Ionizable amphiphilic janus dendrimers, delivery systems comprising the same, and methods of use thereof

Abstract

The invention relates to ionizable amphiphilic Janus dendrimers with different linker lengths. In some embodiments, the different linkers enable delivery of the agent to a specific target of interest (e.g., kidney, heart, lung, liver, lymph nodes, spleen, or any combination thereof). In some embodiments, the invention relates to nanoparticles comprising said ionizable amphiphilic Janus dendrimers. In some embodiments, the invention relates to compositions comprising said ionizable amphiphilic Janus dendrimers. In some aspects, the present invention also relates to methods of inducing an adaptive immune response in a subject comprising administering to the subject an effective amount of said nanoparticle or said composition.

Description

TITLE OF THE INVENTION IONIZABLE AMPHIPHILIC JANUS DENDRIMERS, DELIVERY SYSTEMS COMPRISING THE SAME, AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No.63 / 709,287, filed October 18, 2024, which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under 1720530 and 2104554 awarded by the National Science Foundation. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] Viral and synthetic vectors facilitated the success of Covid-19 vaccines (Whitehead, et al., Nat. Rev. Drug Discov.2009, 8, 129–138; Cullis, et al., Mol. Ther.2017, 25, 1467–1475; Buschmann, et al., Vaccines 2021, 9, 65; Wilson, Hum. Gene Ther.2014, 25, 257–261; Nguyen, et al., Acc. Chem. Res.2012, 45, 1152–1162; Lacroix, et al., ACS Nano 2021, 15, 3631–3645; Billingsley, et al., Nano Lett.2020, 20, 1578−1589; Huang, et al., Nat. Med.2022, 28, 2273–2287; Yin, et al., Nat. Rev. Genet.2014, 15, 541−555). This achievement transformed the four-component lipid nanoparticles (LNPs) into the leading nonviral vectors for mRNA delivery to liver (Yin, et al., Nat. Rev. Genet.2014, 15, 541−555).

[0004] These viral and synthetic vectors exhibit advantages and disadvantages. The stable viral vectors show high transfection efficacy (95%) (Luo, et al., Nat. Biotechnol.2000, 18, 33–37), and cell targeting (Wickham, et al., Nat. Med.2003, 9, 135–139). LNPs are less stable (Hajj, et al., Nat. Rev. Mater.2017, 2, 1–17) and less transfection efficient (1–2%) (Ramamoorth, et al., J. Clin. Diagn. Res. 2015, 9, GE01-06). However, viral vectors have drawbacks, including immunogenicity, cytotoxicity, difficult assembly, inflammatory responses to repeated administration, and insertional mutagenesis (Baum, et al., Hum. Gene. Ther.2006, 17, 253–263; Bessis, et al., Gene. Ther.2004, 11, S10–S17; Bouard, et al., Br. J. Pharmacol.2009, 157, 153–165).

[0005] Advantages of LNPs include higher biosafety, lower toxicity, and immunogenicity, with drawbacks such as the need for microfluidic or T-tube technology for assembly (Chen, et al., J. Am. Chem. Soc.2012, 134, 6948−6951; Zhigaltsev, et al., Langmuir 2012, 28, 3633−3640; Leung, et al., J. Phys. Chem. C 2012, 116, 18440−18450; Leung, et al., J. Phys. Chem. B 2015, 119, 8698−8706), along with low-temperature for storage (–70oC) for stability in time (Pardi, et al., Nat. Rev. Drug Discov.2018,17, 261–279). Some disadvantages of LNPs arise from the unknown distribution of their 4-components within the LNP structure (Jayaraman, et al., Angew. Chem. Int. Ed.2012, 51, 8529–8533; Kulkarni, et al., Acc. Chem. Res.2019, 52, 2435–2444; Ramezanpour, et al., Nanoscale 2019, 11, 14141–14146; Kulkarni, et al., ACS Nano.2018, 12, 4787–4795). At neutral pH, ionizable amines (IA) segregate as an oil phase in the core of the LNPs, limiting transfection efficacy to 1-2%. “PEG dilemma,” mediated by PEG-conjugated lipids is another drawback (Hatakeyama, et al., Adv. Drug Deliv. Rev.2011, 63, 152– 160; Sato, et al., J. Control. Release 2012, 163, 267–276; Choi, et al., Bioconjugate Chem.2003, 14, 420–429; Pelegri-O’Day, et al., J. Am. Chem. Soc.2014, 136, 14323−14332; Whitfield, et al., Chem. Rev.2021, 121, 11030–11084).

[0006] Permanently charged cationic lipids and dendrimers have pioneered DNA delivery but are toxic and unsuitable to deliver less stable mRNA, which requires encapsulation to protect from enzymatic degradation (Kukowska-Latallo, et al., Proc. Natl. Acad. Sci. U.S.A.1996, 93, 4897–4902; Zhou, et al., Chem. Commun.2006, No.22, 2362; Posadas, et al., Nanomed 2010, 5, 1219–1236; Rodrigo, et al., Biomacromolecules 2011, 12, 1205–1213; Majoral, et al., Chem. Rev.1999, 99, 845–880; Janiszewska, et al., PLoS ONE 2016, 11, e0165704; Haensler, J.; et al., Bioconjugate Chem.1993, 4, 372–379; Loup, et al., Chem. Eur. J 1999, 5, 3644–3650; Pedziwiatr, et al., Eur. J. Clin. Invest.2007, 37, 79–79; Zinselmeyer, et al., Pharm. Res.2002, 19, 960–967).

[0007] Thus, there is a need in the art for compositions and methods for the delivery of various cargos (e.g., mRNA) to a variety of specific targets. The present invention satisfies this unmet need. SUMMARY OF THE INVENTION

[0008] In one aspect, the present invention relates to an ionizable amphiphilic Janus dendrimer represented by Formula (I):Formula (I), or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or derivative thereof, wherein X is a hydrophilic group comprising at least one amine; Y1, Y2, and Y3each independently represent C1-C30-alkyl; and m is an integer having the value of 1, 2, 4, or 5.

[0009] In one aspect, the present invention relates to a composition comprising the ionizable amphiphilic Janus dendrimer represented by Formula (I) or a nanoparticle comprising the ionizable amphiphilic Janus dendrimer represented by Formula (I)

[0010] In one aspect, the present invention relates to a method of preventing or treating a disease or disorder in a subject in need thereof, wherein the method comprises administering to the subject at least one nanoparticle encapsulating an agent; wherein the nanoparticle comprises an ionizable amphiphilic Janus dendrimer represented by Formula (I):

[0011] In one aspect, the present invention relates to a method of inducing an immune response in a subject in need thereof, wherein the method comprises administering to the subject at least one nanoparticle encapsulating an agent; wherein the nanoparticle comprises an ionizable amphiphilic Janus dendrimer represented by Formula (I) BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0013] Figure 1 depicts a schematic representation of the synthesis of pentaerythritol (PE)-based ionizable amphiphilic Janus dendrimers (IAJDs) with different linkers interconnecting IAs to hydrophobic domains. Reaction conditions are: (i) RBr, NaOH, tetrabutylammonium bromide (TBAB), H2O, 80 °C, 5 h; (ii) SOCl2, DMF (cat.), dry CH2Cl2, 23 °C, 1 h, then 4-dimethylaminopyridine (DMAP), dry NEt3, dry CH2Cl2, 0-23 °C, 2 h; (iii) K2CO3, MeCN, 95 °C, 3 h.

[0014] Figure 2 depicts HPLC traces for IAJDs 313, 314, 93, and 288.

[0015] Figure 3 depicts HPLC traces for IAJDs 325, 326, 333, and 334.

[0016] Figure 4 depicts HPLC traces for IAJDs 317, 318, 249, and 312.

[0017] Figure 5 depicts HPLC traces for IAJDs 319, 320, 331, and 332.

[0018] Figure 6 depicts MALDI-TOF plots for IAJDs 313, 314, 93, and 288.

[0019] Figure 7 depicts MALDI-TOF plots for IAJDs 325, 326, 333, and 334.

[0020] Figure 8 depicts MALDI-TOF plots for IAJDs 317, 318, 249, and 312.

[0021] Figure 9 depicts MALDI-TOF plots for IAJDs 319, 320, 331, and 332.

[0022] Figure 10 depicts DLS data of DNPs assembled from IAJDs 313, 93, 325, 314, 288, and 326.

[0023] Figure 11 depicts DLS data of DNPs assembled from IAJDs 317, 249, 319, 318, 312, and 320.

[0024] Figure 12 depicts DLS data of DNPs assembled from IAJDs 331, 332, 333, and 334.

[0025] Figure 13 depicts titration curves showing changes in pH in response to addition of a strong acid, and calculated pKa, for IAJDs 93, 249, 288, and 312.

[0026] Figure 14 depicts titration curves showing changes in pH in response to addition of a strong acid, and calculated pKa, for IAJDs 313, 314, 317, and 318.

[0027] Figure 15 depicts titration curves showing changes in pH in response to addition of a strong acid, and calculated pKa, for IAJDs 319, 320, 325, and 326.

[0028] Figure 16 depicts titration curves showing changes in pH in response to addition of a strong acid, and calculated pKa, for IAJDs 331, 332, 333, and 334.

[0029] Figure 17 is a comparison of representative in-vivo images of luciferase activities of EH- based PE IAJDs 333, 313, 93, and 325 with two-, three-, four- and five-carbon linkers. The results marked with an asterisk (*) are from previously published data.

[0030] Figure 18 is a comparison of representative in-vivo images of luciferase activities of EH- based PE IAJDs 334, 314, 288, and 326 with two-, three-, four- and five-carbon linkers. The results marked with an asterisk (*) are from previously published data.

[0031] Figure 19 is a comparison of in-vivo images of luciferase activities of n-octyl-based pentaerythritol IAJDs 331, 317, 249, and 319 with two-, three-, four- and five-carbon linkers. The results marked with an asterisk (*) are from previously published data.

[0032] Figure 20 is a comparison of in-vivo images of luciferase activities of n-octyl-based pentaerythritol IAJDs 332, 318, 312, and 320 with two-, three-, four- and five-carbon linkers.

[0033] Figure 21 depicts molecular models for IAJDs 331 (2-Linker), 317 (3-Linker), and 249, showing the increase the rotational freedom and flexibility with the increase in linker lengths from 2 to 5.

[0034] Figure 22 depicts molecular models for IAJD 319 (5-Linker) further showing the increase the rotational freedom and flexibility with the increase in linker lengths from 2 to 5.

[0035] Figure 23 depicts ball-and-stick molecular models for IAJDs 331 (2-Linker), 317 (3- Linker), 249 (4-Linker), and 319 (5-Linker).

[0036] Figure 24 depicts space-filling molecular models for IAJDs 331 (2-Linker), 317 (3- Linker), 249 (4-Linker), and 319 (5-Linker).

[0037] Figure 25 is a comparison of in-vivo luciferase activity of pentaerythritol IAJDs with different linker length. Results marked with an asterisk (*) are from previous published data.

[0038] Figure 26 depicts structures of IAJDs containing two different ionizable amines attached to the pentaerythritol hydrophobic fragments through four different linkers. IAJD numbers and pKa corresponding to IAJDs are shown. DETAILED DESCRIPTION

[0039] The present invention is based, in part, on the unexpected results that nanoparticlescomprising at least one ionizable amphiphilic Janus dendrimer having the structure of Formula (I) with different linkers (e.g., different linker lengths, functional groups, etc.) effectively and efficiently delivered an agent to a target of interest. Thus, in one aspect, the present invention relates to an ionizable amphiphilic Janus dendrimer having the structure of Formula (I), or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or derivative thereof, comprising a specific linker of interest. Definitions

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

[0041] As used herein, each of the following terms has the meaning associated with it in this section.

[0042] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0043] “About” as used herein when referring to a measurable value, for example numerical values and / or ranges, such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. For example, “about 40 [units]” may mean within ± 25% of 40 (e.g., from 30 to 50), within ± 20%, ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1%, less than ± 1%, or any other value or range of values therein or therebelow. Furthermore, the phrases “less than about [a value]” or “greater than about [a value]” should be understood in view of the definition of the term “about” provided herein.

[0044] The term “compound,” as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein. In one embodiment, the term also refers to stereoisomers and / or optical isomers (including racemic mixtures) or enantiomerically enriched mixtures of disclosed compounds.

