydroxyl (-OH), or thiol (-SH), pyranosyl, cycloalkyl, aryl, heteroaryl, and heterocycle, each of which may be substituted with halogen, hydroxyl (-OH), and alkyl groups; A is an amine linker; B is a hydrophobic unit; and m is 1 to 20.

[0028] In one embodiment, the core may be a three-way core, where c is 3 in Formula II. The three-way core may be trimethylolpropane or 1,1,1-tris(hydroxyphenylethane). For reference, the structure of the above core is presented pictorially as the following scaffold: [ka]

[0029] In one embodiment, the core may be a four-way core, where c is 4 in Formula II.

[0030] The four-way core is composed of pentaerythritol, adamantane-1,3,5,7-tetraol or 5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphine, [1,1'-biphenyl]-3,3',5,5'-tetraol, 2,3,6,7-tetrahydroxy-9,10-dimethyl-anthracene, 9,10-dimethyl-9,10-dihydro-9,10-ethanoanthracene-2,3,6,7-tetraol, 6,13-di These four-way cores may be, but are not limited to, hydropentacene-5,7,12,14-tetraol, hexahydro-[1,4]dioxino[2,3-b][1,4]dioxin-2,3,6,7-tetraol, anthracene-1,4,9,10-tetraol, pyrene-1,3,6,8-tetraol, or 3,3,3',3'-tetramethyl-2,2',3,3'-tetrahydro-1-1'-spirobi[indene]-5,5',6,6'-tetraol. These four-way cores are exemplified by the following scaffolds: [ka]

[0031] The amine linker A is a moiety that contains one or more nitrogen atoms with lone electron pairs, thereby imparting proton-accepting functionality to the nucleic acid carrier molecule. Therefore, the amine linker can accept a free proton (H+) under acidic conditions. In a preferred embodiment, the nitrogen atom is present in the form of a secondary or tertiary amine. Examples of amine linkers include 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. 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 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) 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,The amine may be derived from 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)butan-1-ol, 4,4'-azanediylbis(butan-1-ol), or N1,N1'-(butane-1,4-diyl)bis(propane-1,3-diamine). For reference, the structures of the above amines are presented pictorially as the following structures:

[0032] [ka] [ka]

[0033] As mentioned above, protonatable dendrimers / dendrons may have acid dissociation constants (pKa) of the protonatable groups ranging from approximately 3.3 to approximately 10.4. These delivery molecules become cationic during formulation with nucleic acids under acidic pH conditions. Under these conditions, ionic interactions allow these delivery molecules to condense with negatively charged nucleic acids. Analysis of the structure-activity relationship of ionizable polyester dendrimer / dendron molecules revealed an unexpected relationship between the pKa of the ionizable delivery molecules and the ability of the nanoparticles to deliver functionally active replicon RNA. As an example, shown below, delivery molecules containing amine 1 and amine 2, with predicted pKa values ​​of 6.7 and 7.7, respectively, were able to deliver mRNA into cells (as seen in the SEAP expression in Figure 12), whereas dendrimer molecules containing amine 3 (predicted pKa of 3.3) were unable to deliver the payload. pKa values ​​were calculated using the ACD / Perceptor pKa prediction tool. [ka]

[0034] The hydrophobic unit B is C1-C 22 Alkyl group or C2-C 22 It may be an alkenyl group. 22 Alkyl group or C2-C 22 Each alkenyl group may be optionally substituted with 1 to 4 substituents selected from halogen, -CN, -NO, -N, C-C alkyl, halo(C-C alkyl), -OR, -NR, -COR, -OC(O)R, -CON(R), -OC(O)N(R), -NHC(O)N(R), -NHC(NH)N(R), C-C cycloalkyl, C-C cycloalkenyl, aryl, heteroaryl, or heterocycle. Each R may be independently selected from hydrogen, C-C alkyl, halo(C-C alkyl), C-C cycloalkyl, C-C 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).

[0035] The hydrophobic unit B of formula Ia and formula Ib may be introduced by contacting the PE dendrimer or dendron with a functional reagent such as a fatty acid or its derivative. 28The fatty acid may be a saturated or unsaturated fatty acid having a chain length, such as, but not limited to, arachidonic acid, oleic acid, eicosapentaenoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, or linolenic acid. The fatty acid derivative may be, but is not limited to, 12-hydroxy-9-cis-octadecenoic acid, 12-methyltetradecanoic acid, 12-methyltridecanoic acid, 14-methylhexadecanoic acid, 14-methylhexadecanoic acid, 18-methylnonadecanoic acid, 19-methylarachidonic acid, isopalmitic acid, isostearic acid, phytanic acid, (±)-2-hydroxyoctanoic acid, (±)-3-hydroxydecanoic acid, (±)-3-hydroxyoctanoic acid, 10-hydroxydecanoic acid, 12-hydroxyoctadecanoic acid, 15-hydroxypentadecanoic acid, 16-hydroxyhexadecanoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxytetradecanoic acid, 2-hydroxydodecanoic acid, DL-α-hydroxystearic acid, DL-β-hydroxylauric acid, DL-β-hydroxymyristic acid, or DL-β-hydroxypalmitic acid.

[0036] The hydrophobic unit B may be 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 decyl group, an undecyl 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, or a 16-hydroxyhexadecyl group.

[0037] The hydrophobic unit B of the nucleic acid carrier of formula I can be an unsaturated alkyl group. The presence of an unsaturated alkyl group in the nucleic acid carrier can prevent the nanoparticles from recycling when the carrier is formulated into a nanoparticle composition. Compared to saturated alkyl groups, unsaturated alkyl groups can have higher mobility and a lower crystallization temperature, and therefore can have the ability to change the morphology of the nanoparticles containing the nucleic acid carrier into a fused form when interacting with the phospholipid bilayer of the cell membrane and rupturing endosomes. Therefore, the nanoparticles can be restricted and remain intracellularly only at the injection site and not migrate to other locations.

[0038] In one embodiment, the nucleic acid carrier may include functional groups suitable for tracking the delivery material in vitro and in vivo. 13 C or 2 The nucleic acid carrier may also have fatty acids containing stable isotopes of carbon (C) or hydrogen (H), such as H (also referred to herein as deuterium, D, or d). When the nucleic acid carrier is formulated into nanoparticles with nucleic acids such as replicon RNA, the nanoparticles may be tracked in vitro and in vivo after administration by techniques such as mass spectrometry or nuclear magnetic resonance imaging. These stable isotopes are enriched in tissues. 12 C and 1 The inclusion of a stable isotope may be beneficial for the identification of the delivery molecule, as it is different from the H isotope. Tracking is useful for identifying the biodistribution, material clearance, and molecular stability of 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, Octane 2 H15 acid, decanoic acid-1- 13 C, Decanoic acid-10- 13 C, Decane-10,10,10- 2 H3 acid, decane 2 H19 acid, undecanoic acid-1- 13 C, lauric acid-12,12,12- 2 H3, laurin- 2 H23 acid, lauric acid-1- 13 C, lauric acid-1,12-13 C2, tridecane-2,2- 2 H2 acid, myristic acid-14- 13 C, myristic acid-1- 13 C, Myristic acid-14,14,14- 2 H3, myristic acid-d27, palmitic acid-1- 13 C, palmitic acid-16- 13 C, palmitic acid-16- 13 C,16,16,16- 2 H3, palmitic acid- 2 H31, stearic acid-1- 13 C, stearic acid-18- 13 C, stearic acid-18,18,18- 2 H3, stearin 2 H35 acid, 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 acid or eicosane 2 It may be, but is not limited to, H39 acid.

[0039] The linker unit P of formula IB may be a homobifunctional linker having two azide groups. The linker unit P may be used in the synthesis of dimeric molecules. In one embodiment, P may have formula IV: [ka] In the formula, m is in the range of 1 to 20.

[0040] In one embodiment, there is provided a nanoparticle composition comprising any of the nucleic acid carriers described herein. The nanoparticle compositions herein may be useful for introducing a drug into a cell. The drug may be a nucleic acid. The nanoparticle compositions herein may be useful as a transfection agent. The nanoparticle compositions herein may be useful in therapeutic methods.

[0041] In one embodiment, the nanoparticle composition may include a mixture of nucleic acid carriers, each containing a different amine density or side chain. These nucleic acid carriers may be mixed in a fixed ratio. For example, in a mixture of three dendrimers, 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 value between any two of these.

[0042] In one embodiment, the nanoparticle composition may comprise one or more therapeutic or immunogenic nucleic acid agents. As used herein, the term "nucleic acid" refers to natural or synthetic DNA or RNA molecules. The therapeutic or immunogenic nucleic acid agents of the compositions herein may be complexed with or encapsulated in the nucleic acid carrier of the nanoparticle composition.

[0043] In one embodiment, the therapeutic or immunogenic nucleic acid agent may be an RNA or DNA molecule. The terms "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refer to a polymer of deoxyribonucleotides. A DNA molecule may be a polynucleotide, an oligonucleotide, DNA, or cDNA. A DNA molecule may encode a wild-type or recombinant protein, peptide, or polypeptide, such as an antigen. The terms "RNA" or "RNA molecule" or "ribonucleic acid molecule" refer to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). An RNA molecule may be a replicon RNA (repRNA), a small interfering RNA (siRNA), a miRNA, a single-stranded guide RNA (sgRNA), a messenger RNA (mRNA), or a transfer RNA (tRNA). Replicon RNA (repRNA) refers to a replicative 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 mRNA and a replication template. Small interfering RNA (siRNA) refers to RNA (or RNA analog) containing approximately 10-50 nucleotides (or nucleotide analogs) that can direct or mediate RNA interference. MicroRNA (miRNA) refers to small (20-24 nt) regulatory non-coding RNAs involved in the post-transcriptional regulation of eukaryotic gene expression by affecting either or both the stability and translation of coding mRNA. Messenger RNA (mRNA) is typically 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. DNA or RNA molecules may be chemically modified.

[0044] An RNA molecule may be monocistronic or polycistronic mRNA. Monocistronic mRNA refers to an mRNA that contains only one sequence encoding a protein, polypeptide, or peptide. Polycistronic mRNA usually refers to two or more sequences encoding two or more proteins, polypeptides, or peptides. The mRNA may encode a protein, polypeptide, or peptide that acts as an antigen.

[0045] In one embodiment, the DNA molecule may be a polynucleotide, an oligonucleotide, DNA, or cDNA. The RNA molecule may be a replicon RNA (repRNA), a small interfering RNA (siRNA), a miRNA, a single-stranded guide RNA (sgRNA), a messenger RNA (mRNA), or a transfer RNA (tRNA). The therapeutic or immunogenic nucleic acid agent may be non-covalently or covalently bound to the nucleic acid carrier. The therapeutic or immunogenic nucleic acid agent may be electrostatically bound to the charged nucleic acid carrier via an ionic bond.

[0046] In one embodiment, the nanoparticle compositions described herein may include an immunogenic or therapeutic nucleic acid agent that encodes an antigen.

[0047] As used herein, "encapsulation" can refer to nanoparticles that provide active or therapeutic agents, such as nucleic acids (e.g., messenger RNA), with complete encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is completely 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).

[0048] As used herein, an "antigen" is defined as a molecule that provokes 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 macromolecules such as proteins, peptides, or polypeptides. An antigen may be a structural component of a pathogen or a cancer cell. Antigens 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.

[0049] The antigen may be, but is not limited to, a vaccine antigen, a parasite 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 deliver an antigen to stimulate the adaptive immune response of a subject's body. The immune response induced by vaccination usually leads to the development of immunological memory, which subsequently provides the organism with the ability to respond quickly when encountering an antigen or infectious agent.

[0050] In this specification, it is preferable to use a "nucleic acid carrier" as a carrier for nucleic acids, and therefore the term "nucleic acid carrier" is used. However, embodiments also include a combination of the nucleic acid carrier herein with a drug containing a negative or partially negative charge. The drug may be a drug, a protein, or a lipid complex.

[0051] In one embodiment, the nanoparticle composition may be formulated to contain a drug containing a negative or partial negative charge. The nanoparticles may be formulated via electrostatic binding between the negative and positive charges of protonated amine groups within the nucleic acid carrier. The negatively charged drug may be an ionic drug. The term "ionic drug" refers to an electrically asymmetric molecule that is water-soluble and ionizable in a distilled water solution. The ionic drug may contain a phosphate, phosphonate, or phosphinate functional group. The drug containing a phosphate group may be a phosphate-containing nucleotide analog, such as a drug used in cancer treatment and viral chemotherapy. The phosphate-containing drug may be, but is not limited to, a purine and pyrimidine nucleoside analog, arabinosylcytosine (ara-C), Ara-C monophosphate (ara-CMP), azidothymidine (AZT), AZT monophosphate (AZTMP), 2'3'-dideoxycytidine (ddCD), cyclic adenosine monophosphate (cAMP), tenofovir, or adefovir.

[0052] In one embodiment, the nanoparticle composition may comprise one or more proteins. Non-limiting examples of the one or more proteins include an antibody or antibody fragment, a cytokine such as interferon (IFN)-α or interleukin (IL)-2, an antigen derived from a pathogen such as the SARS-CoV spike protein or its receptor binding domain (RBD), an antigen derived from a cancer such as a mutant form of Kirsten rat sarcoma 2 viral oncogene homolog (KRAS), or other therapeutic biologics such as insulin, factor VIII, or erythropoietin. The proteins in the compositions herein may be included in the bulk composition and / or complexed with or encapsulated in the nucleic acid carrier of the nanoparticle composition.

[0053] In one embodiment, the nanoparticle compositions described herein may contain lipid complexes. 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, PEG-lipid complexes. Non-limiting examples of PEG-lipids include PEG conjugated to lipids such as DMG-PEG 2000, PEG conjugated to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to cholesterol or its derivatives, and mixtures thereof. In some cases, PEG may be optionally substituted with an alkyl group, an alkoxy group, an acyl group, or an aryl group.

[0054] PEG is a linear, water-soluble polymer of repeating ethylene PEG units with two terminal hydroxyl groups. PEG is classified by molecular weight; for example, PEG 2000 has an average molecular weight of approximately 2000 daltons, and PEG 5000 has an average molecular weight of approximately 5000 daltons. PEG is commercially available from Avanti Polar Lipids. The PEG portion of the PEG-lipid conjugates described herein may have an average molecular weight ranging from approximately 550 daltons to approximately 10,000 daltons.

[0055] Phosphatidylethanolamines with various acyl chain groups of different chain lengths and saturations can be conjugated to PEG to form lipid conjugates. Phosphatidylethanolamines can be commercially available or 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. Contemplated phosphatidylethanolamines include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoylphosphatidylethanolamine (DSPE).

[0056] The PEG-lipid may comprise PEG conjugated to cholesterol or a cholesterol derivative, including, but not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0057] The size, relative amount, and distribution of PEG-lipids in a nanoparticle composition can affect the physical properties of the nanoparticle composition. Controllable physical properties can include, but are not limited to, the diameter of the nanoparticles, the tendency of the nanoparticles to aggregate, the number of nucleic acid molecules within each nanoparticle, 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 nanoparticle uptake by cells.

[0058] 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 integers above. The nanoparticle composition containing PEG-lipid may contain nanoparticles with a smaller diameter than nanoparticles in a composition lacking PEG-lipid. The nanoparticle composition may contain nanoparticles with a higher tendency to aggregate than nanoparticles in a composition lacking PEG-lipid.

[0059] The nanoparticle composition may contain "amphiphilic lipids." As used herein, "amphiphilic lipid" refers to any material having a nonpolar, hydrophobic "tail" and a polar "head." Polar groups may include phosphate, carboxyl, sulfato, amino, sulfhydryl, nitro, hydroxyl, or other groups. Nonpolar groups may include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclic groups. Examples of amphiphilic lipids include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.

[0060] The nanoparticle composition may contain amphiphilic lipid in an amount ranging from 10 mol % to 15 mol % per nanoparticle composition.

[0061] In one embodiment, the nanoparticle composition may comprise cholesterol or a cholesterol derivative. Examples of cholesterol derivatives 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 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. Cholesterol derivatives may contain sugar moieties such as mannose and galactose. Cholesterol derivatives may contain sugar moieties and / or amino acids such as serine, threonine, lysine, histidine, arginine, or derivatives thereof. The nanoparticle composition may contain cholesterol or a cholesterol derivative in an amount ranging from 50 mol % to 75 mol % per nanoparticle composition.

[0062] The pharmaceutical compositions herein may be sterilized by conventional, well-known sterilization techniques. The aqueous solutions may be packaged for use or lyophilized. The lyophilized preparation may be combined with a sterile aqueous solution prior to administration.

[0063] In one embodiment, the nanoparticle composition may include a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, zinc stearate, or stearic acid), or solvent encapsulating material, involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient.Pharmaceutically 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 and derivatives thereof such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and / or cellulose acetate; (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; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and / or soybean oil; and (10) protease inhibitors. Glycols such as pyrene 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 PEG 400, (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 (80) and / or surfactants such as poloxamers, 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," "carrier," "pharmaceutically acceptable carrier," and the like are used interchangeably.

[0064] One embodiment includes a method of treating or preventing a disease or condition in a subject. The method can include providing any of the nanoparticle compositions described herein. The method can include administering a therapeutically effective amount of the nanoparticle composition to the subject.

[0065] 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 in at least a subset of cells in an animal. The therapeutic effect may be achieved at a reasonable benefit / risk ratio applicable to any treatment. A "therapeutically effective amount" may refer to an amount sufficient to produce antigen-specific antibodies in serum. A "therapeutically effective amount" may refer to an amount sufficient to alleviate 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, alleviation of symptoms of a disease may be assessed by a reduction in virus in feces, body fluids, or secretions. The nanoparticle composition may be administered at a dosage and via a route of administration effective to generate an immune response.

[0066] Therapeutic efficacy may depend on the effective amount of active agent and the duration of administration necessary to achieve the desired result. Administration of the nanoparticle composition may be a preventative measure. Administration of the nanoparticle composition may be a therapeutic measure to promote immunity against infectious agents and minimize complications associated with slow immune development, particularly in patients with weakened immune systems, the elderly, or infants.

[0067] 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, drug combinations, and individual response to therapy.

[0068] The therapeutic efficacy and toxicity of active agents in nanoparticle compositions may be determined by standard pharmaceutical procedures, for example, by determining the therapeutically effective dose (ED50) for 50% of a population and the lethal dose (LD50) for 50% of a population 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.

[0069] A therapeutically effective dose may be initially estimated from cell culture assays. A therapeutically effective dose may be formulated in animal models to achieve a circulating plasma concentration range that contains the IC50 (i.e., the concentration of the therapeutic agent that achieves a 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.