[0045] As used herein, the term “analog,” “analogue,” or “derivative” is meant to refer to a chemical compound or molecule made from a parent compound or molecule by one or more chemical reactions. As such, an analog can be a structure having a structure similar to that of the small molecule therapeutic agents described herein or can be based on a scaffold of a small molecule therapeutic agents described herein, but differing from it in respect to certain components or structural makeup, which may have a similar or opposite action metabolically. An analog or derivative can also be a small molecule that differs in structure from the reference molecule, but retains the essential properties of the reference molecule. An analog or derivative may change its interaction with certain other molecules relative to thereference molecule. An analog or derivative molecule may also include a salt, an adduct, tautomer, isomer, prodrug, or other variant of the reference molecule.

[0046] As used herein, the term “prodrug” refers to an agent that is converted into the parent drug in vivo. For example, the term “prodrug” refers to a derivative of a known direct acting drug, which derivative has enhanced delivery characteristics and therapeutic value as compared to the drug, and is transformed into the active drug by an enzymatic or chemical process. In some embodiments, “prodrug” refers to an inactive or relatively less active form of an active agent that becomes active by undergoing a chemical conversion through one or more metabolic processes. In one embodiment, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically, or therapeutically active form of the compound. In another embodiment, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically, or therapeutically active form of the compound. For example, the present compounds can be administered to a subject as a prodrug that includes an initiator bound to an active agent, and, by virtue of being degraded by a metabolic process, the active agent is released in its active form.

[0047] The term “tautomers” are constitutional isomers of organic compounds that readily interconvert by a chemical process (tautomerization).

[0048] The term “isomers” or “stereoisomers” refers to compounds, which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0049] As used herein, the term “alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e., C1-50means one to fifty carbon atoms) and including straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl.

[0050] As used herein, the term “substituted alkyl” means alkyl as defined above, substituted by one, two or three substituents selected from the group consisting of halogen, -OH, alkoxy, -NH2, amino, azido, -N(CH3)2, -C(=O)OH, trifluoromethyl, -C≡N, -C(=O)O(C1-C4)alkyl, -C(=O)NH2, -SO2NH2, - C(=NH)NH2, and -NO2. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

[0051] As used herein, the term “heteroalkyl” by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well asattached to the most distal carbon atom in the heteroalkyl group. Examples include -O-CH2-CH2-CH3, -CH2-CH2-CH2-OH, -CH2-CH2-NH-CH3, -CH2-S-CH2-CH3, and -CH2CH2-S(=O)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3, or -CH2-CH2-S-S-CH3.

[0052] The term “amino” refers to a group of the formula -NRaRa, -NHRa, or -NH2, where each Ra is, independently, an alkyl, alkenyl or alkynyl group as defined above containing 1 to 20 carbon atoms. Unless stated otherwise specifically in the specification, an alkylamino group can be optionally substituted.

[0053] The term “hydroxy” or “hydroxyl” refers to a group of the formula -OH group.

[0054] As used herein, the term “alkoxy” employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers.

[0055] As used herein, the term “halo”, “halide”, or “halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0056] “Haloalkyl” or “alkylhalide” refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2 trifluoroethyl, 1,2 difluoroethyl, 3 bromo 2 fluoropropyl, 1,2 dibromoethyl, and the like. Unless stated otherwise specifically in the specification, a haloalkyl group can be optionally substituted.

[0057] As used herein, the term “cycloalkyl” refers to a mono cyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. In one embodiment, the cycloalkyl group is saturated or partially unsaturated. In another embodiment, the cycloalkyl group is fused with an aromatic ring. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties:

[0058] Monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Dicyclic cycloalkyls include, but are not limited to, tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycyclic cycloalkyls include adamantine and norbornane. The term cycloalkyl includes “unsaturated nonaromatic carbocyclyl” or “nonaromatic unsaturated carbocyclyl” groups, both of which refer to a nonaromatic carbocycle as defined herein, which contains at least one carbon double bond or one carbon triple bond.

[0059] As used herein, the term “heterocycloalkyl” or “heterocyclyl” refers to a cyclic group containing one to four ring heteroatoms each selected from O, S, and N. In one embodiment, each heterocycloalkyl group has from 4 to 10 atoms in its ring system, with the proviso that the ring of said group does not contain two adjacent O atoms. In another embodiment, the heterocycloalkyl group is fused with an aromatic ring. In one embodiment, the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quaternized. The heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure. A heterocycle may be aromatic or non-aromatic in nature. In one embodiment, the heterocycle is a heteroaryl.

[0060] An example of a 3-membered heterocycloalkyl group includes, and is not limited to, aziridine. Examples of 4-membered heterocycloalkyl groups include, and are not limited to, azetidine and a beta lactam. Examples of 5-membered heterocycloalkyl groups include, and are not limited to, pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6-membered heterocycloalkyl groups include, and are not limited to, piperidine, morpholine and piperazine. Other non-limiting examples of heterocycloalkyl groups are:

[0061] Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, pyrazolidine, imidazoline, dioxolane,sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6- tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-1,3-dioxepin, and hexamethyleneoxide.

[0062] As used herein, the term “aromatic” refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e. having (4n + 2) delocalized π (pi) electrons, where n is an integer.

[0063] As used herein, the term “aryl,” employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings), wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples of aryl groups include phenyl, anthracyl, and naphthyl.

[0064] “Aralkyl” or “arylalkyl” refers to a radical of the formula -Rb-Rcwhere Rbis an alkylene group as defined above and Rcis one or more aryl radicals as defined above, for example, benzyl, diphenylmethyl and the like. Unless stated otherwise specifically in the specification, an aralkyl group can be optionally substituted. For example, as used herein, the term “aryl-(C1-C3)alkyl” means a functional group wherein a one- to three-carbon alkylene chain is attached to an aryl group, e.g., -CH2CH2- phenyl, – CH2-phenyl (benzyl), aryl-CH2- and aryl-CH(CH3)-. The term “substituted aryl-(C1-C3)alkyl” means an aryl-(C1-C3)alkyl functional group in which the aryl group is substituted. Similarly, the term “heteroaryl- (C1-C3)alkyl” means a functional group wherein a one to three carbon alkylene chain is attached to a heteroaryl group, e.g., -CH2CH2-pyridyl. The term “substituted heteroaryl-(C1-C3)alkyl” means a heteroaryl-(C1-C3)alkyl functional group in which the heteroaryl group is substituted.

[0065] As used herein, the term “heteroaryl” or “heteroaromatic” refers to a heterocycle having aromatic character. A polycyclic heteroaryl may include one or more rings that are partially saturated. Examples include the following moieties:

[0066] Examples of heteroaryl groups also include pyridyl, pyrazinyl, pyrimidinyl (particularly2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.

[0067] Examples of polycyclic heterocycles and heteroaryls include indolyl (particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (particularly 2-benzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.

[0068] As used herein, the term “substituted” means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group. The term “substituted” further refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In one embodiment, the substituents vary in number between one and four. In another embodiment, the substituents vary in number between one and three. In yet another embodiment, the substituents vary in number between one and two.

[0069] As used herein, the term “optionally substituted” means that the referenced group may be substituted or unsubstituted. In one embodiment, the referenced group is optionally substituted with zero substituents, i.e., the referenced group is unsubstituted. In another embodiment, the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from groups described herein.

[0070] In one embodiment, the substituents are independently selected from the group consisting of oxo, halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, alkyl (including straight chain, branched and / or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoro alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, - S-alkyl, S(=O)2alkyl, S(=O)2N[H, alkyl, or aryl], -C(=O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], -C(=O)N[H or substituted or unsubstituted alkyl or aryl]2, - OC(=O)N[substituted or unsubstituted alkyl]2, -NHC(=O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], -NHC(=O)alkyl, -N[substituted or unsubstituted alkyl]C(=O)[substituted or unsubstituted alkyl], -NHC(=O)[substituted or unsubstituted alkyl], - C(OH)[substituted or unsubstituted alkyl]2, and -C(NH2)[substituted or unsubstituted alkyl]2. In anotherembodiment, by way of example, an optional substituent is selected from oxo, fluorine, chlorine, bromine, iodine, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -CH3, -CH2CH3, -CH(CH3)2, -CF3, -CH2CF3, -OCH3, - OCH2CH3, -OCH(CH3)2, -OCF3, - OCH2CF3, -S(=O)2-CH3, -C(=O)NH2, -C(=O)-NHCH3, - NHC(=O)NHCH3, -C(=O)CH3, -ON(O)2, and -C(=O)OH. In yet one embodiment, the substituents are independently selected from the group consisting of C1-6alkyl, -OH, C1-6alkoxy, halo, amino, acetamido, oxo and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic.

[0071] Several references to integers and R, R1, R2, R3, R4, R5, R6, etc. are made in chemical structures and moieties disclosed and described herein. Any description of integers and R, R1, R2, R3, R4, R5, R6, etc. in the specification is applicable to any structure or moiety reciting integers and R, R1, R2, R3, R4, R5, R6, etc. respectively.

[0072] The term “nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm), which includes one or more amphiphilic Janus dendrimer of Formula (I). In some embodiments, nanoparticles are included in a formulation comprising a nucleoside-modified RNA as described herein. In some embodiments, such nanoparticles an ionizable hydrophilic group and a lipophilic (hydrophobic) group. In one embodiment, the nanoparticles further comprise one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids. In one embodiment, the nanoparticles do not comprise additional excipients. In one embodiment, the nanoparticles do not comprise any of additional lipids, additional cationic polymers, steroids, neutral lipids, charged lipids, or polymer conjugated lipids, besides the at least one compound of Formula (I). In some embodiments, the nucleoside-modified RNA is encapsulated in the lipid portion of the nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response.

[0073] The term “antibody,” as used herein, refers to an immunoglobulin molecule, which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0074] The term “antibody fragment” refers to a portion of an intact antibody and refers to theantigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0075] An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0076] An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. k and l light chains refer to the two major antibody light chain isotypes.

[0077] By the term “synthetic antibody” as used herein, is meant an antibody, which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art. The term should also be construed to mean an antibody, which has been generated by the synthesis of an RNA molecule encoding the antibody. The RNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the RNA has been obtained by transcribing DNA (synthetic or cloned) or other technology, which is available and well known in the art.

[0078] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an adaptive immune response. This immune response may involve either antibody production, or the activation of specific immunogenically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. A skilled artisan will understand that any DNA or RNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an adaptive immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0079] The term “adjuvant” as used herein is defined as any molecule to enhance an antigen-specific adaptive immune response.

[0080] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0081] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0082] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0083] “Homologous” as used herein, refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximumhomology.

[0084] “Immunogen” refers to any substance introduced into the body in order to generate an immune response. That substance can be a physical molecule, such as a protein, or can be encoded by a vector, such as DNA, mRNA, or a virus.

[0085] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0086] In the context of the invention, the following abbreviations for the commonly occurring nucleosides (nucleobase bound to ribose or deoxyribose sugar via N-glycosidic linkage) are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0087] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0088] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translation by translational machinery in a cell. For example, an mRNA where all of the uridines have been replaced with pseudouridine, 1-methyl psuedouridine, or another modified nucleoside.

[0089] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0090] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids arepolynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0091] In certain instances, the polynucleotide or nucleic acid of the invention is a “nucleoside- modified nucleic acid,” which refers to a nucleic acid comprising at least one modified nucleoside. A “modified nucleoside” refers to a nucleoside with a modification. For example, over one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

[0092] In certain embodiments, “pseudouridine” refers, in another embodiment, to m1acp3Ψ (1- methyl-3-(3-amino-3-carboxypropyl) pseudouridine. In another embodiment, the term refers to m1Ψ (1- methylpseudouridine). In another embodiment, the term refers to Ψm (2’-O-methylpseudouridine). In another embodiment, the term refers to m5D (5-methyldihydrouridine). In another embodiment, the term refers to m3Ψ (3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In another embodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the invention.

[0093] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0094] As used herein, the terms “amino acid”, “amino acidic monomer”, or “amino acid residue” refer to any of the twenty naturally occurring amino acids including synthetic amino acids with unnatural side chains and including both D and L optical isomers.

[0095] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologicallyactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0096] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. For example, the promoter that is recognized by bacteriophage RNA polymerase and is used to generate the mRNA by in vitro transcription.