[0070] A therapeutically effective dose may be 0.0001 μg to 1 mg of therapeutic or immunogenic nucleic acid per kg body weight of the subject, or 0.00001 μg to 1 mg (μg) units per dose per subject, and may be administered daily. However, doses greater than 1 mg may be provided. For example, a dose may be at least 1 milligram, or about 3×1 mg, or about 10×1 mg of nucleic acid units per dose per subject. Because nanoparticle vaccines are easy to manufacture and potentially inexpensive to process, design, and store, larger doses for large animal subjects may be economically feasible. For animal subjects several orders of magnitude larger than the experimental animals used in the examples herein, dosages are more easily adjusted; for example, dosages may be about 3×10×1 μg, about 3×20×1 μg, or about 3×30×1 μg for animals such as humans or small agricultural animals. However, for example, for high-value zoo or agricultural animals such as elephants, the dosage may be about 3 x 40 x 1 μg, about 3 x 50 x 1 μg, or even about 3 x 60 x 1 μg. For prophylactic immunization, routine treatment, or treatment of small wild animals, the dosage may be less than about 3 x 1 μg, less than about 1 μg, less than about 500 ng, less than about 250 ng, less than about 100 ng, less than about 50 ng, less than about 25 ng, less than about 10 ng, less than about 5 ng, less than about 1 ng, less than about 500 pg, less than about 250 pg, less than about 100 pg, or any range therebetween. The therapeutic and immunogenic nucleic acids may be a combination of different nucleic acids used per treatment dose. The terms "subject" and "individual" are used interchangeably herein and refer to either a human or an animal. Preferably, the animal is a vertebrate, such as a primate, rodent, livestock, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques such as rhesus monkeys. Rodents may be selected from mice, rats, guinea pigs, woodchucks, ferrets, rabbits, and hamsters. Domestic or game animals may be selected from cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, foxes, wolves, avian species such as chickens, emus, and ostriches, and fish such as trout, catfish, and salmon.The patient or subject may be selected from the patients described above or a subset thereof. The patient or subject may be selected from all of the above, but may be selected exclusively from 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 or human. The terms "patient" and "subject" are used interchangeably herein. The terms "patient" and "subject" are used interchangeably herein.

[0071] 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, or a cow. The non-human mammal may represent an animal model of a disease or disorder. Additionally, the methods described herein may be intended for the treatment of livestock and / or pets. The subject may be male or female.

[0072] As used herein, the terms "administer," "administering," "administration," and the like refer to the placement of a composition into a subject. Administration may be carried out by any method or route that at least partially localizes the composition to the desired site to achieve a desired effect. The nanoparticle compositions described herein may be administered by any suitable route known in the art, including, but not limited to, intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), pulmonary, nasal, rectal, or oral or parenteral routes, including topical (including buccal and sublingual) administration.

[0073] 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.

[0074] The nanoparticle composition may be used to deliver therapeutic or immunogenic nucleic acids for gene targeting. 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 contain a sequence complementary to a sequence contained in an mRNA transcript of a target gene when expressed in a host cell. The antisense oligonucleotide may be a morpholino antisense oligonucleotide. The antisense oligonucleotide may contain a sequence complementary to a sequence contained in an mRNA transcript of a target gene. The therapeutic or immunogenic nucleic acid may be an interfering RNA (iRNA) directed against a specific target gene in a specific target organism. The iRNA may downregulate or prevent gene expression by inducing sequence-specific silencing of the 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 double-stranded RNA (dsRNA). The dsRNA may be 25 nucleotides or more in length, or may be longer. The dsRNA may contain a sequence complementary to the sequence of the target gene(s).

[0075] In one embodiment, the therapeutic or immunogenic nucleic acid may be or encode an agent that completely or partially reduces, inhibits, prevents, or modulates the activity or synthesis of one or more genes encoding target proteins. 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.

[0076] 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 described herein. As used herein, the term "alteration" refers to a genomic modification in a subject's cells. The alteration may be an insertion or deletion of nucleotides in the sequence of a target gene. "Insertion" refers to the addition of one or more nucleotides to the sequence of a target gene. "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 more favorable expression of the gene, manifested by an improvement in the genotype and / or phenotype of the host organism.

[0077] Alterations to 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.

[0078] An exemplary genome editing system is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, as described, for example, in International Publication No. WO 2018 / 154387, published August 30, 2018, and incorporated by reference as if fully set forth herein. Generally, a "CRISPR system" refers to transcripts and other elements involved in the expression of CRISPR-associated (Cas) genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences, tracr mate sequences, guide sequences, or other sequences and transcripts from a CRISPR locus. One or more tracr mate sequences may be operably linked 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 a 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. A single fusion crRNA-tracrRNA construct is also referred to herein 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.

[0079] 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 the actual gene products, proteins, or other molecules.

[0080] In one embodiment, the nanoparticle compositions may be used for gene targeting in a subject in vivo or ex vivo, for example, by isolating cells from the subject, gene editing, and transplanting the edited cells into the subject. One embodiment includes a method comprising administering a nanoparticle composition herein to cells isolated from a subject. The method may include gene targeting. The method may include transplanting the edited cells into the subject.

[0081] One embodiment includes a method of introducing a drug into a cell. The method may include exposing the cell to a nanoparticle composition herein. The drug may be a nucleic acid. The drug may be as described above. The method may be a method of transfection when the drug is a nucleic acid. The drug 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. Examples are provided below.

[0082] The following list of embodiments includes specific embodiments of the present invention, but this list is not intended to be limiting or to exclude alternative embodiments or embodiments otherwise described herein.

[0083] List of embodiments 1. A nucleic acid carrier having a structure represented by formula Ia or formula Ib, [ka] wherein PE is a polyester dendrimer or dendron comprising a core and a plurality of monomeric polyester units forming one or more generations, A is an amine linker, B is a hydrophobic unit, and z is the number of surface groups.

[0084] 2. PE has formula II: [(core) c -Gn-O] II In the formula, c is the core multiplicity or the number of wedges derived from the core, and its value independently ranges from 1 to 6; G is the layer or generation of the dendrimer or dendron; and n is the number of generations, and ranges from 1 to 10.

[0085] 3. A nucleic acid carrier according to claim 1 or 2, wherein the plurality of monomer polyester units are 2,2-bis(hydroxymethyl)propionic acid or 2,2-bis(hydroxymethyl)butyric acid.

[0086] 4. z has formula III, z=cb n III In the formula, b is the branch point multiplicity or the number of branches at each branch point, c is the core multiplicity or the number of wedges derived from the core, and is in the range of 1 to 6, and n is the number of generations, and is in the range of 1 to 10.

[0087] 5. A nucleic acid carrier according to any one or more of embodiments 1 to 4, wherein c is 1 and said core is a unidirectional core.

[0088] 6. A nucleic acid carrier according to embodiment 5, wherein the unidirectional core is a carboxylic acid or a derivative thereof.

[0089] 7. The above core is [ka] is selected from the group consisting of 6. The nucleic acid carrier according to embodiment 5, wherein Y is selected from methyl, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, neopentyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, azide (N), halogen (Cl, Br or I), acetylene (C2H2), hydroxyl (-OH), or thiol (-SH), -pyranosyl, cycloalkyl, aryl, heteroaryl and heterocycle; A is an amine linker; B is a hydrophobic unit; and m is 1 to 20.

[0090] 8. The nucleic acid of embodiment 7, wherein the cycloalkyl, aryl, heteroaryl, and heterocycle are substituted with at least one group selected from a halogen group, a hydroxyl group (—OH), and an alkyl group.

[0091] 9. A nucleic acid carrier according to any one or more of embodiments 1 to 5, wherein c is 3 and said core is a three-directional core.

[0092] 10. The three-way core is trimethylolpropane or 1,1,1-tris(hydroxyphenylethane), and [ka] 10. The nucleic acid carrier according to embodiment 9, having a structure represented by:

[0093] 11. A nucleic acid carrier according to any one or more of embodiments 1 to 5, wherein c is 4 and said core is a four-directional core.

[0094] 12. The four-way core is selected from the group consisting of pentaerythritol, adamantane-1,3,5,7-tetraol or 5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphine, [1,1'-biphenyl]-3,3',5,5'-tetraol, 2,3,6,7-tetrahydroxy-9,10-dimethyl-anthracene, 3,9,10-dimethyl-9,10-dihydro-9,10-ethanoanthracene-2,3,6,7-tetraol, 4,6, selected from the group consisting of 13-dihydropentacene-5,7,12,14-tetraol, hexahydro-[1,4]dioxino[2,3-b][1,4]dioxine-2,3,6,7-tetraol, anthracene-1,4,9,10-tetraol, pyrene-1,3,6,8-tetraol, and 3,3,3',3'-tetramethyl-2,2',3,3'-tetrahydro-1-1'-spirobi[indene]-5,5',6,6'-tetraol, [ka] [ka] 12. The nucleic acid carrier according to embodiment 11, having a structure represented by:

[0095] 13. 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-hydroxypropyl)(methyl)amino)butan-1-ol, 4-((3-hydroxypropyl)(methyl)amino)butan-1-ol, N1-(4-aminopropyl)-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,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(propane-1,3-diamine), respectively: [ka] [ka] [ka] [ka] 13. The nucleic acid carrier according to any one or more of embodiments 1 to 12, having a structure represented by the following formula:

[0096] 14. B is C1-C 22 Alkyl group or C2-C 22 13. The nucleic acid carrier according to any one or more of embodiments 1 to 12, wherein the group is an alkenyl group.

[0097] 15. C1-C above 22 Alkyl group or C2-C 22 The nucleic acid carrier of embodiment 14, 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.

[0098] 16. The nucleic acid carrier of claim 15, wherein the 1 to 4 substituents are selected from OR, -NR2, -CO2R, -OC(O)R, -CON(R)2, -OC(O)N(R)2, -NHC(O)N(R)2 and -NHC(NH)N(R)2.

[0099] 17. The nucleic acid carrier of claim 15, wherein each cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle is further substituted with R', and R' is independently selected from the group consisting of halogen, -CN, -NO2, -N3, C1-C6 alkyl, and halo(C1-C6 alkyl).

[0100] 18. The nucleic acid carrier according to any one or more of embodiments 1 to 17, wherein B is an unsaturated alkyl group.