[0097] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0098] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0099] The phrase “under transcriptional control” or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0100] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, or eradication of at least one sign or symptom of a disease or disorder state.

[0101] As used herein, the terms “therapeutic compound”, “therapeutic agent”, “drug”, “active pharmaceutical”, and “active pharmaceutical ingredient” are used interchangeably to refer to chemical entities that display certain pharmacological effects in a body and are administered for such purpose.Non-limiting examples of therapeutic agents include, but are not limited to, hydrophilic therapeutic agents, hydrophobic therapeutic agents, antibiotics, antibodies, small molecules, anti-cancer agents, chemotherapeutic agents, immunomodulatory agents, RNA molecules, siRNA molecules, DNA molecules, gene editing agents, gene-silencing agents, CRISPR-associated agents (e.g., guide RNA molecules, endonucleases, and variants thereof), analgesics, vaccines, anticonvulsants; anti-diabetic agents, antifungal agents, antineoplastic agents, anti-parkinsonian agents, anti-rheumatic agents, appetite suppressants, biological response modifiers, cardiovascular agents, central nervous system stimulants, contraceptive agents, dietary supplements, vitamins, minerals, lipids, saccharides, metals, amino acids (and precursors), nucleic acids and precursors, contrast agents, diagnostic agents, dopamine receptor agonists, erectile dysfunction agents, fertility agents, gastrointestinal agents, hormones, immunomodulators, antihypercalcemia agents, mast cell stabilizers, muscle relaxants, nutritional agents, ophthalmic agents, osteoporosis agents, psychotherapeutic agents, parasympathomimetic agents, parasympatholytic agents, respiratory agents, sedative hypnotic agents, skin and mucous membrane agents, smoking cessation agents, steroids, sympatholytic agents, urinary tract agents, uterine relaxants, vaginal agents, vasodilator, anti-hypertensive, hyperthyroids, anti-hyperthyroids, anti-asthmatics and vertigo agents. In certain embodiments, the one or more therapeutic agents are water-soluble, poorly water-soluble drug or a drug with a low, medium or high melting point. The therapeutic agents may be provided with or without a stabilizing salt or salts.

[0102] Some examples of active ingredients suitable for use in the pharmaceutical formulations and methods of the present invention include hydrophilic, lipophilic, amphiphilic or hydrophobic, and that can be solubilized, dispersed, or partially solubilized and dispersed, on or about the nanocluster. The active agent-nanocluster combination may be coated further to encapsulate the agent-nanocluster combination and may be directed to a target by functionalizing the nanocluster with, e.g., aptamers and / or antibodies. Alternatively, an active ingredient may also be provided separately from the solid pharmaceutical composition, such as for co-administration. Such active ingredients can be any compound or mixture of compounds having therapeutic or other value when administered to an animal, particularly to a mammal, such as drugs, nutrients, cosmeceuticals, nutraceuticals, diagnostic agents, nutritional agents, and the like. The active agents described herein may be found in their native state, however, they will generally be provided in the form of a salt. The active agents described herein include their isomers, analogs and derivatives.

[0103] An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[0104] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is beingsought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0105] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0106] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0107] In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0108] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0109] As used herein, “treating a disease or disorder” means reducing the frequency with which a symptom of the disease or disorder is experienced by a patient. Disease and disorder are used interchangeably herein.

[0110] A disease or disorder is “alleviated” if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

[0111] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0112] “Parenteral” administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

[0113] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges aswell as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Compounds

[0114] In one aspect, the present invention relates to an ionizable amphiphilic Janus dendrimer represented by Formula (I):or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or derivative thereof, wherein X is a hydrophilic group comprising at least one amine; Y1, Y2, and Y3each independently a lipophilic group; and m is an integer having the value of 1, 2, 4, or 5.

[0115] In one embodiment, m is 2. In very select embodiments, m has the value of 3. For example, the present invention relates in part to IAJD 312, in which m is 3, Y1, Y2, and Y3are each n- octyl, and X represents 2-(2-(piperazin-1-yl)ethoxy)ethan-1-ol.

[0116] In one embodiment, X comprises at least one tertiary amine. In one embodiment, X comprises at least two tertiary amines. In one embodiment, X is selected from the group consisting ofwherein dashed lines indicate the connection to Formula (I); each occurrence of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10is independently selected from the group consisting of hydrogen, deuterium, halide, alkyl, aryl, cycloalkyl, amine, heterocycloalkyl, carbonyl, and combinations thereof; wherein any two of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10optionally together form a ring; and each occurrence of Rx, Ry, and Rzis independently selected from the group consisting of hydrogen, deuterium, halide, alkyl, alkoxy, hydroxy, ester, ether, and combinations thereof.

[0117] In one embodiment, at least one of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, Rx, Ry, and Rzrepresents the following structure:wherein n is 0, 1, 2, or 3

[0118] In one embodiment, X is selected from the group consisting of:wherein dashed lines indicate the connection to Formula (I); and each occurrence of Rx, Ry, and Rzis independently selected from the group consisting of hydrogen, deuterium, halide, alkyl, alkoxy, hydroxy, ester, ether, and combinations thereof.

[0119] In one embodiment, Y1, Y2, and Y3each independently represent a C1-C30alkyl group. In one embodiment, each of Y1, Y2, and Y3independently represents a branched alkyl group or a linear alkyl group. In one embodiment, Y1, Y2, and Y3independently represent a linear C6-C16-alkyl chain or branched C6-C16-alkyl chain. In one embodiment, Y1, Y2, and Y3independently represent n-octyl or 2- ethylhexyl groups. In one embodiment, each of Y1, Y2, and Y3are the same. In one embodiment, Y1differs in structure from Y2and Y3. In one embodiment, Y1, Y2, and Y3each have a different structure.

[0120] In one embodiment, Y1, Y2, and Y3are each a lipophilic group further comprising at least one linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, poly(alkyl ether), heteroatom, or any combination thereof. In one embodiment, one or more of Y1, Y2, and Y3comprises alkylene, arylene, alkenylene, alkynylene, disulfide, ether, or any combination thereof.

[0121] In one embodiment, Y1, Y2, and Y3independently represent a C6alkyl chain, a C7alkyl chain, a C8alkyl chain, a C9alkyl chain, a C10alkyl chain, a C11alkyl chain, a C12alkyl chain, a C13alkyl chain, a C14alkyl chain, a C15alkyl chain, a C20alkyl chain, a C21alkyl chain, a C23alkyl chain, a C27alkyl chain, a C30alkyl chain, a C35alkyl chain, a C40alkyl chain, or a C50alkyl chain. In one embodiment, one or more of Y1, Y2, and Y3comprises a carbohydrate.

[0122] In one embodiment, the ionizable amphiphilic Janus dendrimer has one of the following structures:

[0123] In one embodiment, the ionizable amphiphilic Janus dendrimer has one of the following structures:.

[0124] In some embodiments, the ionizable amphiphilic Janus dendrimer has a pKa of between about 5.0 to about 7.0. For example, in some embodiments, the ionizable amphiphilic Janus dendrimer has a pKa of between about 5.50 to about 6.50.

[0125] In one aspect, the present invention relates to a nanoparticle comprising the ionizable amphiphilic Janus dendrimer of Formula (I). In one aspect, the present invention relates to a composition comprising the nanoparticle of claim 12, wherein the composition further comprises a second agent, a buffer, an adjuvant, a stabilizer, a solvent, or a polymer. In one embodiment, the nanoparticle further comprises an agent encapsulated within the nanoparticle; wherein the agent is selected from the group consisting of a diagnostic agent, detectable agent, therapeutic agent, nucleic acid molecule, or a combination thereof.

[0126] In one embodiment, the agent is selected from the group consisting of a linear mRNA, circular mRNA, DNA, RNA, cDNA, cRNA, CirRNA, mRNA, miRNA, siRNA, microRNA, CRISPR- Cas9, sgRNA, modified RNA, tRNA, antagomir, antisense molecule, nucleic acid molecule, targeted nucleic acid, small molecule, protein, antibody, peptide, protein, and any combination thereof. In one embodiment, the agent is a nucleic acid molecule encoding at least one selected from the group consisting of an antigen, antibody, gene editing molecule, chimeric antigen receptor (CAR), and any combination thereof.

[0127] In one aspect, the present invention relates to a method of delivering an agent to a subject in need thereof, wherein the method comprises administering to the subject the inventive nanoparticle. In one embodiment, the invention relates to a method of preventing or treating a disease or disorder in a subject in need thereof, wherein the method comprises administering to the subject at least one nanoparticle encapsulating an agent; wherein the nanoparticle comprises an ionizable amphiphilic Janus dendrimer represented by Formula (I). In one embodiment, the disease or disorder is selected from the group consisting of a viral infection, bacterial infection, fungal infection, parasitic infection, cancer,disease or disorder associated with cancer, autoimmune disease or disorder, and any combination thereof.

[0128] In one aspect, the present invention relates to a method of inducing an immune response in a subject in need thereof, wherein the method comprises administering to the subject at least one nanoparticle encapsulating an agent; wherein the nanoparticle comprises an ionizable amphiphilic Janus dendrimer represented by Formula (I). In one embodiment, the nanoparticle targets the mucosal surface of the subject, gastrointestinal tract of the subject, the stomach of the subject, the small intestine of the subject, the large intestine of the subject, the kidney of the subject, the heart of the subject, the liver of the subject, the spleen of the subject, the lungs of the subject, the lymph nodes of the subject, or any combination thereof. Compositions

[0129] In another aspect, the present invention relates to a nanoparticle comprising at least one ionizable amphiphilic Janus dendrimer of the present invention. In some embodiments, the nanoparticle further comprises at least one agent. In some embodiment, the nanoparticle further comprises at least one agent that is encapsulated by the ionizable amphiphilic Janus dendrimer of the present invention.

[0130] In another aspect, the present invention relates to a composition comprising at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle thereof. In some embodiments, the composition is a vaccine. In some embodiments, the composition has a pH of above about 4.0. For example, in one embodiment, the composition has a pH of about 7.4.

[0131] In various aspects, the present invention provides a one-component delivery system, two- component delivery system, three-component delivery system, or four-component delivery system comprising at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof.

[0132] In one aspect, the present invention relates to methods of delivering an agent to a target of interest (e.g., mucosal surface, gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, spleen, etc.) using at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof. In another aspect, the present invention relates to methods of delivering an agent to two or more targets of interest (e.g., a combination of mucosal surface, gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, spleen, etc.) using at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof. In some embodiments, the method comprises a one-component delivery system, two-component delivery system, three-component delivery system, four-component delivery system comprising at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof, or five-component deliverysystem comprising at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof.

[0133] In one aspect, the present invention relates to methods of preventing or treating a disease or disorder in a subject using at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof. In another aspect, the present invention relates to methods of inducing an adaptive immune response in a subject using at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof. In another aspect, the present invention relates to methods of administering at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof to a subject. Additional Compounds

[0134] In one aspect, the invention relates to amphiphilic Janus dendrimers of Formula (II): Formula (II), or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or derivative thereof. In various embodiments, the amphiphilic Janus dendrimer is an ionizable amphiphilic Janus dendrimer. In some embodiments, A is a polyvalent group comprising structure selected from:some embodiments, A is optionally substituted.

[0135] In some embodiments, dashed lines represent a binding site of X or Y.

[0136] In some embodiments, X is a hydrophilic group comprising at least one amine and atleast one linker. In some embodiments, the hydrophilic group is optionally substituted. In some embodiments, the linker is optionally substituted.

[0137] In some embodiments, X is a hydrophilic group comprising at least one tertiary amine and at least one linker. In some embodiments, X is a hydrophilic group comprising at least two tertiary amine and at least one linker. In some embodiments, the amine is tertiary amine. In some embodiments, the amine is optionally substituted.

[0138] In some embodiments, the linker comprises at least one C0-C20-alkyl chain, at least one electron withdrawing group, at least one electron donating group, or any combination thereof.