[0101] 19. The nucleic acid carrier of claim 1, wherein B is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, 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, and arachidonyl.

[0102] 20. The nucleic acid carrier according to any one or more of embodiments 1 to 17, wherein B is derived from a fatty acid or a derivative thereof.

[0103] 21. A nucleic acid carrier described in embodiment 20, wherein the fatty acid is selected from the group consisting of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid and eicosapentanoic acid.

[0104] twenty two. 21. The nucleic acid carrier according to embodiment 20, wherein the fatty acid derivative is selected from the group consisting of 12-hydroxy-9-cis-octadecenoic acid, 12-methyltetradecanoic acid, 12-methyltridecanoic acid, 14-methylhexadecanoic acid, 14-methylhexadecanoic acid, 18-methylnonadecanoic acid, 19-methylarachidonic acid, isopalmitic acid, isostearic acid, phytanic acid, (±)-2-hydroxyoctanoic acid, (±)-3-hydroxydecanoic acid, (±)-3-hydroxyoctanoic acid, 10-hydroxydecanoic acid, 12-hydroxyoctadecanoic acid, 15-hydroxypentadecanoic acid, 16-hydroxyhexadecanoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxytetradecanoic acid, 2-hydroxydodecanoic acid, DL-α-hydroxystearic acid, DL-β-hydroxylauric acid, DL-β-hydroxymyristic acid, and DL-β-hydroxypalmitic acid.

[0105] 23. A nucleic acid carrier according to any one or more of embodiments 20 to 22, wherein the fatty acid comprises one or more stable isotopes.

[0106] 24. A nucleic acid carrier described in embodiment 23, wherein the stable isotope is a stable isotope of carbon or hydrogen.

[0107] 25. The stable isotopes of carbon mentioned above are 13 25. The nucleic acid carrier of embodiment 24, wherein said nucleic acid carrier is C.

[0108] 26. The stable isotopes of hydrogen mentioned above are 2 The nucleic acid carrier according to claim 24, wherein the nucleic acid carrier is H.

[0109] 27. The above fatty acid containing the above stable isotope is octanoic acid-1- 13 C, Octanoic acid-8- 13 C, Octanoic acid-8,8,8-d3, Octane- 2 H15 acid, decanoic acid-1- 13 C, Decanoic acid-10- 13 C, decanoic acid-10,10,10-d3 acid, decanoic acid-d19 acid, undecanoic acid-1- 13 C, lauric acid-12,12,12-2 H3, laurin- 2 H23 acid, lauric acid-1- 13 C, lauric acid-1,12- 13 C2, tridecane-2,2- 2 H2 acid, myristic acid-14- 13 C, myristic acid-1- 13 C, Myristic acid-14,14,14- 2 H3, myristic- 2 H27 Acid, 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, stearin 2 H35 acid, 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 acid and eicosane 2 27. The nucleic acid carrier according to any one or more of embodiments 23 to 26, wherein the nucleic acid carrier is selected from the group consisting of H39 acids.

[0110] 28. P is a homobifunctional linker having two azide groups and has the structure represented by formula IV: [ka] 28. The nucleic acid carrier according to any one or more of embodiments 1 to 27, wherein m is a number in the range of 1 to 20.

[0111] 29. A nanoparticle composition comprising a nucleic acid carrier according to any one of embodiments 1 to 29 and a therapeutic or immunogenic nucleic acid agent encapsulated in the nucleic acid carrier.

[0112] 30. The nanoparticle composition described in embodiment 29, 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.

[0113] 31. The nanoparticle composition of embodiment 29 or 30, wherein the therapeutic or immunogenic nucleic acid agent encodes one or more antigens selected from the group consisting of infectious diseases, pathogens, cancer, autoimmune diseases, and allergic diseases.

[0114] 32. The nanoparticle composition of embodiment 29 or 30, wherein the therapeutic or immunogenic nucleic acid agent comprises RNA or DNA capable of silencing, suppressing or modifying the activity of a gene.

[0115] 33. A nanoparticle composition described in any one of embodiments 29 to 32, further comprising a PEG-lipid.

[0116] 34. The nanoparticle composition of embodiment 33, 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.

[0117] 35. A nanoparticle composition described in embodiment 33 or 34, comprising the above PEG-lipid in the range of 1 mol% to 10 mol% per nanoparticle composition.

[0118] 36. The nanoparticle composition of any one of embodiments 33 to 35, further comprising a phospholipid and cholesterol or a derivative thereof.

[0119] 37. The nanoparticle composition of embodiment 36, wherein the phospholipid is dioleoylphosphatidylcholine (DOPC) or distearoylphosphatidylcholine (DSPC).

[0120] 38. The nanoparticle composition described in embodiment 37, comprising the phospholipid in an amount ranging from 10 mol% to 15 mol% per nanoparticle composition.

[0121] 39. The nanoparticle composition described in embodiment 36, which contains 50 mol % to 75 mol % of the cholesterol or its derivative per nanoparticle composition.

[0122] 40. 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 nanoparticle composition described in any one of embodiments 29 to 39.

[0123] 41. The method of embodiment 40, 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.

[0124] 42. The method of embodiment 41, wherein the subject is a mammal.

[0125] 43. The method of embodiment 42, wherein the mammal is selected from the group consisting of chickens, rodents, dogs, primates, horses, high-value agricultural animals, and humans. [Example]

[0126] The following non-limiting examples are intended to illustrate particular embodiments, and the entire embodiment may be supplemented with one or more details from one or more of the following examples, and / or one or more elements of the embodiment may be substituted with one or more details from one or more of the following examples.

[0127] Example 1. Nanoparticle Compositions Containing Fatty Acid-Modified Biodegradable Dendrimers The characteristics of the surface groups of dendrimers determine their physicochemical properties, biological activity, and biocompatibility. An ideal gene delivery vehicle should be biodegradable to prevent bioaccumulation and subsequent cytotoxicity. Large dendrimers are highly packaged and therefore less likely to biodegrade. There is a need for lower generation dendrimers that can complex with nucleic acids and translocate across cell membranes while maintaining biodegradability and avoiding cytotoxicity.

[0128] Preferably, the chemical bonds present in the monomer units are ester bonds, and the terminal layers of the low-generation polyester dendrimers are substituted with endogenous / essential fatty acid side chains via amide bonds, making them susceptible to hydrolysis in plasma by esterases and amidases, thus making them biodegradable. Such low-generation dendrimers with fatty acid chains can non-covalently bind to nucleic acids to form nanoparticles through the properties of their dynamic equilibrium.

[0129] The modified dendrimers in this example consisted of (1) bis-MPA-OH or 2,2-bis(hydroxymethyl)propionic acid dendrimers with a trimethylolpropane core (generations 1 and 2), (2) bis-MPA-OH dendrimers with a pentaerythritol core (generations 1 and 2), (3) bis-MPA-OH dendrimers with a 1,1,1-tris(hydroxyphenylethane) core, or (4) bis-MPA-OH dendrimers with an adamantane core, as follows:

[0130] (1) Bis-MPA-OH dendrimer with a trimethylolpropane core (Generations 1 and 2) [ka]

[0131] (2) Bis-MPA-OH dendrimer with a pentaerythritol core (Generations 1 and 2) [ka]

[0132] (3) Bis-MPA-OH dendrimers (generations 1 and 2) with a 1,1,1-tris(hydroxyphenylethane) core [ka]

[0133] (4) Bis-MPA-OH dendrimer with an adamantane core (Generations 1 and 2) [ka]

[0134] FIG. 1 is a schematic diagram of a generation 1 modified polyester dendrimer and the fatty acid side chains (B) that can be used for modification. In the diagram, the fatty acid side chains B are C4-C 28 The fatty acids may be selected from any of the fatty acids.

[0135] An example of the synthesis of PE-linoleic acid is as follows. [ka]

[0136] [ka]

[0137] [ka]

[0138] [ka]

[0139] Compound 1: The starting material, bis-MPA-OH dendrimer trimethylolpropane core, generation 1 (300 mg, 0.62 mmol), was dissolved in dry DCM (6 mL). Pyridine (0.9 mL, 4.96 mmol) was added, followed by p-nitrophenyl chloroformate (2.1 g, 10 mmol) dissolved in dry DCM (25 mL). The reaction mixture was stirred overnight (16 h) at 0 °C to room temperature (23 °C). The next day, TLC showed the formation of the product. The reaction mixture was diluted with 1.33 M NaHSO and extracted with EtOAc. The organic layer was washed with brine and evaporated. The crude reaction mixture was loaded onto a 40 g silica gel column. The loaded compound was then purified by flash chromatography using DCM / EtOAc. The compound began to elute with 8% EtOAc, affording the desired product as a pale yellow oil (870 mg, 65%). 1 H NMR (301MHz, chloroform-d) δ ppm 0.93-1.02(m,3H),1.34-1.42(m,9H),1.54-1.65(m,2H),4.19-4.25(m,6H),4.42-4.56(m,12H),7.27-7.37(m,12H),8.15-8.24(m,12H).

[0140] Compound 2: A solution of compound 1, PNP carbonate (450 mg, 0.31 mmol) dissolved in dry DCM (6 mL) was added to excess mono-Boc-DAPMA (453 mg, 1.85 mmol) dissolved in dry DCM (6 mL). A solution of DMAP (76 mg, 0.62 mmol) and DIPEA (0.32 mL, 1.86 mmol) in dry DCM (4 mL) was added, and the reaction mixture was stirred overnight at 23 °C for 16 hours under an argon atmosphere. TLC confirmed completion of the reaction. The crude product was then purified by flash chromatography using mobile phase a (DCM) / mobile phase b (CHCl / MeOH / NHOH aq). The compound began to elute at 40% mobile phase b (Rf = 0.1 (1:1 mobile phase a / mobile phase b)) to give the desired product as a pale yellow oil (400 mg, 62%). 1H NMR (301 MHz, chloroform-d) δ ppm 0.66-0.78 (m, 3H), 0.98-1.07 (m, 9H), 1.18-1.28 (m, 54H), 1.38-1.52 (m, 24H), 1.97-2.06 (m, 18H), 2.12-2.24 (m, 24H), 2.85-3.00 (m, 24H), 3.17-3.25 (m, 12H); 13 C NMR (76 MHz, methanol-d4) δ ppm 17.92, 27.95, 28.04, 28.71, 39.58, 40.11, 42.18, 42.59, 48.01, 54.69, 56.01, 56.08, 64.76, 66.72, 79.66, 158.03, 158.25, 174.08.

[0141] Compound 5: 171 mg of compound 4 (0.082 mmol) was dissolved in 3 mL of MeOH, and then treated with 34 equivalents of AcCl (0.2 mL). The reaction was stirred at 0-23 °C for 16 hours, evaporated to dryness, dissolved in 2 mL of DMF, and 0.1 mL of EtN (0.73 mmol, 9 equivalents) was added. 365 mg of linoleic-NHS (synthesized according to published procedures: Talukder et al., WO 2020 / 132196, incorporated by reference as fully set forth herein) dissolved in 2 mL of DMF was then added. The reaction mixture was stirred at 23 °C for 24 hours, then concentrated under reduced pressure in a Genevac and purified by flash chromatography on a silica column using a gradient elution from 100% CHCl to 75:22:3 CHCl / MeOH / NHOH (by volume) over 40 minutes. The desired product was eluted with 50:7:1 CH2Cl2 / MeOH / NH4OH aq. The product-containing fractions were combined and dried under gradient high vacuum for 12 h to give the desired product as a pale yellow oil (40 mg, 16%), which was stored at 4 °C until use. 1H NMR (300MHz, chloroform-d) δ ppm 0.85-0.94(m,21H),1.13-1.40(m,93H),1.44-1.51(m,2H),1.51-1.71(m,43H),1.94-2.06(m,24H),2.09-2.2 2(m,34H),2.34-2.44(m,24H),2.69-2.79(m,9H),3.09-3.33(m,26H),3.97-4.26(m,17H),5.22-5.46(m,22H).

[0142] Example 2. Compound of formula (I) The following compounds may be prepared according to the procedures described above, varying the starting materials when necessary to provide the desired product. [ka]

[0143] [ka]

[0144] [ka]

[0145] [ka]

[0146] [ka]

[0147] [ka]

[0148] [ka]

[0149]

change

[0150]

change

[0151]

change

[0152]

change

[0153]

change

[0154]

change

[0155]

change

[0156]

change

[0157]

change

[0158]

change

[0159]

change

[0160] [ka]

[0161] [ka]

[0162] Example 3. Nanoparticle Formulations Figure 2 shows the preparation method for nanoparticle compositions designed for improved self-assembly. Nanoparticles were formulated by directly mixing 30 μl of an ethanol phase containing the modified dendrimer (PE-stearin) combined with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-lipid, Avanti Polar Lipids) with 90 μl of luciferase mRNA diluted in ultrapure, DNase- / RNase-free, and endotoxin-free distilled water (Invitrogen) and sterile 100 mM (pH 5.0) QB citrate buffer (Teknova) to a final citrate concentration of 10 mM. The resulting nanoparticles had a mass ratio of modified dendrimer:PEG-lipid:RNA of 6:1:2. The formulation was diluted 1000-fold for analysis of particle size distribution, Z-average and induced count rate using a Zetasizer Nano ZS (Malvern Panalytical).

[0163] Example 4. Hydrodynamic size measurements Figure 3 shows the distribution of nanoparticle composition measured as intensity based on nanoparticle size (d.nm, diameter in nm). Referring to Figure 3, "Z-average" is the intensity-weighted average hydrodynamic size of the entire population of particles measured by dynamic light scattering (DLS). Referring to Figure 3, the strongest intensity was observed for nanoparticles with a size of 248.4 d.nm.

[0164] Example 5. Gel retardation assay Agarose gel electrophoresis was performed to assess the binding of the modified dendrimers to RNA according to published methods (Geall et al., 10.1073 / pnas.1209367109, incorporated by reference as if fully set forth herein). Figure 4 is a photograph of an agarose gel showing the binding of the modified dendrimers to RNA. The gel was stained with ethidium bromide (EB), and the gel image was captured using a Syngene G Box Imaging System (Syngene, USA). Referring to Figure 4, lane 1 contained unformulated luciferase mRNA, lane 2 contained a formulation of PE-palmitin dendrimer and luciferase mRNA, lane 3 contained a formulation of PE-heptadecane and luciferase mRNA, lane 4 contained a formulation of PE-stearin and luciferase mRNA, lane 5 contained a formulation of PE-olein and luciferase mRNA, and lane 6 contained a formulation of PE-linolein and luciferase mRNA. Prior to loading, the samples were incubated with formaldehyde loading dye, denatured at 65°C for 10 minutes, and cooled to room temperature. The gel was run at 90V, and gel images were captured using a Syngene G Box Imaging System (Syngene, USA). For RNA detection, the gel was stained with ethidium bromide. Referring to FIG. 4, the lower band corresponds to small-sized free RNA (lane 1), and the upper band represents large-sized nanoparticles formed by binding the RNA to the dendrimer carrier.

[0165] Example 6. Material stability Polyester dendrimers constitute a unique class of materials due to their susceptibility to degradation due to the hydrolytic susceptibility of ester bonds. However, an ideal nucleic acid carrier should be stable not only at formulation pH but also at storage pH. Therefore, the stability of the dendrimer material was confirmed at different pHs. Figure 5 shows LCMS chromatograms of the dendrimer at neutral pH and pH 5. The chromatograms show a single peak (elution time 13.42 min) at both neutral pH and pH 5 at room temperature, indicating that the polyester dendrimer structure remained intact while formulating RNA at pH 5.

[0166] Example 7. Colloidal stability of nanoparticle compositions containing fatty acid-modified polyester dendrimers Nanoparticle stability was determined by comparing particle size distributions after 21 days of formulation and storage at 4°C with those measured immediately after 2 hours of dialysis. The same polyester dendrimer showed good stability in PBS at room temperature as well as at 37°C without significant aggregation. Figures 6A-6E show the stability of RNA-PE-stearin nanoparticles in PBS as measured by DLS and agarose gel electrophoresis. Figure 6A shows the distribution of nanoparticle composition after dialysis, measured as intensity based on nanoparticle size (d). Figure 6B shows the stability of PE-stearin PR8 HA mRNA as measured by DLS after 3 weeks of storage at 4°C. Figure 6C shows the stability of PE-stearin PR8 HA mRNA as measured by DLS after 3 days of storage at room temperature (RT). Figure 6D shows the stability of PE-stearin PR8 HA mRNA as measured by DLS after 2 hours of storage at 37°C. Figure 6E shows the stability results based on the gel retention assay: lane 1—PR8 HA mRNA; lane 2—PE-stearin PR8 HA mRNA, 4°C, 3 weeks; lane 3—PE-stearin PR8 HA mRNA, rt, 3 days; lane 4—PE-stearin PR8 HA mRNA, 37°C, 1 hour; lane 5—PE-stearin PR8 HA mRNA, 37°C, 2 hours. The particle size distributions were observed to be the same, indicating stability under these storage conditions.

[0167] Example 8. Cell-based luciferase mRNA expression analysis A luciferase cDNA expression cassette consisting of a 5' UTR, luciferase ORF, 3' UTR, and poly(A) tail was designed. This cassette was synthesized using GenScript and cloned into the pcDNA3.1 vector at the Nhel / Kpnl site downstream of the T7 promoter. The derived expression vector was used as a DNA template to synthesize luciferase mRNA using in vitro T7-based transcription (Hongene, #ON-040) and the vaccinia capping system (Hongene, #ON-028). After purification by lithium chloride precipitation, the luciferase mRNA was formulated with polyester dendrimers to form nanoparticles for further analysis. RAW264.7 (ATCC, TIB-71) and A549 (ATCC, CCL-185) cells obtained from ATCC were grown and maintained according to the protocol suggested by ATCC. To test the ability of the newly formulated nanoparticles to deliver mRNA to mammalian cells and induce protein expression, monolayers of RAW264.7 or A549 cells grown in 96-well plates were transfected with 50 ng of luciferase mRNA nanoparticles per well in 50 μl of PBS. After 1 h of incubation at 37 °C, 50 μl of growth medium was added per well. At the indicated time points posttransfection, cells were lysed, and luciferase activity was measured using a Luciferase One-Step Glow Assay Kit (ThermoFisher, #88263). Figures 7A and 7B show luciferase expression in cell culture from luciferase mRNA delivered to mammalian cells by modified polyester dendrimer-based nanoparticles and measured by quantification of intracellular luciferase activity using a luminescence assay. Figure 7A shows the cell culture expression of luciferase mRNA delivered by nanoparticles containing PE-linolenic acid, PE-stearic acid, and PE-palmitic acid compared to naked luciferase mRNA, and Figure 7B shows the cell culture expression of luciferase mRNA delivered by nanoparticles containing PE-heptadecane, PE-stearic acid, PE-oleic acid, and PE-16-hydroxypalmitic acid compared to naked luciferase mRNA.Referring to these figures, it was observed that all RNA nanoparticles were able to be taken up by RAW264.7 cells, leading to gene expression.