[0139] In some embodiments, the linker comprises at least one C0-C5-alkyl chain, at least one C1-C3-alkyl chain, or any combination thereof. In some embodiments, the linker comprises at least one C0-alkyl chain, C1-alkyl chain, C2-alkyl chain, C3-alkyl chain, C4-alkyl chain, C5-alkyl chain, C6-alkyl chain, C7-alkyl chain, C8-alkyl chain, C9-alkyl chain, C10-alkyl chain, C11-alkyl chain, C12-alkyl chain, C13-alkyl chain, C14-alkyl chain, C15-alkyl chain, C16-alkyl chain, C17-alkyl chain, C18-alkyl chain, C19- alkyl chain, C20-alkyl chain, or any combination thereof. For example, in one embodiment, the linker comprises at least one C0-alkyl chain. In another embodiment, the linker comprises at least one C1-alkyl chain. In another embodiment, the linker comprises at least one C2-alkyl chain. In another embodiment, the linker comprises at least one C3-alkyl chain. In another embodiment, the linker comprises at least one C4-alkyl chain. In another embodiment, the linker comprises at least one C5-alkyl chain.

[0140] In some embodiments, the linker comprises at least one C0-C5-alkyl chain, at least one C1-C3-alkyl chain, or any combination thereof to enable delivery of the agent to a target of interest (e.g., mucosal surface, gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, spleen, or any combination thereof). In some embodiments, the linker comprises at least one C0-alkyl chain, C1-alkyl chain, C2-alkyl chain, C3-alkyl chain, C4-alkyl chain, C5-alkyl chain, C6-alkyl chain, C7-alkyl chain, C8-alkyl chain, C9-alkyl chain, C10-alkyl chain, C11-alkyl chain, C12-alkyl chain, C13-alkyl chain, C14-alkyl chain, C15-alkyl chain, C16-alkyl chain, C17-alkyl chain, C18-alkyl chain, C19-alkyl chain, C20-alkyl chain, or any combination thereof to enable delivery of the agent to a target of interest (e.g., mucosal surface, gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, spleen, or any combination thereof).

[0141] In some embodiments, the linker comprises at least one electron withdrawing group. In some embodiments, the linker comprises at least one electron withdrawing group to enable delivery of the agent to a target of interest (e.g., mucosal surface, gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, spleen, or any combination thereof). Examples of such electron withdrawing group include any electron withdrawing group known in the art.

[0142] In some embodiments, the linker comprises at least one electron donating group. In someembodiments, the linker comprises at least one electron donating group to enable delivery of the agent to a target of interest (e.g., mucosal surface, gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, spleen, or any combination thereof). Examples of such electron donating group include any electron donating group known in the art.

[0143] In some embodiments, each occurrence of X is independently selected from:, or any combination thereof.

[0144] In some embodiments, each occurrence of u is independently an integer from 0 to 20. In some embodiments, each occurrence of u is independently an integer from 0 to 10. In some embodiments, each occurrence of u is independently an integer from 0 to 5. In some embodiments, each occurrence of u is independently an integer from 0 to 3. For example, in one embodiment, u is an integer of 0. In one embodiment, u is an integer of 1. In one embodiment, u is an integer of 2. In one embodiment, u is an integer of 3. In one embodiment, u is an integer of 4. In one embodiment, u is an integer of 5. In one embodiment, u is an integer of 6. In one embodiment, u is an integer of 7. In one embodiment, u is an integer of 8. In one embodiment, u is an integer of 9. In one embodiment, u is an integer of 10. In one embodiment, u is an integer of 11. In one embodiment, u is an integer of 12. In one embodiment, u is aninteger of 13. In one embodiment, u is an integer of 14. In one embodiment, u is an integer of 15. In one embodiment, u is an integer of 16. In one embodiment, u is an integer of 17. In one embodiment, u is an integer of 18. In one embodiment, u is an integer of 19. In one embodiment, u is an integer of 20.

[0145] In some embodiments, the dashed lines indicate the connection to A.

[0146] In one embodiment, Y is a lipophilic (hydrophobic) group comprising at least one C1-C50alkyl chain. In one embodiment, Y is a lipophilic (hydrophobic) group comprising at least one C1-C30alkyl chain. In one embodiment, Y is a lipophilic (hydrophobic) group comprising at least one C4-C30alkyl chain. For example, in one embodiment, Y is a lipophilic (hydrophobic) group comprising at least two C1-C30alkyl chains. In one embodiment, Y is a lipophilic (hydrophobic) group comprising at least two C1-C30alkyl chain having differing numbers of carbon atoms.

[0147] In some embodiments, the lipophilic group is optionally substituted. In some embodiments, the C1-C50-alkyl chain is optionally substituted. In some embodiments, Y is a lipophilic group comprising a linear C1-C50-alkyl chain, branched C1-C50-alkyl chain, or a combination thereof. For example, in some embodiments, Y is a lipophilic group comprising a linear C1-C30-alkyl chain, branched C1-C30-alkyl chain, or a combination thereof. In some embodiments, Y is a lipophilic group comprising at least two C1-C50-alkyl chains having same numbers of carbon atoms. For example, in some embodiments, Y is a lipophilic group comprising at least two C1-C30-alkyl chains having same numbers of carbon atoms. In some embodiments, Y is a lipophilic group comprising at least two C1-C50-alkyl chains having differing numbers of carbon atoms. For example, in some embodiments, Y is a lipophilic group comprising at least two C1-C30-alkyl chains having differing numbers of carbon atoms. In some embodiments, the ionizable amphiphilic Janus dendrimer comprises a first Y and a second Y, wherein the first Y comprises an alkyl chain having an even number of carbon atoms, and the second Y comprises an alkyl chain having an odd number of carbon atoms.

[0148] In one embodiment, Y is a lipophilic group further comprising at least one linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, poly(alkyl ether), heteroatom, or any combination thereof. In one embodiment, Y comprises alkylene, arylene, alkenylene, alkynylene, disulfide, ether, or any combination thereof. In one embodiment, Y does not comprise an amine. In one embodiment, Y is a lipophilic group comprising at least one C6alkyl chain, at least one C7alkyl chain, at least one C8alkyl chain, at least one C9alkyl chain, at least one C10alkyl chain, at least one C11alkyl chain, at least one C12alkyl chain, at least one C13alkyl chain, at least one C14alkyl chain, at least one C15alkyl chain, at least one C20alkyl chain, at least one C21alkyl chain, at least one C23alkyl chain, at least one C27alkyl chain, at least one C30alkyl chain, at least one C35alkyl chain, at least one C40alkyl chain,or at least one C50alkyl chain. In one embodiment, Y comprises at least one carbohydrate.

[0149] In some embodiments, each occurrence of Z is independently selected from C(R19)(R20), C=O, O, N(R19), or any combination thereof. For example, in one embodiment, Z is C(R19)(R20).

[0150] In some embodiments, each of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, and R20is independently hydrogen, deuterium, halide, hydroxy, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, alkyl halide, alkoxy, alkoxy halide, aryl, amine, carbonyl, ester, ether, or any combination thereof. In some embodiments, R1is selected from hydrogen, deuterium, halide, hydroxy, C1-C50-alkyl, C1-C50-alkyl halide, C1-C50-alkoxy, C1-C50-alkoxy halide, ester, ether, or any combination thereof. In some embodiments, each of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, and R20is independently hydrogen, deuterium, halide, hydroxy, C1-C30-alkyl, C1-C30-alkyl halide, C1-C30-alkoxy, C1-C30-alkoxy halide, or any combination thereof. In some embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, and R20are optionally substituted. For example, in some embodiments, the hydroxy, C1-C50-alkyl, C1-C50-alkyl halide, C1-C50-alkoxy, and / or C1-C50-alkoxy halide are optionally substituted.

[0151] In some embodiments, any two of R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18may together form a ring.

[0152] For example, in some embodiments, each occurrence of R1, R2, R3, R4, R5, R6, R7, and R8is hydrogen. Thus, in some embodiments, A is a polyvalent comprising the structure selected from:, any combination thereof.

[0153] In some embodiments, each occurrence of R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, and R20is hydrogen. Thus, in some embodiments, each occurrence of X is independently selected from:, or any combination thereof.

[0154] In some embodiments, each occurrence of u is independently an integer from 0 to 20. In some embodiments, each occurrence of u is independently an integer from 0 to 10. In some embodiments, each occurrence of u is independently an integer from 0 to 5. In some embodiments, each occurrence of u is independently an integer from 0 to 3. For example, in one embodiment, u is an integer of 0. In one embodiment, u is an integer of 1. In one embodiment, u is an integer of 2. In one embodiment, u is aninteger of 3. In one embodiment, u is an integer of 4. In one embodiment, u is an integer of 5. In one embodiment, u is an integer of 6. In one embodiment, u is an integer of 7. In one embodiment, u is an integer of 8. In one embodiment, u is an integer of 9. In one embodiment, u is an integer of 10. In one embodiment, u is an integer of 11. In one embodiment, u is an integer of 12. In one embodiment, u is an integer of 13. In one embodiment, u is an integer of 14. In one embodiment, u is an integer of 15. In one embodiment, u is an integer of 16. In one embodiment, u is an integer of 17. In one embodiment, u is an integer of 18. In one embodiment, u is an integer of 19. In one embodiment, u is an integer of 20.

[0155] In some embodiments, Rx, Ry, and Rzare independently hydrogen, deuterium, halide, hydroxy, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, alkyl halide, alkoxy, alkoxy halide, aryl, amine, carbonyl, ester, ether, or any combination thereof. In some embodiments, Rxis selected from hydrogen, deuterium, halide, hydroxy, C1-C50-alkyl, C1-C50-alkyl halide, C1-C50-alkoxy, C1-C50-alkoxy halide, ester, ether, or any combination thereof. In some embodiments, Rxis selected from hydrogen, deuterium, halide, hydroxy, C1-C30-alkyl, C1-C30-alkyl halide, C1-C30-alkoxy, C1-C30-alkoxy halide, or any combination thereof. In some embodiments, Rxis optionally substituted. For example, in some embodiments, the hydroxy, C1-C50-alkyl, C1-C50-alkyl halide, C1-C50-alkoxy, and / or C1-C50-alkoxy halide are optionally substituted.

[0156] In some embodiments, s is an integer from 0 to 4. In some embodiments, s is an integer from 1 to 4. In some embodiments, s is an integer from 2 to 4. For example, in one embodiment, s is an integer 4. In one embodiment, s is an integer 3. In one embodiment, s is an integer 2. In one embodiment, s is an integer 1. In one embodiment, s is an integer 0.

[0157] In some embodiments, t is an integer from 0 to 4. In some embodiments, t is an integer from 1 to 4. In some embodiments, t is an integer from 2 to 4. For example, in one embodiment, t is an integer 4. In one embodiment, t is an integer 3. In one embodiment, t is an integer 2. In one embodiment, t is an integer 1. In one embodiment, t is an integer 0.

[0158] In some embodiments, the sum of s and t is equal to the valency of A. For example, in one embodiment, the valency of A is 4, and the sum of s and t is 4. In one embodiment, s and t are each 2. In one embodiment, s is 1 and t is 3. In one embodiment, s is 3 and t is 1.

[0159] In some embodiments, the amphiphilic Janus dendrimer is an amphiphilic Janus dendrimer having a structure selected from at least one structure of Figure 6, or any combination thereof.

[0160] In some embodiments, the amphiphilic Janus dendrimer comprises an amphiphilic Janus dendrimer having a structure selected from:, or any combination thereof.

[0161] In some embodiments, the ionizable amphiphilic Janus dendrimer comprises at least one amine having a pKa of between about 5.0 to about 7.0. In some embodiments, the pKa is between about5.0 to about 6.5. In some embodiments, the pKa is between about 5.0 to about 6.05. In some embodiments, the pKa is between about 5.8 to about 6.05.

[0162] In some embodiments, the ionizable amphiphilic Janus dendrimer comprises a homochiral, racemic, or achiral branding points. Thus, in some embodiments, the ionizable amphiphilic Janus dendrimer is a homochiral ionizable amphiphilic Janus dendrimer, racemic ionizable amphiphilic Janus dendrimer, or achiral ionizable amphiphilic Janus dendrimer.

[0163] In some embodiments, the ionizable amphiphilic Janus dendrimer is a symmetric ionizable amphiphilic Janus dendrimer. In some embodiments, the ionizable amphiphilic Janus dendrimer is an asymmetric ionizable amphiphilic Janus dendrimer. In some embodiments, the ionizable amphiphilic Janus dendrimer is a nonsymmetric ionizable amphiphilic Janus dendrimer.