[0168] Example 9. Western blot analysis of HA expression An influenza HA expression cassette consisting of the full-length HA ORF (PR8 HA) flanked by 5' UTR and 3' UTR and followed by a poly(A) tail was synthesized and cloned into the pcDNA3.1 vector downstream of a T7 promoter. This HA expression vector was used as a DNA template for in vitro T7-based transcription (Hongene, #ON-040) and vaccinia capping system (Hongene, #ON-028) to synthesize HA mRNA. After purification by lithium chloride precipitation, the HA mRNA was formulated to form dendrimer-based nanoparticles. For Western blot analysis, A549 cells grown in 12-well plates were transfected with 2 μg of HA mRNA nanoparticles per well in 1 ml of PBS. After 1 hour of incubation at 37°C, 1 ml of growth medium was added to each well. 24 hours after transfection, cells were harvested and lysed in RIPA buffer (Biovision, #2114) according to the manufacturer's protocol. The cell lysate was mixed with Laemmli sample buffer, and the mixture was analyzed by SDS-PAGE (10% Bis-Tris Plus Gels, ThermoFisher, #NW00100BOX) and electroblotted onto a PVDF membrane (ThermoFisher, #IB24002). After blocking overnight with 5% nonfat dry milk and 0.1% Tween-20 in PBS, the membrane was incubated with influenza A virus H1N1 HA antibody (GeneTex, #GTX127357) diluted in the same buffer for 1 hour at room temperature. Figure 8 shows the Western blot analysis of HA expression. HA protein (indicated by an arrow in Figure 8) was visualized using a Chemi-XRS system (SynGene) with a sheep anti-rabbit IgG (H+L) secondary antibody conjugated to HRP (ThermoFisher, #A12172) and a chemiluminescence detection system (ProSignal® Femto ECL Reagent, Prometheus #20-302). Referring to Figure 8, it was observed that some of the RNA nanoparticles could be taken up by A549 cells, leading to gene expression.

[0169] Example 10. Hemagglutinin Inhibition Assay (HAI) HAI tests were performed on vaccinated animal sera according to the standard recommended WHO protocol [World Health Organization (2002). WHO manual on animal influenza diagnosis and surveillance 2002.5 Rev.1.2002]. Sera were treated with receptor-destroying enzyme (Denka Seiken Co., Ltd., Tokyo, Japan) overnight at 37°C and heat-inactivated at 56°C for 30 minutes. After cooling to room temperature, these serum samples were mixed with two volumes of a 25% suspension of turkey red blood cells (RBCs, Rockland Immunochemicals), incubated at room temperature for 2 hours, and then centrifuged to remove the RBCs. The treated sera, estimated at a 1:10 dilution, were serially diluted and incubated with 4 HA units of inactivated influenza A virus (PR / 8 / 34, Virusys #IAV210) at room temperature for 30 minutes. An equal volume of 0.5% turkey RBCs was added to each well and incubated for 30 minutes at room temperature. HAI titers were read as the highest dilution of serum that completely inhibited hemagglutination.

[0170] After the prime vaccination, a humoral immune response, as measured by HI titers, was observed in the HA mRNA group immunized with 5 μg of the nanoparticle vaccine formulated with PE-olein (Figure 9). At week 4, the mean HI titer in this group was 40, and one week after a booster immunization with the same vaccine dose, it continued to rise to a mean HI titer of 192 at week 5. This is in contrast to the group immunized with naked mRNA, whose HI titers remained at a baseline of 10 at all time points.

[0171] Example 11. Nanoparticle compositions containing DNA SEAP DNA was generated by cloning the SEAP sequence into the pcDNA3 plasmid. This plasmid contains the necessary origin of replication and ampicillin resistance genes required for maintenance and propagation in bacterial culture, a mammalian CMV promoter upstream of the gene cloning site to drive expression in mammalian cells in tissue culture, and a bacteriophage T7 transcription promoter downstream of the CMV promoter to transcribe mRNA encoding the cloned gene sequence in vitro, terminated with a BspQI restriction site. Plasmids were constructed from commercially available DNA fragments using the In-Fusion (Clontech Laboratories) cloning kit.

[0172] PE-linoleic-SEAP DNA nanoparticles were formulated by directly mixing 20 μl of an ethanol phase containing the modified dendrimer (PE-linoleic) combined with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-lipid, Avanti Polar Lipids) with 60 μl of ultrapure, DNAase / RNase-free, and endotoxin-free distilled water (Invitrogen) and sterile 100 mM (pH 5.0) QB citrate buffer (Teknova) to a final citrate concentration of 10 mM and a final DNA concentration of 0.38 mg / mL. The ethanol and citrate streams were mixed in a 1:3 ethanol:citric acid volume ratio to produce nanoparticles. The resulting nanoparticles had a mass ratio of 6:1:2 modified dendrimer:PEG-lipid:DNA. The formulations were diluted 1000-fold for analysis of particle size distribution, Z-average and induced count rate using a Zetasizer Nano ZS (Malvern Panalytical).

[0173] FIG. 10 shows the particle size distribution of nanoparticles produced by mixing PE-linoleic modified dendrimers with SEAP DNA.

[0174] [Table 1]

[0175] To test the ability of these nanoparticles to express SEAP in vitro, 293T cells were treated with the nanoparticles. Each well of a 96-well 293T dish was treated with 10 μL (approximately 0.25 μg) of each formulation diluted in a 1:1 mixture of Optimem:PBS to a final volume of 200 μL. Untreated wells received 200 μL of 50 / 50 PBS / OptiMEM. Twenty-four hours after treatment or transfection, conditioned medium was collected from each culture. The medium was analyzed using the QuantiBlue assay (InvivoGen). 180 μL of QuantiBlue reagent was combined with 50 μL of medium from each well. Readings were taken 40 minutes later by measuring absorbance at 650 nm, according to the manufacturer's protocol. For a no-treatment negative control, 50 μL of medium from a well not treated with nanoparticles was added to 180 μL of QuantiBlue reagent. These samples were treated identically to all other samples at all steps of the assay.

[0176] Figure 11 shows in vitro SEAP expression in nanoparticle formulations with modified dendrimers used to treat 293T cells. SEAP DNA formulated with PE-heptadecane, PE-olein, and PE-linoleic acid produced nanoparticles that conferred SEAP expression.

[0177] Example 12. Nanoparticle compositions containing fatty acid-modified polyester dendrons Nanoparticles containing PE dendrons modified to contain terminal fatty acid tails (e.g., PE dendron-G2-A1-ricinol):DSPC:cholesterol:DMG-PEG2k in a molar ratio of 1:0.5:2.4:0.035 were formulated using a NanoAssemblr Benchtop (Precision NanoSystems Inc., Vancouver, British Columbia, 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 12 mL per minute with a 3:1 ratio of aqueous to organic phase. The nanoparticles were dialyzed against sterile, endotoxin-free PBS using a 20,000 molecular weight cutoff dialysis solution, using glassware that had been washed with 1.0 M NaOH for 24 hours and sterilized in a steam autoclave or depyrogenated by heating at 250°C for 24 hours to remove endotoxins. The dialyzed nanoparticles were sterile filtered through a 0.2 micron poly(ethersulfone) filter and characterized using a Zetasizer NanoZS machine (Malvern). The size distribution was characterized by a single peak with a low polydispersity index, indicating a relatively monodisperse size. Using the Ribogreen® assay (Geall et al., 10.1073 / pnas.1209367109, incorporated by reference as if fully set forth herein), the encapsulation efficiency was measured to be 95% for the nanoparticle composition (formulated at pH 5) containing PE dendron_G2-A1-ricinol and SEAP replicon RNA.

[0178] To test the formulation of modified PE dendrons, we used the secreted embryonic alkaline phosphatase SEAP reporter system. For in vivo testing, mice were injected with nanoparticles at a dose of 5 μg of SEAP replicon RNA, and serum was collected from the mice 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 measured with a BioTek Synergy HTX microplate reader is reported in arbitrary units (AU). Error bars are ± SEM. Referring to Figure 12, we observed higher SEAP amounts in the pH 5.0 formulation compared to the pH 4.0 formulation, possibly due to faster release of RNA from the pH 5.0 formulation due to weaker binding.

[0179] Example 13. Western blot analysis of spike expression Using the method described in Example 12, BHK cells at approximately 80% confluency in 12-well dishes were treated with spiked replicon RNA formulated with polyester dendron and dendrimer molecules. The medium was removed, and the cells were treated with 5 μg of nanoparticles. The cells were incubated overnight at 37°C and harvested approximately 16 hours after treatment by scraping. The cell pellet was centrifuged at 13,000 rpm for 3 minutes, resuspended in 100 μL of RIPA (supplemented with HALT™ protease, phosphatase inhibitor cocktail, and Pierce™ Universal Nuclease), and then 25 μL of 6X SDS-Laemmli buffer was added. The samples were boiled for 10 minutes and then centrifuged to remove particulates. 20 μL of each sample was electrophoretically separated on a 10-well Bolt Bis-Tris SDS-PAGE gel. A sample from an unrelated experiment previously used to verify spike expression was included as a positive control. Proteins were transferred from the gel to a PVDF membrane using the iBlot2 dry transfer system. After transfer, the membrane was blocked with TBST + 10% milk for 30 minutes before adding rabbit anti-spike antibody at a dilution of 1:1000 in TBST + 10% milk. The membrane was incubated at room temperature for 45 minutes and then washed three times with TBST. Next, the membrane was incubated with sheep anti-rabbit HRP antibody at a dilution of 1:2000 in TBST + 10% milk for 30 minutes. The membrane was washed three times with TBST and then developed with Prometheus™ ProSignal™ Dura chemiluminescent substrate. The chemiluminescent reaction was visualized using a GeneSys imaging system. Referring to Figure 13, we observed that RNA nanoparticles formulated using PE dendron_G2-A1-ricinol were able to be taken up by BHK cells, leading to gene expression.

[0180] Example 14. COVID-19 Spike Trimer Direct Serum ELISA Using the method described in Example 12, BALB / c mice were vaccinated with 10 μg of spike replicon RNA formulated with PE dendron-G2-ricinol via bilateral IM injection into the leg muscles (total volume of 100 μL PBS). At 21 and 28 days post-injection, mice were bled, and serum was isolated from the whole blood by centrifugation of clotted samples at 10,000 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 containing the recombinant spike trimeric protein, pH 9.5. After blocking the wells with PBS + 1% BSA, serum was added to the wells starting at a 1:100 dilution and serially diluted 1:2 in PBS + 1% BSA up to 1:12,800. Samples were incubated for 1 hour at room temperature and washed three times with PBST. Afterward, goat anti-mouse IgG HRP was added at a dilution of 1:3000 in PBS + 1% BSA and incubated for 1 hour. Plates were washed again five times with PBST and developed using the chromogenic HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB). The reaction was stopped by adding H2SO4, and absorbance was measured at 450 nm and 570 nm. The endpoint titer was determined as the highest dilution yielding an absorbance at 450 nm minus an absorbance at 570 nm of ≥ 0.08. At week 3, the endpoint dilution titers in the group immunized with 10 μg of nanoparticle vaccine formulated with PE Dendron_G2-A1-ricinol (Figure 14) ranged from 512 to 1024 and continued to increase to a mean value of > 2048 at week 4. This is in contrast to the non-immunized seronegative group, whose titers remained at baseline 100 at all time points.

[0181] Example 15. RNA delivery to heart and spleen tissue Six BALB / c mice per experimental group were intravenously administered the presented nanoparticle formulation of replicon RNA encoding a luciferase reporter gene. For the DlinMC3DMA LNP control formulation, 31.4 μg of RNA was injected, and for the PE dendron G2-5A2-5 ricinol formulation, 7.2 μg of RNA was injected. Two mice were sacrificed at 6, 16, and 42 hours post-injection, and the hearts and spleens were removed and stored in liquid nitrogen until all time points were met. To quantify luciferase gene expression in isolated hearts and spleens, each organ was homogenized in 1 ml of PBS for 30 seconds using a VWR® Mini Bead Mill Homogenizer, and the crude homogenates were centrifuged at 16,000 RCF for 10 minutes at 4°C. After centrifugation, 150 μL of each supernatant (clarified homogenate) sample was placed into a white-walled 96-well assay plate, and 150 μL of Pierce™ Firefly Luc One-Step Glow Assay Reagent was added. The relative light units (RLU) from each well were measured using a BioTek Synergy HTX microplate reader. The background signal was defined as the RLU measured in organs from control mice of the same genotype and age that had not received RNA injections. Imaging data show high luciferase expression in the heart and spleen for intravenously injected nanoparticles (Figure 15).

[0182] Example 16. Nanoparticle composition containing a nucleic acid carrier of formula [I]b Dendritic-linear block copolymers are typically hybrids in which linear chains are end-functionalized with dendritic segments. Several groups have reported facile synthetic approaches for the delivery of hybrid structures containing two MPA dendritic moieties (PE) along with a linear polymer (P) in excellent yields. These PEG-based hybrids were successfully constructed via CuAAC click chemistry. For these materials, a primary azide intermediate containing a PEG end group was used in a convergent coupling reaction to a dendron containing a single complementary click group in the core. Representative structures of these hybrids are shown below. [ka] A is an amine linker, B is a hydrophobic unit, and PEG200 is a linker connecting two polyester dendrons.

[0183] An example of the synthesis of PE dendron G2 A1 (lysine)2 PEG 200 is as follows. [ka]

[0184] [ka]

[0185] [ka]

[0186] [ka]

[0187] Compound 6: The starting material, PE dendron G2 acetylene-OH (300 mg, 0.62 mmol) was dissolved in dry DCM (6 mL), pyridine (0.9 mL, 4.96 mmol) was added, followed by p-nitrophenyl chloroformate (2.1 g, 10 mmol) dissolved in dry DCM (15 mL), and the reaction mixture was stirred at 0°C to 23°C for 16 hours. The next day, TLC showed the formation of the product. The reaction mixture was diluted with 1.33 M NaHSO4 and extracted with EtOAc. The organic layer was washed with brine and evaporated in a Rotavap. The crude material was then purified by flash chromatography using DCM / EtOAc. This compound was purified by 45% EtOAc (R f =0.9, 1:9 EtOAc / DCM) to give the desired product as a pale yellow oil (553 mg, 70%).

[0188] Compound 7: A solution of compound 6 (438 mg, 0.41 mmol) dissolved in dry DCM (6 mL) was added to excess mono-Boc-DAPMA (0.45 mL, 1.64 mmol) dissolved in dry DCM (6 mL). A solution of DMAP (100 mg, 0.82 mmol) and DIPEA (0.29 mL, 1.64 mmol) in dry DCM (1 mL) was added, and the reaction mixture was stirred at 23 °C for 16 h under an argon atmosphere. TLC confirmed the reaction was complete. The crude product was concentrated under reduced pressure on a Rotavap and purified by flash chromatography on a silica column with a gradient elution from 100% CHCl (mobile phase a) to 75:22:3 CHCl / MeOH / NHOH aq (by volume, mobile phase b) over 40 min. The desired product was obtained by elution with 45% mobile phase b (R f =0.4, 1:1 mobile phase a / mobile phase b) to give the desired product as a pale yellow oil (404 mg, 66%). 1 H NMR (301MHz, chloroform-d) δ ppm 1.10-1.27(m,9H),1.33-1.43(m,36H),1.52-1.65(m,15H)2.08-2.16(m,12H),2.26-2.37(m,16H),2.51-2.54(m,1H),3.02-3 .20(m,15H),3.36-3.42(m,3H),4.01-4.26(m,10H),4.66-4.70(m,2H)5.24-5.27(m,3H),5.28-5.39(m,3H)5.88-5.97(m,3H).

[0189] Compound 8: PEG-200-azide (MW: 200, 11.4 mg, 57 μmol) was placed in a 50 mL RBF, followed by compound 7 (MW: 1490, 170 mg, 114 μmol) dissolved in THF (0.6 mL) along with CuSO4.5HO (3 mg, 11.4 μmol, 10 mol%, MW 249.69) and sodium ascorbate (4.5 mg, 22.8 μmol, 20 mol%, MW 198.11) in degassed THF:HO (2 mL, 1:1). The reaction mixture was stirred at 23 °C for 16 h. The next day, TLC confirmed completion of the reaction. The reaction mixture was purified by flash chromatography on a silica column using a gradient elution from 100% CHCl2 (mobile phase a) to 75:22:3 CHCl2 / MeOH / NH4OH aq (by volume, mobile phase b) over 40 min. The desired product was isolated using 76% mobile phase b (R f Elution with HCl (=0.65, 75:22:3 CH2Cl2 / MeOH / NH4OH aq) gave the desired product as a yellow oil (71 mg, 20%). MS (ESI) C 146 H 268 N 30 O 46 Calculated for [M+4H]4+ m / z 795.5, found 794.9, [M+3H]3+ m / z 1059.96, found 1060.2.

[0190] Compound 9: 70 mg of compound 8 (0.022 mmol) was dissolved in 3 ml of MeOH, and then treated with 20 equivalents of AcCl (0.03 ml, 0.44 mmol). The reaction was stirred at 23 °C for 5 hours, evaporated to dryness, dissolved in 2 ml of DMF, and 0.06 ml of EtN (0.44 mmol) was added, followed by the addition of 104 mg of ricinol-NHS (synthesized according to published procedures: Talukder et al., WO 2020 / 132196, incorporated by reference as fully set forth herein) dissolved in 2 ml of DMF. The reaction mixture was stirred at 23 °C for 24 hours and then purified by flash chromatography on a silica column using a gradient elution from 100% CHCl (mobile phase a) to 75:22:3 CHCl / MeOH / NHOH aq (by volume, mobile phase b) over 40 minutes. The desired product was obtained from 55% mobile phase b (R f Elution with HCl (=0.85, 70:24:6 CH2Cl2 / MeOH / NH4OH aq) gave the desired product as a yellow oil (60 mg, 43%). 1 H NMR (301MHz, chloroform-d) δ ppm 0.72-1.00(m,28H)1.03-1.22(m,26H),1.22-1.46(m,148H),1.51-1.59(m,13H),1.91-2.07(m,20H), 2.08-2.25(m,60H),2.30-2.46(m,33H),3.11-3.34(m,33H),3.45(s,3H),3.52-3.72(m,17H),3.87(br s,4H),3.98-4.32(m,25H),4.54(br s,3H),5.22(s,3H),5.33-5.58(m,16H),6.12-6.36(m,7H),6.86(br s,7H),7.83(s,2H).

[0191] Nanoparticles containing a nucleic acid carrier of Formula Ib (e.g., [PE dendron_G2-A1-ricinol]2PEG200):DSPC:cholesterol:DMG-PEG2k at a molar ratio of 1:0.5:2.4:0.035 were formulated using a NanoAssemblr Benchtop (Precision NanoSystems Inc, Vancouver, British Columbia, Canada). The 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 12 mL per minute with a 3:1 ratio of aqueous to organic phase. The nanoparticles were dialyzed against sterile, endotoxin-free PBS using a 20,000 molecular weight cutoff dialysis system, using glassware that had been cleaned with 1.0 M NaOH for 24 hours and sterilized by steam autoclaving or depyrogenated by heating at 250 °C for 24 hours for endotoxin removal. The dialyzed nanoparticles were sterile filtered through a 0.2 micron 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, indicating a relatively monodisperse size (Figure 16).