[0164] In some embodiments, the ionizable amphiphilic Janus dendrimer maintains stability and activity at between about -10 °C to about 30 °C, between about 0 °C to about 20 °C, or between about 0 °C to about 10 °C. For example, in one embodiment, the ionizable amphiphilic Janus dendrimer maintains stability and activity at about 4 °C, 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 27 °C, 28 °C, 29 °C, or 30 °C.

[0165] In some embodiments, amphiphilic Janus dendrimer having the structure of Formula (I) or Formula (II), or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or derivative thereof, comprising an amine (the ionizable amine) in X that is sufficient to provide the dual hydrophilic and binding role in the IAJD. For example, co-assembly of all IAJDs with Luc-mRNA may be performed by the injection of an ethanol solution of IAJD into an acetate buffer of pH = 4. In one embodiment, the acetate buffer may comprise mRNA added to the buffer from neutral water. Further details are found in the Example section below. The co-assembly forms a dendrimersome nanoparticle (DNP). Nanoparticles

[0166] In one aspect, the invention relates to nanoparticles comprising at least one amphiphilic Janus dendrimer of the present invention. In various embodiments, the nanoparticle is a one-component nanoparticle. In various embodiments, the nanoparticle is a two-component nanoparticle. In various embodiments, the nanoparticle is a three-component nanoparticle. In various embodiments, the nanoparticle is a four-component nanoparticle. In various embodiments, the nanoparticle is a five- component nanoparticle.

[0167] In some embodiments, the nanoparticle comprises a homochiral ionizable amphiphilic Janus dendrimer, achiral ionizable amphiphilic Janus dendrimer, or any combination thereof.

[0168] In some embodiments, the nanoparticle is a unilamellar nanoparticle. In someembodiments, the nanoparticle is a multilamellar nanoparticle. In some embodiments, the nanoparticle is a racemic ionizable amphiphilic Janus dendrimer. In some embodiments, the nanoparticle is a dendrimersome nanoparticle (DNP).

[0169] In some embodiments, the nanoparticle comprises at least two amphiphilic Janus dendrimers. Thus, in some embodiments, the nanoparticle comprises a first ionizable amphiphilic Janus dendrimer and a second ionizable amphiphilic Janus dendrimer. In some embodiments, the first ionizable amphiphilic Janus dendrimer has a different structure than the second ionizable amphiphilic Janus dendrimer.

[0170] In some embodiments, the nanoparticles further comprise at least one amphiphilic Janus dendrimer disclosed in Wang, et al., J. Am. Chem. Soc.2020, 142, 9525−9536; Xiao et al., J. Am. Chem. Soc.2016, 138, 12655–12663; Torre et al., Proc. Natl. Acad. Sci. U.S.A.2019, 116, 15378–15385; Percec et al., J. Am. Chem. Soc.2021, 143, 17724−17743; Wilson et al., J. Polym. Sci. Part A: Polymer Chemistry, 2010, 2498-2508; Xiao et al., Proc. Natl. Acad. Sci. U. S. A.2017, E7045−E7053; and U.S. Patent Application Publication No.2012277460; and U.S. Patent No.8,614,347; each of which is hereby incorporated by reference in their entireties.

[0171] In various embodiments, the nanoparticle has a mean diameter of from about 10 nm to about 100,000 nm, about 30 nm to about 1000 nm, about 30 nm to about 500 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 200 nm, 250 nm, 300 nm, 310 nm, 375 nm, 400 nm, 500 nm, 800 nm, 1000 nm, 1250 nm, 1400 nm, or 1500 nm. For example, in some embodiments, the nanoparticle has a mean diameter of from about 10 nm to about 1,000 nm. In some embodiments, the nanoparticle has a mean diameter of about 605 nm.

[0172] In various embodiments, the nanoparticle is substantially non-toxic. In various embodiments, the nanoparticle is biodegradable.

[0173] In one aspect of the invention, the nanoparticle comprises at least one cargo. In various aspects, the invention is not limited to any particular cargo or otherwise agent for which the nanoparticle is able to carry or transport. Rather, the invention includes any agent that can be carried by the nanoparticle. For example, agents that can be carried by the nanoparticle of the invention include, but are not limited to, diagnostic agents, detectable agents, and therapeutic agents. Thus, in various embodiments, the nanoparticle comprises at least one agent. In other embodiments, the nanoparticle encapsulates at leastone agent.

[0174] In some embodiments, the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 1 : 1 to about 10,000 : 1. In some embodiments, the nanoparticle comprises, or encapsulates, at least one agent. In some embodiments, the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 2 : 1 to about 1,000 : 1. In some embodiments, the nanoparticle comprises, or encapsulates, at least one agent. In some embodiments, the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 3 : 1 to about 10 : 1. In some embodiments, the nanoparticle comprises, or encapsulates, at least one agent. In some embodiments, the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 4 : 1 to about 10 : 1. In some embodiments, the nanoparticle comprises, or encapsulates, at least one agent. In some embodiments, the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 5 : 1 to about 10 : 1. In some embodiments, the nanoparticle comprises, or encapsulates, at least one agent. In some embodiments, the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 6 : 1 to about 10 : 1. In some embodiments, the nanoparticle comprises, or encapsulates, at least one agent. In some embodiments, the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 7 : 1 to about 10 : 1. In some embodiments, the nanoparticle comprises, or encapsulates, at least one agent. In some embodiments, the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 8 : 1 to about 10 : 1. In some embodiments, the nanoparticle comprises, or encapsulates, at least one agent. In some embodiments, the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 9 : 1 to about 10 : 1. In some embodiments, the nanoparticle comprises, or encapsulates, at least one agent. In some embodiments, the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 9.5 : 1 to about 10 : 1.

[0175] In some embodiments, the composition comprises between about 0.01 µg to about 1,000 µg of the agent. In some embodiments, the composition comprises between about 0.05 µg to about 1,000 µg of the agent. In some embodiments, the composition comprises between about 0.1 µg to about 1,000 µg of the agent. In some embodiments, the composition comprises between about 0.25 µg to about 1,000 µg of the agent. In some embodiments, the composition comprises between about 0.5 µg to about 1,000 µg of the agent. In some embodiments, the composition comprises between about 1 µg to about 1,000 µg of the agent. In some embodiments, the composition comprises between about 1 µg to about 900 µg of the agent. In some embodiments, the composition comprises between about 1 µg to about 800 µg of the agent. In some embodiments, the composition comprises between about 1 µg to about 700 µg of the agent. In some embodiments, the composition comprises between about 1 µg to about 600 µg of the agent. In some embodiments, the composition comprises between about 1 µg to about 500 µg of theagent. In some embodiments, the composition comprises between about 1 µg to about 250 µg of the agent. In some embodiments, the composition comprises between about 1 µg to about 200 µg of the agent. In some embodiments, the composition comprises between about 1 µg to about 100 µg of the agent. In some embodiments, the composition comprises between about 1 µg to about 50 µg of the agent. For example, in some embodiments, the composition comprises about 1.25 µg of the agent. In some embodiments, the composition comprises about 1.5 µg of the agent. In some embodiments, the composition comprises about 2.5 µg of the agent. In some embodiments, the composition comprises about 5 µg of the agent. In some embodiments, the composition comprises about 10 µg of the agent. In some embodiments, the composition comprises about 50 µg of the agent.

[0176] In various embodiments, the nanoparticle is suitable for delivering at least one cargo to a target of interest. Examples of such targets include, but are not limited to, mucosal surface, gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, spleen, and any combination thereof.

[0177] For example, in some embodiments, the nanoparticle is suitable for delivering at least one cargo to mucosal surface. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to gastrointestinal tract. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to stomach. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to small intestine. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to large intestine. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to kidney. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to heart. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to lung. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to liver. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to lymph nodes. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to spleen. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to mucosal surface, gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, and spleen.

[0178] In some embodiments, the nanoparticle is suitable for delivering at least one cargo to a cell of interest. Examples of such cells include, but are not limited to, epithelial cell, connective tissue cell, muscle cell, nerve cell, endothelial cell, parenchymal cell, blood cell, T cell, macrophages, neutrophils, dendritic cell (DC), monocytes, and any combination thereof.

[0179] For example, in some embodiments, the nanoparticle is suitable for delivering at least one cargo to at least one cell having mucosal surface. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to at least one gastrointestinal tract cell. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to at least one stomach cell. In someembodiments, the nanoparticle is suitable for delivering at least one cargo to at least one small intestine cell. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to at least one large intestine cell. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to at least one kidney cell. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to at least one heart cell. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to at least one lung cell. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to at least one liver cell. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to at least one lymph node cell. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to at least one spleen cell. In some embodiments, the nanoparticle is suitable for delivering at least one cargo to at least one cell having mucosal surface, at least one gastrointestinal tract cell, at least one stomach cell, at least one small intestine cell, at least one large intestine cell, at least one kidney cell, at least one heart cell, at least one lung cell, at least one liver cell, at least one lymph node cell, and at least one spleen cell.

[0180] In some embodiments, the cargo is any cargo known in the art. For example, in some embodiments, the cargo is at least one agent comprising a diagnostic agent, detectable agent, therapeutic agent, nucleic acid molecule, gene editing agent, vaccine, composition for protein replacement therapy, or any combination thereof. In some embodiments, the at least one agent is selected from a RNA, linear RNA, circular RNA, mRNA, linear mRNA, circular mRNA, siRNA, microRNA, CRISPR-Cas9, sgRNA, small molecule, protein, antibody, peptide, protein, or any combination thereof. In some embodiments, the at least one agent comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule encodes at least one selected from an antigen, antibody, gene editing molecule, chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the nucleic acid molecule is a DNA molecule or an RNA molecule. In some embodiments, the nucleic acid molecule is selected from cDNA, cRNA, CirRNA, RNA, linear RNA, circular RNA, mRNA, linear mRNA, circular mRNA, miRNA, siRNA, sgRNA, modified RNA, tRNA, antagomir, antisense molecule, targeted nucleic acid, or any combination thereof. In some embodiments, the modified RNA is a nucleoside-modified RNA. In some embodiments, the nucleoside-modified RNA comprises pseudouridine. In some embodiments, the nucleoside-modified RNA comprises pseudouridine plus 5-methyl-cytosine. In some embodiments, the nucleoside-modified RNA comprises 5-methyl-uridine. In some embodiments, the nucleoside-modified RNA comprises 1- methyl-pseudouridine.

[0181] Thus, in one embodiment, the nanoparticles may be used for the delivery of nucleoside- modified RNA to a subject in need thereof. In certain embodiments, delivery of a nucleoside-modified RNA to a subject comprises mixing the nucleoside-modified RNA with at least one dendrimer of Formula (I) or Formula (II), or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, orderivative thereof, prior to the step of contacting. In another embodiment, a method of invention further comprises administering nucleoside-modified RNA together with at least one dendrimer of Formula (I) or Formula (II), or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or derivative thereof.

[0182] In some embodiments, additional customizable targeting can be achieved based on the identity of the linking group A. Additionally, the identity of the linking group A affects the delivery of the nanoparticle cargo. This allows the nanoparticle to be tailored to facilitate delivery to the desired target organ. One example of which would be the delivery of anti-inflammatory drugs to the lungs.

[0183] In another embodiment, the transfection reagent forms a nanoparticle, which is a liposome. Liposomes, in another embodiment, increase intracellular stability, increase uptake efficiency and improve biological activity. In another embodiment, liposomes are hollow spherical vesicles composed of dendrimers arranged in a similar fashion as those lipids which make up the cell membrane. They have, in another embodiment, an internal aqueous space for entrapping water-soluble compounds and range in size from 0.05 to several microns in diameter. In another embodiment, nanoparticle liposomes can deliver RNA to cells in a biologically active form.

[0184] In various embodiments, the nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, the nucleoside-modified RNA, when present in the nanoparticles, is resistant in aqueous solution to degradation with a nuclease.

[0185] In some embodiments, the nanoparticle further comprises at least one second agent, buffer, adjuvant, stabilizer, solvent, polymer, or any combination thereof. Exemplary second agents include, but are not limited to, a diagnostic agent, detectable agent, therapeutic agent, nucleic acid molecule, or any combination thereof.

[0186] In some embodiments, the buffer, adjuvant, stabilizer, solvent, and polymer is any buffer, adjuvant, stabilizer, solvent, and polymer known in the art.

[0187] Exemplary buffers include, but are not limited to, acetate buffer, phosphate-buffered saline, sodium phosphate buffer, or any combination thereof.

[0188] In some embodiments, the buffer has a concentration between about 1 mM to about 100 mM. In some embodiments, the buffer has a concentration between about 1 mM to about 50 mM. In someembodiments, the buffer has a concentration between about 1 mM to about 20 mM. In some embodiments, the buffer has a concentration between about 5 mM to about 20 mM. In some embodiments, the buffer has a concentration between about 10 mM to about 15 mM. For example, in some embodiments, the buffer has a concentration of about 10 mM. In some embodiments, the buffer has a concentration of about 11 mM. In some embodiments, the buffer has a concentration of about 12 mM. In some embodiments, the buffer has a concentration of about 13 mM. In some embodiments, the buffer has a concentration of about 14 mM. In some embodiments, the buffer has a concentration of about 15 mM. In some embodiments, the buffer has a concentration of about 16 mM. In some embodiments, the buffer has a concentration of about 17 mM. In some embodiments, the buffer has a concentration of about 18 mM. In some embodiments, the buffer has a concentration of about 19 mM. In some embodiments, the buffer has a concentration of about 20 mM.

[0189] Exemplary polymers include, but are not limited to, PEG.

[0190] In some embodiments, the nanoparticle comprises about 0.01 mol% to about 50 mol% polymer. In some embodiments, the nanoparticle comprises about 0.05 mol% to about 50 mol% polymer. In some embodiments, the nanoparticle comprises about 0.25 mol% to about 50 mol% polymer. In some embodiments, the nanoparticle comprises about 0.5 mol% to about 50 mol% polymer. In some embodiments, the nanoparticle comprises about 0.01 mol% to about 40 mol% polymer. In some embodiments, the nanoparticle comprises about 0.01 mol% to about 30 mol% polymer. In some embodiments, the nanoparticle comprises about 0.01 mol% to about 20 mol% polymer. In some embodiments, the nanoparticle comprises about 0.01 mol% to about 10 mol% polymer. In some embodiments, the nanoparticle comprises about 0.01 mol% to about 5 mol% polymer. In some embodiments, the nanoparticle comprises about 0.25 mol% to about 10 mol% polymer. In some embodiments, the nanoparticle comprises about 0.5 mol% to about 10 mol% polymer. In some embodiments, the nanoparticle comprises about 1 mol% to about 10 mol% polymer. In some embodiments, the nanoparticle comprises about 1.5 mol% to about 10 mol% polymer. In some embodiments, the nanoparticle comprises about 2 mol% to about 10 mol% polymer. In some embodiments, the nanoparticle comprises about 4 mol% to about 10 mol% polymer. For example, in some embodiments, the nanoparticle comprises about 0.25 mol% to polymer. In some embodiments, the nanoparticle comprises about 0.5 mol% polymer. In some embodiments, the nanoparticle comprises about 1 mol% polymer. In some embodiments, the nanoparticle comprises about 1.5 mol% polymer. In some embodiments, the nanoparticle comprises about 2 mol% polymer. In some embodiments, the nanoparticle comprises about 3 mol% polymer. In some embodiments, the nanoparticle comprises about 4 mol% polymer. In some embodiments, the nanoparticle comprises about 5 mol% polymer.

[0191] In some embodiments, the nanoparticle comprises an N / P ratio of between about 1 toabout 100. In some embodiments, the nanoparticle comprises an N / P ratio of between about 1 to about 90. In some embodiments, the nanoparticle comprises an N / P ratio of between about 1 to about 80. In some embodiments, the nanoparticle comprises an N / P ratio of between about 1 to about 70. In some embodiments, the nanoparticle comprises an N / P ratio of between about 1 to about 60. In some embodiments, the nanoparticle comprises an N / P ratio of between about 1 to about 50. In some embodiments, the nanoparticle comprises an N / P ratio of between about 1 to about 40. In some embodiments, the nanoparticle comprises an N / P ratio of between about 5 to about 40. In some embodiments, the nanoparticle comprises an N / P ratio of between about 1 to about 30. In some embodiments, the nanoparticle comprises an N / P ratio of between about 1 to about 20. In some embodiments, the nanoparticle comprises an N / P ratio of between about 1 to about 10. For example, in some embodiments, the nanoparticle comprises an N / P ratio of about 5. In some embodiments, the nanoparticle comprises an N / P ratio of about 10. In some embodiments, the nanoparticle comprises an N / P ratio of about 20. In some embodiments, the nanoparticle comprises an N / P ratio of about 30. In some embodiments, the nanoparticle comprises an N / P ratio of about 40. Small Molecule Therapeutic Agents

[0192] In various embodiments, the agent is a therapeutic agent. In various embodiments, the therapeutic agent is a small molecule. When the therapeutic agent is a small molecule, a small molecule may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art. In one embodiment, a small molecule therapeutic agent comprises an organic molecule, inorganic molecule, biomolecule, synthetic molecule, and the like.

[0193] Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art, as are method of making the libraries. The method may use a variety of techniques well-known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development. In some embodiments of the invention, the therapeutic agent is synthesized and / or identified using combinatorial techniques.

[0194] In a general method for small library synthesis, an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles. The shape and rigidity of the core determine the orientation of the building blocks inshape space. The libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure (“focused libraries”) or synthesized with less structural bias using flexible cores. In some embodiments of the invention, the therapeutic agent is synthesized via small library synthesis.

[0195] The small molecule and small molecule compounds described herein may be present as salts even if salts are not depicted, and it is understood that the invention embraces all salts and solvates of the therapeutic agents depicted here, as well as the non-salt and non-solvate form of the therapeutic agents, as is well understood by the skilled artisan. In some embodiments, the salts of the therapeutic agents of the invention are pharmaceutically acceptable salts.

[0196] Where tautomeric forms may be present for any of the therapeutic agents described herein, each and every tautomeric form is intended to be included in the invention, even though only one or some of the tautomeric forms may be explicitly depicted. For example, when a 2-hydroxypyridyl moiety is depicted, the corresponding 2-pyridone tautomer is also intended.

[0197] The invention also includes any or all of the stereochemical forms, including any enantiomeric or diastereomeric forms of the therapeutic agents described. The recitation of the structure or name herein is intended to embrace all possible stereoisomers of therapeutic agents depicted. All forms of the therapeutic agents are also embraced by the invention, such as crystalline or non-crystalline forms of the therapeutic agent. Compositions comprising a therapeutic agent of the invention are also intended, such as a composition of substantially pure therapeutic agent, including a specific stereochemical form thereof, or a composition comprising mixtures of therapeutic agents of the invention in any ratio, including two or more stereochemical forms, such as in a racemic or non-racemic mixture.

[0198] The invention also includes any or all active analog or derivative, such as a prodrug, of any therapeutic agent described herein. In one embodiment, the therapeutic agent is a prodrug. In one embodiment, the small molecules described herein are candidates for derivatization. As such, in certain instances, the analogs of the small molecules described herein that have modulated potency, selectivity, and solubility are included herein and provide useful leads for drug discovery and drug development. Thus, in certain instances, during optimization new analogs are designed considering issues of drug delivery, metabolism, novelty, and safety.

[0199] In some instances, small molecule therapeutic agents described herein are derivatives or analogs of known therapeutic agents, as is well known in the art of combinatorial and medicinal chemistry. The analogs or derivatives can be prepared by adding and / or substituting functional groups at various locations. As such, the small molecules described herein can be converted into derivatives / analogs using well known chemical synthesis procedures. For example, all of the hydrogen atoms or substituents can be selectively modified to generate new analogs. Also, the linking atoms orgroups can be modified into longer or shorter linkers with carbon backbones or hetero atoms. Also, the ring groups can be changed so as to have a different number of atoms in the ring and / or to include hetero atoms. Moreover, aromatics can be converted to cyclic rings, and vice versa. For example, the rings may be from 5-7 atoms and may be carbocyclic or heterocyclic.

[0200] As used herein, the term “analog,” “analogue,” or “derivative” is meant to refer to a chemical compound or molecule made from a parent compound or molecule by one or more chemical reactions. As such, an analog can be a structure having a structure similar to that of the small molecule therapeutic agents described herein or can be based on a scaffold of a small molecule therapeutic agents described herein but differing from it in respect to certain components or structural makeup, which may have a similar or opposite action metabolically. An analog or derivative of any of a small molecule inhibitor in accordance with the invention can be used to treat a disease or disorder.

[0201] In one embodiment, the small molecule therapeutic agents described herein can independently be derivatized, or analogs prepared therefrom, by modifying hydrogen groups independently from each other into other substituents. That is, each atom on each molecule can be independently modified with respect to the other atoms on the same molecule. Any traditional modification for producing a derivative / analog can be used. For example, the atoms and substituents can be independently comprised of hydrogen, an alkyl, aliphatic, straight chain aliphatic, aliphatic having a chain hetero atom, branched aliphatic, substituted aliphatic, cyclic aliphatic, heterocyclic aliphatic having one or more hetero atoms, aromatic, heteroaromatic, polyaromatic, polyamino acids, peptides, polypeptides, combinations thereof, halogens, halo-substituted aliphatics, and the like. Additionally, any ring group on a compound can be derivatized to increase and / or decrease ring size as well as change the backbone atoms to carbon atoms or hetero atoms. Nucleic Acid Therapeutic Agents

[0202] In other related aspects, the therapeutic agent is an isolated nucleic acid. In certain embodiments, the isolated nucleic acid molecule is one of a DNA molecule or an RNA molecule. In certain embodiments, the isolated nucleic acid molecule is a cDNA, RNA, linear RNA, circular RNA, mRNA, linear mRNA, circular mRNA, siRNA, shRNA or miRNA molecule. In one embodiment, the isolated nucleic acid molecule encodes a therapeutic peptide such a thrombomodulin, endothelial protein C receptor (EPCR), anti-thrombotic proteins including plasminogen activators and their mutants, antioxidant proteins including catalase, superoxide dismutase (SOD) and iron-sequestering proteins. In some embodiments, the therapeutic agent is an siRNA, miRNA, shRNA, or an antisense molecule, which inhibits a targeted nucleic acid including those encoding proteins that are involved in aggravation of the pathological processes.

[0203] In one embodiment, the nucleic acid comprises a promoter / regulatory sequence such that the nucleic acid is capable of directing expression of the nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous nucleic acid into cells with concomitant expression of the exogenous nucleic acid in the cells such as those described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York) and as described elsewhere herein.

[0204] In one embodiment, siRNA is used to decrease the level of a targeted protein. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA- induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No.6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432:173-178) describe a chemical modification to siRNAs that aids in intravenous systemic delivery. Optimizing siRNAs involves consideration of overall G / C content, C / T content at the termini, Tm and the nucleotide content of the 3’ overhang. See, for instance, Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al., 2003, Cell 115:209-216. Therefore, the invention also includes methods of decreasing levels of PTPN22 using RNAi technology.

[0205] In one aspect, the invention includes a vector comprising an siRNA or an antisense polynucleotide. Preferably, the siRNA or antisense polynucleotide is capable of inhibiting the expression of a target polypeptide. The incorporation of a desired polynucleotide into a vector and the choice of vectors are well-known in the art as described in, for example, Sambrook et al. (2012), and in Ausubel et al. (1997), and elsewhere herein.

[0206] In certain embodiments, the expression vectors described herein encode a short hairpin RNA (shRNA) therapeutic agents. shRNA molecules are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certaininstances, the cell possesses native enzymes (e.g., dicer) that cleave the shRNA to form siRNA.

[0207] In order to assess the expression of the siRNA, shRNA, or antisense polynucleotide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification of expressing cells from the population of cells sought to be transfected or infected using a the delivery vehicle of the invention. In other embodiments, the selectable marker may be carried on a separate piece of DNA and also be contained within the delivery vehicle. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like.

[0208] Therefore, in one aspect, the delivery vehicle may contain a vector, comprising the nucleotide sequence or the construct to be delivered. The choice of the vector will depend on the host cell in which it is to be subsequently introduced. In a particular embodiment, the vector of the invention is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In specific embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Prokaryote- and / or eukaryote-vector based systems can be employed for use with the invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.

[0209] By way of illustration, the vector in which the nucleic acid sequence is introduced can be a plasmid, which is or is not integrated in the genome of a host cell when it is introduced in the cell. Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the invention or the gene construct of the invention can be inserted include a tet-on inducible vector for expression in eukaryote cells.