[0192] To test the formulation of PE dendron G2 A1 lysinol PEG 200, we used the secreted embryonic alkaline phosphatase SEAP reporter system. For in vivo testing, mice were injected with 2.5 μg of SEAP replicon RNA nanoparticles, and serum samples were collected from the mice 1, 3, 5, and 7 days later. The amount of SEAP was quantified using the Invitrogen NovaBright™ Phospha-Light™ EXP Assay kit for SEAP detection according to the manufacturer's protocol. The amount of SEAP in mouse serum samples measured with a BioTek Synergy HTX microplate reader is reported in arbitrary units (AU). Error bars are ± SEM. Referring to Figure 17, it was observed that the amount of SEAP was higher with dimeric PE dendron G2 A1 lysinol PEG 200 than with the monomeric PE dendron G2 A1 lysinol.

[0193] Example 17. Tracking nanoparticle composition To facilitate tracking of delivered materials in vitro and in vivo, modified dendrimers may be 13 C or 2 The modified dendrimers can have cores containing stable isotopes of carbon (C) or hydrogen (H), such as H. When the modified dendrimers are formulated into nanoparticles with nucleic acids, such as replicon RNA, the nanoparticles can be tracked in vitro and in vivo after administration by known techniques, such as mass spectrometry or nuclear magnetic resonance imaging. These stable isotopes are abundant and are found prominently in tissues. 12 C and 1 The inclusion of a stable isotope facilitates identification of the delivery molecule, as it is different from the H isotope. Tracking may be useful in identifying the biodistribution, material clearance, and molecular stability of nanoparticles after administration, and related issues.

[0194] References References cited throughout this application are incorporated herein for all purposes apparent in the specification and those references, as if each reference were fully set forth. For illustrative purposes, specific ones of these references are cited herein at specific locations. The citation of a reference at a specific location indicates the manner in which the teachings of the reference are incorporated. However, the citation of a reference at a specific location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.

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[0196] It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but is intended to include all modifications that are within the spirit and scope of the present invention as defined by the appended claims, the above specification, and / or as illustrated in the accompanying drawings.

Claims

1. A nucleic acid carrier having a structure represented by formula Ia or formula Ib, 【Chemistry 1】 wherein PE is a polyester dendrimer or dendron comprising a core and a plurality of monomeric polyester units forming one or more generations; the plurality of monomeric polyester units are 2,2-bis(hydroxymethyl)propionic acid or 2,2-bis(hydroxymethyl)butyric acid; A is an amine linker; The amine linker may be 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 N1-(3-aminopropyl)-N1-methylpropane-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) 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)butan-1,4-diamine, 3,3'-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(propane-1,3-diamine), and 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 and B is derived from a fatty acid or a derivative thereof; the fatty acid is selected from the group consisting of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, and eicosapentanoic acid; the fatty acid derivative is selected from the group consisting of 12-hydroxy-9-cis-octadecenoic acid, 12-methyltetradecanoic acid, 12-methyltridecanoic acid, 14-methylhexadecanoic acid, 14-methylhexadecanoic acid, 18-methylnonadecanoic acid, 19-methylarachidonic acid, isopalmitic acid, isostearic acid, phytanic acid, (±)-2-hydroxyoctanoic acid, (±)-3-hydroxydecanoic acid, (±)-3-hydroxyoctanoic acid, 10-hydroxydecanoic acid, 12-hydroxyoctadecanoic acid, 15-hydroxypentadecanoic acid, 16-hydroxyhexadecanoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxytetradecanoic acid, 2-hydroxydodecanoic acid, DL-α-hydroxystearic acid, DL-β-hydroxylauric acid, DL-β-hydroxymyristic acid and DL-β-hydroxypalmitic acid; P is a linker connecting two polyester dendrons; The nucleic acid carrier, wherein z is the number of surface groups.

2. PE has formula II: [(core) ｃ -Gn-O] II In the formula, c is the core multiplicity or the number of wedges derived from the core, and its value is independently in the range of 1 to 6; G is the layer or generation of the dendrimer or dendron; and n is the number of generations, and is in the range of 1 to 10.

3. z has formula III: z=cb ｎ ΙΙΙ In the formula, b is the branch point multiplicity or the number of branches at each branch point, c is the core multiplicity or the number of wedges derived from the core and is in the range of 1 to 6, and n is the number of generations and is in the range of 1 to 10.

4. The nucleic acid carrier according to claim 2, wherein c is 1 and the core is a unidirectional core.

5. The nucleic acid carrier according to claim 4 , wherein the unidirectional core is a carboxylic acid or a derivative thereof.

6. The core is 【Transformation 6】 is selected from the group consisting of In the formula, Y is methyl, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, neopentyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, azide (N3), halogen (Cl, Br or I), acetylene (C ２ H ２ 5. The nucleic acid carrier according to claim 4, wherein m is selected from the group consisting of hydroxyl (-OH), hydroxyl (-OH), thiol (-SH), -pyranosyl, cycloalkyl, aryl, heteroaryl and heterocycle, and m is 1 to 20.

7. The nucleic acid carrier according to claim 6, wherein the cycloalkyl, aryl, heteroaryl and heterocycle are substituted with at least one group selected from a halogen group, a hydroxyl group (—OH) and an alkyl group.

8. The nucleic acid carrier according to claim 2, wherein c is 3 and the core is a three-directional core.

9. The three-way core is trimethylolpropane or 1,1,1-tris(hydroxyphenylethane), and 【Transformation 7】 The nucleic acid carrier according to claim 8, having a structure represented by the formula:

10. The nucleic acid carrier according to claim 2, wherein c is 4 and the core is a four-directional core.

11. The four-way core may be composed of pentaerythritol, 5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphine, [1,1'-biphenyl]-3,3',5,5'-tetraol, 2,3,6,7-tetrahydroxy-9,10-dimethyl-anthracene, 9,10-dimethyl-9,10-dihydro-9,10-ethanoanthracene-2,3,6,7-tetraol, 6,13-dihydropentacene-5 ,7,12,14-tetraol, hexahydro-[1,4]dioxino[2,3-b][1,4]dioxin-2,3,6,7-tetraol, anthracene-1,4,9,10-tetraol, pyrene-1,3,6,8-tetraol and 3,3,3',3'-tetramethyl-2,2',3,3'-tetrahydro-1-1'-spirobi[indene]-5,5',6,6'-tetraol, 【Transformation 8】 【Chemistry 9】 The nucleic acid carrier according to claim 10, having a structure represented by the formula:

12. A is, 【Chemistry 10】 The nucleic acid carrier according to claim 1, having a structure represented by any one of the following:

13. The nucleic acid carrier of claim 1 , wherein the fatty acid contains one or more stable isotopes.

14. The nucleic acid carrier according to claim 13, wherein the stable isotope is a stable isotope of carbon or hydrogen.

15. The stable isotope of carbon is １３ The nucleic acid carrier according to claim 14, wherein the nucleic acid carrier is C.

16. The stable isotope of hydrogen is ２ The nucleic acid carrier according to claim 14, wherein the nucleic acid carrier is H.

17. The fatty acid containing the stable isotope is octanoic acid-1- １３ C, Octanoic acid-8- １３ C, Octanoic acid-8,8,8-d3, Octane- ２ H15 acid, decanoic acid-1- １３ C, Decanoic acid-10- １３ C, decane-10,10,10-d3 acid, decane-d19 acid, undecanoic acid-1- １３ C, lauric acid-12,12,12- ２ H3, laurin ２ H23 acid, lauric acid-1- １３ C, lauric acid-1,12- １３ C ２ , tridecane-2,2- ２ H2 acid, myristic acid-14- １３ C, myristic acid-1- １３ C, Myristic acid-14,14,14- ２ H3, myristin ２ H27 acid, 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, stearin ２ H35 acid, 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 acid and eicosane ２ 14. The nucleic acid carrier of claim 13, wherein the nucleic acid carrier is selected from the group consisting of H39 acids.

18. P is a homobifunctional linker having two azide groups and has the structure represented by formula IV: 【Chemistry 11】 The nucleic acid carrier according to claim 1, wherein m is a number in the range of 1 to 20.

19. A nanoparticle composition comprising a nucleic acid carrier according to any one of claims 1 to 18 and a therapeutic or immunogenic nucleic acid agent encapsulated in the nucleic acid carrier.

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

21. 20. The nanoparticle composition of claim 19, 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.

22. 20. The nanoparticle composition of claim 19, wherein the therapeutic or immunogenic nucleic acid agent comprises RNA or DNA capable of silencing, inhibiting or altering the activity of a gene.

23. 20. The nanoparticle composition of claim 19, further comprising a PEG-lipid.

24. 24. The nanoparticle composition of claim 23, wherein the PEG-lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-ethylene glycol)-2000] or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

25. The nanoparticle composition of claim 23, comprising the PEG-lipid in the range of 1 mol% to 10 mol% per nanoparticle composition.

26. 24. The nanoparticle composition of claim 23, further comprising a phospholipid and cholesterol or a derivative thereof.

27. 27. The nanoparticle composition of claim 26, wherein the phospholipid is dioleoylphosphatidylcholine (DOPC) or distearoylphosphatidylcholine (DSPC).

28. 27. The nanoparticle composition of claim 26, comprising the phospholipid in the range of 10 mol% to 15 mol% per nanoparticle composition.

29. The nanoparticle composition according to claim 26, wherein the cholesterol or a derivative thereof is contained in the nanoparticle composition in an amount ranging from 50 mol % to 75 mol %.

30. 20. The nanoparticle composition of claim 19 for treating or preventing a disease or condition in a subject.

31. 31. The nanoparticle composition of claim 30, comprising 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.

32. The nanoparticle composition of claim 30 , wherein the subject is a mammal.

33. 33. The nanoparticle composition of claim 32, wherein the mammal is selected from the group consisting of rodents, dogs, primates, horses, high value agricultural animals, and humans.

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