[0210] The vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2012). In a particular embodiment, the vector is a vector useful for transforming animal cells.

[0211] In one embodiment, the recombinant expression vectors may also contain nucleic acid molecules, which encode a peptide or peptidomimetic.

[0212] A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5’ non-coding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologousenhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0213] Naturally, it will be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2012). The promoters employed may be constitutive, tissue-specific, inducible, and / or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0214] The recombinant expression vectors may also contain a selectable marker gene, which facilitates the selection of host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin, which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.

[0215] Following the generation of the siRNA polynucleotide, a skilled artisan will understand that the siRNA polynucleotide will have certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, the siRNA polynucleotide may be further designed to resist degradation by modifying it to include phosphorothioate, or other linkages, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and the like (see, e.g., Agrawal et al., 1987, Tetrahedron Lett.28:3539-3542; Stec et al., 1985 Tetrahedron Lett.26:2191-2194; Moody et al., 1989 Nucleic Acids Res.12:4769-4782; Eckstein, 1989 Trends Biol. Sci.14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp.97-117 (1989)).

[0216] Any polynucleotide may be further modified to increase its stability in vivo. Possiblemodifications include, but are not limited to, the addition of flanking sequences at the 5’ and / or 3’ ends; the use of phosphorothioate or 2’ O-methyl rather than phosphodiester linkages in the backbone; and / or the inclusion of nontraditional bases such as inosine, queuosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0217] In one embodiment of the invention, an antisense nucleic acid sequence, which is expressed by a plasmid vector is used as a therapeutic agent to inhibit the expression of a target protein. The antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of the target protein.

[0218] Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule thereby inhibiting the translation of genes.

[0219] The use of antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal. Biochem.172:289). Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No.5,190,931.

[0220] Alternatively, antisense molecules of the invention may be made synthetically and then provided to the cell. Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, are preferred, since they are easily synthesized and introduced into a target cell. Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).

[0221] In one embodiment of the invention, a ribozyme is used as a therapeutic agent to inhibit expression of a target protein. Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure, which are complementary, for example, to the mRNA sequence encoding the target molecule. Ribozymes targeting the target molecule, may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them.

[0222] In one embodiment, the therapeutic agent may comprise one or more components of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding a target molecule, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene. In one embodiment, the therapeutic agent comprises a gRNA or a nucleic acid molecule encoding a gRNA. Inone embodiment, the therapeutic agent comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.

[0223] In one embodiment, the agent comprises a miRNA or a mimic of a miRNA. In one embodiment, the agent comprises a nucleic acid molecule that encodes a miRNA or mimic of a miRNA.

[0224] MiRNAs are small non-coding RNA molecules that are capable of causing post- transcriptional silencing of specific genes in cells by the inhibition of translation or through degradation of the targeted mRNA. A miRNA can be completely complementary or can have a region of noncomplementarity with a target nucleic acid, consequently resulting in a “bulge” at the region of non- complementarity. A miRNA can inhibit gene expression by repressing translation, such as when the miRNA is not completely complementary to the target nucleic acid, or by causing target RNA degradation, which is believed to occur only when the miRNA binds its target with perfect complementarity. The disclosure also can include double-stranded precursors of miRNA. A miRNA or pri-miRNA can be 18- 100 nucleotides in length, or from 18-80 nucleotides in length. Mature miRNAs can have a length of 19-30 nucleotides, or 21-25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides. MiRNA precursors typically have a length of about 70-100 nucleotides and have a hairpin conformation. miRNAs are generated in vivo from pre- miRNAs by the enzymes Dicer and Drosha, which specifically process long pre-miRNA into functional miRNA. The hairpin or mature microRNAs, or pri-microRNA agents featured in the disclosure can be synthesized in vivo by a cell-based system or in vitro by chemical synthesis.

[0225] In various embodiments, the agent comprises an oligonucleotide that comprises the nucleotide sequence of a disease-associated miRNA. In certain embodiments, the oligonucleotide comprises the nucleotide sequence of a disease-associated miRNA in a pre -microRNA, mature or hairpin form. In other embodiments, a combination of oligonucleotides comprising a sequence of one or more disease-associated miRNAs, any pre -miRNA, any fragment, or any combination thereof is envisioned.

[0226] MiRNAs can be synthesized to include a modification that imparts a desired characteristic. For example, the modification can improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell -type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism.

[0227] Modifications can also increase sequence specificity and consequently decrease off-site targeting. Methods of synthesis and chemical modifications are described in greater detail below. If desired, miRNA molecules may be modified to stabilize the miRNAs against degradation, to enhance half-life, or to otherwise improve efficacy. Desirable modifications are described, for example, in U.S. Patent Publication Nos.20070213292, 20060287260, 20060035254.20060008822. and 2005028824, each of which is hereby incorporated by reference in its entirety. For increased nuclease resistance and / orbinding affinity to the target, the single- stranded oligonucleotide agents featured in the disclosure can include 2’-O-methyl, 2’-fluorine, 2’-O-methoxyethyl, 2’-O-aminopropyl, 2’-amino, and / or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2’-4’-ethylene- bridged nucleic acids, and certain nucleotide modifications can also increase binding affinity to the target. The inclusion of pyranose sugars in the oligonucleotide backbone can also decrease endonucleolytic cleavage. An oligonucleotide can be further modified by including a 3’ cationic group, or by inverting the nucleoside at the 3’-terminus with a 3 -3’ linkage. In another alternative, the 3 ‘-terminus can be blocked with an aminoalkyl group. Other 3’ conjugates can inhibit 3’-5’ exonucleolytic cleavage. While not being bound by theory, a 3’ may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 3’ end of the oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3’-5’- exonucleases.

[0228] In one embodiment, the miRNA includes a 2’-modified oligonucleotide containing oligodeoxynucleotide gaps with some or all internucleotide linkages modified to phosphorothioates for nuclease resistance. The presence of methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA and thus reduces the IC5Q. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present disclosure may be used in conjunction with any technologies that may be developed to enhance the stability or efficacy of an inhibitory nucleic acid molecule.

[0229] miRNA molecules include nucleotide oligomers containing modified backbones or non- natural internucleoside linkages. Oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this disclosure, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered to be nucleotide oligomers. Nucleotide oligomers that have modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriest- ers, and boranophosphates. Various salts, mixed salts and free acid forms are also included.

[0230] A miRNA described herein, which may be in the mature or hairpin form, may be provided as a naked oligonucleotide. In some cases, it may be desirable to utilize a formulation that aids in the delivery of a miRNA or other nucleotide oligomer to cells (see, e.g., U.S. Pat. Nos.5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).

[0231] In some examples, the miRNA composition is at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the miRNA composition is in an aqueous phase, e.g., in a solution that includes water. The aqueous phase or the crystalline compositions can be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase), or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the miRNA composition is formulated in a manner that is compatible with the intended method of administration. A miRNA composition can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide agent, e.g., a protein that complexes with the oligonucleotide agent. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg), salts, and RNAse inhibitors (e.g., a broad specificity RNAse inhibitor). In one embodiment, the miRNA composition includes another miRNA, e.g., a second miRNA composition (e.g., a microRNA that is distinct from the first). Still other preparations can include at least three, five, ten, twenty, fifty, or a hundred or more different oligonucleotide species.

[0232] In certain embodiments, the composition comprises an oligonucleotide composition that mimics the activity of a miRNA. In certain embodiments, the composition comprises oligonucleotides having nucleobase identity to the nucleobase sequence of a miRNA and are thus designed to mimic the activity of the miRNA. In certain embodiments, the oligonucleotide composition that mimics miRNA activity comprises a double-stranded RNA molecule which mimics the mature miRNA hairpins or processed miRNA duplexes.

[0233] In one embodiment, the oligonucleotide shares identity with endogenous miRNA or miRNA precursor nucleobase sequences. An oligonucleotide selected for inclusion in a composition of the invention may be one of a number of lengths. Such an oligonucleotide can be from 7 to 100 linked nucleosides in length. For example, an oligonucleotide sharing nucleobase identity with a miRNA may be from 7 to 30 linked nucleosides in length. An oligonucleotide sharing identity with a miRNA precursor may be up to 100 linked nucleosides in length. In certain embodiments, an oligonucleotide comprises 7 to 30 linked nucleosides. In certain embodiments, an oligonucleotide comprises 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, or 30 linked nucleotides. In certain embodiments, an oligonucleotide comprises 19 to 23 linked nucleosides. In certain embodiments, an oligonucleotide is from 40 up to 50, 60, 70, 80, 90, or 100 linked nucleosides in length.

[0234] In certain embodiments, an oligonucleotide has a sequence that has a certain identity to a miRNA or a precursor thereof. Nucleobase sequences of mature miRNAs and their corresponding stem- loop sequences described herein are the sequences found in miRBase, an online searchable database of miRNA sequences and annotation. Entries in the miRBase Sequence database represent a predicted hairpin portion of a miRNA transcript (the stem-loop), with information on the location and sequence ofthe mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs) and may in some instances include the pre-miRNA and some flanking sequence from the presumed primary transcript. The miRNA nucleobase sequences described herein encompass any version of the miRNA, including the sequences described in Release 10.0 of the miRBase sequence database and sequences described in any earlier Release of the miRBase sequence database. A sequence database release may result in the re-naming of certain miRNAs. A sequence database release may result in a variation of a mature miRNA sequence. The compositions of the invention encompass oligomeric compound comprising oligonucleotides having a certain identity to any nucleobase sequence version of a miRNAs described herein.

[0235] In certain embodiments, an oligonucleotide has a nucleobase sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the miRNA over a region of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases. Accordingly, in certain embodiments the nucleobase sequence of an oligonucleotide may have one or more non-identical nucleobases with respect to the miRNA.

[0236] In certain embodiments, the composition comprises a nucleic acid molecule encoding a miRNA, precursor, mimic, or fragment thereof. For example, the composition may comprise a viral vector, plasmid, cosmid, or other expression vector suitable for expressing the miRNA, precursor, mimic, or fragment thereof in a desired mammalian cell or tissue. Nucleic Acids

[0237] In one embodiment, the invention includes a nanoparticle comprising or encapsulating one or more nucleic acid molecule. In one embodiment, the nucleic acid molecule is a nucleoside- modified mRNA molecule. In one embodiment, the nucleoside-modified mRNA molecule encodes an antigen. In one embodiment, the nucleoside-modified mRNA molecule encodes a plurality of antigens. In certain embodiments, the nucleoside-modified mRNA molecule encodes an antigen that induces an adaptive immune response against the antigen. In one embodiment, the invention includes a nucleoside- modified mRNA molecule encoding an adjuvant.

[0238] The nucleotide sequences encoding an antigen or adjuvant, as described herein, can alternatively comprise sequence variations with respect to the original nucleotide sequences, for example, substitutions, insertions and / or deletions of one or more nucleotides, with the condition that the resulting polynucleotide encodes a polypeptide according to the invention. Therefore, the scope of the invention includes nucleotide sequences that are substantially homologous to the nucleotide sequences recited herein and encode an antigen or adjuvant of interest.

[0239] As used herein, a nucleotide sequence is “substantially homologous” to any of thenucleotide sequences described herein when its nucleotide sequence has a degree of identity with respect to the nucleotide sequence of at least 60%, advantageously of at least 70%, preferably of at least 85%, and more preferably of at least 95%. A nucleotide sequence that is substantially homologous to a nucleotide sequence encoding an antigen can typically be isolated from a producer organism of the antigen based on the information contained in the nucleotide sequence by means of introducing conservative or non- conservative substitutions, for example. Other examples of possible modifications include the insertion of one or more nucleotides in the sequence, the addition of one or more nucleotides in any of the ends of the sequence, or the deletion of one or more nucleotides in any end or inside the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.

[0240] Further, the scope of the invention includes nucleotide sequences that encode amino acid sequences that are substantially homologous to the amino acid sequences recited herein and preserve the immunogenic function of the original amino acid sequence.

[0241] As used herein, an amino acid sequence is “substantially homologous” to any of the amino acid sequences described herein when its amino acid sequence has a degree of identity with respect to the amino acid sequence of at least 60%, advantageously of at least 70%, preferably of at least 85%, and more preferably of at least 95%. The identity between two amino acid sequences is preferably determined by using the BLASTN algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.20894, Altschul, S., et al., J. Mol. Biol.215: 403-410 (1990)).

[0242] In one embodiment, the invention relates to a construct, comprising a nucleotide sequence encoding an antigen. In one embodiment, the construct comprises a plurality of nucleotide sequences encoding a plurality of antigens. For example, in certain embodiments, the construct encodes 1 or more, 2 or more, 5 or more, 10 or more, 15 or more, or 20 or more antigens. In one embodiment, the invention relates to a construct, comprising a nucleotide sequence encoding an adjuvant. In one embodiment, the construct comprises a first nucleotide sequence encoding an antigen and a second nucleotide sequence encoding an adjuvant.

[0243] In one embodiment, the composition comprises a plurality of constructs, each construct encoding one or more antigens. In certain embodiments, the composition comprises 1 or more, 2 or more, 5 or more, 10 or more, 15 or more, or 20 or more constructs. In one embodiment, the composition comprises a first construct, comprising a nucleotide sequence encoding an antigen; and a second construct, comprising a nucleotide sequence encoding an adjuvant.

[0244] In another particular embodiment, the construct is operatively bound to a translational control element. The construct can incorporate an operatively bound regulatory sequence for the expression of the nucleotide sequence of the invention, thus forming an expression cassette.Vectors

[0245] The nucleic acid sequences encapsulated in the nanoparticle of the invention can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the nucleic acid molecule of interest can be produced synthetically.

[0246] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors and vectors optimized for in vitro transcription.

[0247] In one embodiment, the composition of the invention comprises in vitro transcribed (IVT) RNA encoding an antigen. In one embodiment, the composition of the invention comprises IVT RNA encoding a plurality of antigens. In one embodiment, the composition of the invention comprises IVT RNA encoding an adjuvant. In one embodiment, the composition of the invention comprises IVT RNA encoding one or more antigens and one or more adjuvants. Nucleoside-Modified RNA

[0248] In one embodiment, the nucleic acid molecule comprises a nucleoside-modified RNA. Nucleoside-modified mRNA have particular advantages over non-modified mRNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Nucleoside- modified mRNA useful in the invention is further described in U.S. Patent No.8,278,036, which is incorporated by reference herein in its entirety.

[0249] In certain embodiments, nucleoside-modified mRNA does not activate any pathophysiologic pathways, translates very efficiently and almost immediately following delivery, and serve as templates for continuous protein production in vivo lasting for several days (Karikó et al., 2008, Mol Ther 16:1833-1840; Karikó et al., 2012, Mol Ther 20:948-953). The amount of mRNA required to exert a physiological effect is small and that makes it applicable for human therapy. For example, as described herein, nucleoside-modified mRNA encoding an antigen has demonstrated the ability to induce CD4+ and CD8+ T-cell and antigen-specific antibody production. For example, in certain instances, antigen encoded by nucleoside-modified mRNA induces greater production of antigen-specific antibody production as compared to antigen encoded by non-modified mRNA.

[0250] In certain instances, expressing a protein by delivering the encoding mRNA has manybenefits over methods that use protein, plasmid DNA or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only substance delivered to cells, thus avoiding all the side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA- and viral-based vectors, the mRNA does not carry the risk of being incorporated into the genome and protein production starts immediately after mRNA delivery. For example, high levels of circulating proteins have been measured within 15 to 30 minutes of in vivo injection of the encoding mRNA. In certain embodiments, using mRNA rather than the protein also has many advantages. Half-lives of proteins in the circulation are often short, thus protein treatment would need frequent dosing, while mRNA provides a template for continuous protein production for several days. Purification of proteins is problematic and they can contain aggregates and other impurities that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0251] In certain embodiments, the nucleoside-modified RNA comprises the naturally occurring modified-nucleoside pseudouridine. In certain embodiments, inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (Karikó et al., 2008, Mol Ther 16:1833- 1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Karikó et al., 2011, Nucleic Acids Research 39:e142; Karikó et al., 2012, Mol Ther 20:948-953; Karikó et al., 2005, Immunity 23:165-175).

[0252] It has been demonstrated that the presence of modified nucleosides, including pseudouridines in RNA suppress their innate immunogenicity (Karikó et al., 2005, Immunity 23:165- 175). Further, protein-encoding, in vitro-transcribed RNA containing pseudouridine can be translated more efficiently than RNA containing no or other modified nucleosides (Karikó et al., 2008, Mol Ther 16:1833-1840). Subsequently, it is shown that the presence of pseudouridine improves the stability of RNA (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and abates both activation of PKR and inhibition of translation (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892). A preparative HPLC purification procedure has been established that was critical to obtain pseudouridine-containing RNA that has superior translational potential and no innate immunogenicity (Karikó et al., 2011, Nucleic Acids Research 39:e142). Administering HPLC-purified, pseudourine-containing RNA coding for erythropoietin into mice and macaques resulted in a significant increase of serum EPO levels (Karikó et al., 2012, Mol Ther 20:948-953), thus confirming that pseudouridine-containing mRNA is suitable for in vivo protein therapy.

[0253] The invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside. In certain embodiments, the composition comprises an isolated nucleic acid encoding an antigen, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside. In certain embodiments, the composition comprises a vector, comprising an isolatednucleic acid encoding an antigen, adjuvant, or combination thereof, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside.

[0254] In one embodiment, the nucleoside-modified RNA of the invention is IVT RNA. For example, in certain embodiments, the nucleoside-modified RNA is synthesized by T7 phage RNA polymerase. In another embodiment, the nucleoside-modified mRNA is synthesized by SP6 phage RNA polymerase. In another embodiment, the nucleoside-modified RNA is synthesized by T3 phage RNA polymerase.

[0255] In one embodiment, the modified nucleoside is m1acp3Ψ (1-methyl-3-(3-amino-3- carboxypropyl) pseudouridine. In another embodiment, the modified nucleoside is m1Ψ (1- methylpseudouridine). In another embodiment, the modified nucleoside is Ψm (2’-O- methylpseudouridine. In another embodiment, the modified nucleoside is m5D (5-methyldihydrouridine). In another embodiment, the modified nucleoside is m3Ψ (3-methylpseudouridine). In another embodiment, the modified nucleoside is a pseudouridine moiety that is not further modified. In another embodiment, the modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0256] In another embodiment, the nucleoside that is modified in the nucleoside-modified RNA the invention is uridine (U). In another embodiment, the modified nucleoside is cytidine (C). In another embodiment, the modified nucleoside is adenosine (A). In another embodiment the modified nucleoside is guanosine (G).

[0257] In another embodiment, the modified nucleoside of the invention is m5C (5- methylcytidine). In another embodiment, the modified nucleoside is m5U (5-methyluridine). In another embodiment, the modified nucleoside is m6A (N6-methyladenosine). In another embodiment, the modified nucleoside is s2U (2-thiouridine). In another embodiment, the modified nucleoside is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2’-O-methyluridine).

[0258] In other embodiments, the modified nucleoside is m1A (1-methyladenosine); m2A (2- methyladenosine); Am (2’-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6- isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine); io6A (N6-(cis- hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); ms2t6A (2-methylthio-N6- threonyl carbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6- hydroxynorvalylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2’-O-ribosyladenosine (phosphate)); I (inosine); m1I (1-methylinosine); m1Im (1,2’-O- dimethylinosine); m3C (3-methylcytidine); Cm (2’-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-formylcytidine); m5Cm (5,2’-O-dimethylcytidine); ac4Cm (N4-acetyl-2’-O- methylcytidine); k2C (lysidine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G (7- methylguanosine); Gm (2’-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2’-O- dimethylguanosine); m22Gm (N2,N2,2’-O-trimethylguanosine); Gr(p) (2’-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQ0(7-cyano-7- deazaguanosine); preQ1(7-aminomethyl-7-deazaguanosine); G+(archaeosine); D (dihydrouridine); m5Um (5,2’-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2’-O- methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5- methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (5-methoxycarbonylmethyl-2’-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5- methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5- methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5- carbamoylmethyl-2’-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cmnm5Um (5- carboxymethylaminomethyl-2’-O-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2- thiouridine); m62A (N6,N6-dimethyladenosine); Im (2’-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2’-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5- carboxymethyluridine); m6Am (N6,2’-O-dimethyladenosine); m62Am (N6,N6,O-2’-trimethyladenosine); m2,7G (N2,7-dimethylguanosine); m2,2,7G (N2,N2,7-trimethylguanosine); m3Um (3,2’-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2’-O-methylcytidine); m1Gm (1,2’-O- dimethylguanosine); m1Am (1,2’-O-dimethyladenosine); τm5U (5-taurinomethyluridine); τm5s2U (5- taurinomethyl-2-thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); or ac6A (N6- acetyladenosine).

[0259] In another embodiment, a nucleoside-modified RNA of the invention comprises a combination of 2 or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of 3 or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of more than 3 of the above modifications.

[0260] In another embodiment, between 0.1% and 100% of the residues in the nucleoside- modified of the invention are modified (e.g. either by the presence of pseudouridine or a modified nucleoside base). In another embodiment, 0.1% of the residues are modified. In another embodiment, the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In anotherembodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.

[0261] In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0262] In another embodiment, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In another embodiment, the fraction of the given nucleotide that is modified is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, thefraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.

[0263] In another embodiment, the fraction of the given nucleotide that is modified is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0264] In another embodiment, a nucleoside-modified RNA of the invention is translated in the cell more efficiently than an unmodified RNA molecule with the same sequence. In another embodiment, the nucleoside-modified RNA exhibits enhanced ability to be translated by a target cell. In another embodiment, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In another embodiment, translation is enhanced by a 3-fold factor. In another embodiment, translation is enhanced by a 5-fold factor. In another embodiment, translation is enhanced by a 7-fold factor. In another embodiment, translation is enhanced by a 10-fold factor. In another embodiment, translation is enhanced by a 15-fold factor. In another embodiment, translation is enhanced by a 20-fold factor. In another embodiment, translation is enhanced by a 50-fold factor. In another embodiment, translation is enhanced by a 100-fold factor. In another embodiment, translation is enhanced by a 200-fold factor. In another embodiment, translation is enhanced by a 500-fold factor. In another embodiment, translation is enhanced by a 1000-fold factor. In another embodiment, translation is enhanced by a 2000-fold factor. In another embodiment, the factor is 10-1000-fold. In another embodiment, the factor is 10-100-fold. In another embodiment, the factor is 10-200-fold. In another embodiment, the factor is 10-300-fold. In another embodiment, the factor is 10-500-fold. In another embodiment, the factor is 20-1000-fold. In another embodiment, the factor is 30-1000-fold. In another embodiment, the factor is 50-1000-fold. In another embodiment, the factor is 100-1000-fold. In another embodiment, the factor is 200-1000-fold. In another embodiment, translation is enhanced by any other significant amount or range of amounts.

[0265] In another embodiment, the nucleoside-modified antigen-encoding RNA of the invention induces significantly more adaptive immune response than an unmodified in vitro-synthesized RNA molecule with the same sequence. In another embodiment, the modified RNA molecule exhibits anadaptive immune response that is 2-fold greater than its unmodified counterpart. In another embodiment, the adaptive immune response is increased by a 3-fold factor. In another embodiment the adaptive immune response is increased by a 5-fold factor. In another embodiment, the adaptive immune response is increased by a 7-fold factor. In another embodiment, the adaptive immune response is increased by a 10-fold factor. In another embodiment, the adaptive immune response is increased by a 15-fold factor. In another embodiment the adaptive immune response is increased by a 20-fold factor. In another embodiment, the adaptive immune response is increased by a 50-fold factor. In another embodiment, the adaptive immune response is increased by a 100-fold factor. In another embodiment, the adaptive immune response is increased by a 200-fold factor. In another embodiment, the adaptive immune response is increased by a 500-fold factor. In another embodiment, the adaptive immune response is increased by a 1000-fold factor. In another embodiment, the adaptive immune response is increased by a 2000-fold factor. In another embodiment, the adaptive immune response is increased by another fold difference.

[0266] In another embodiment, “induces significantly more adaptive immune response” refers to a detectable increase in an adaptive immune response. In another embodiment, the term refers to a fold increase in the adaptive immune response (e.g., 1 of the fold increases enumerated above). In another embodiment, the term refers to an increase such that the nucleoside-modified RNA can be administered at a lower dose or frequency than an unmodified RNA molecule with the same species while still inducing an effective adaptive immune response. In another embodiment, the increase is such that the nucleoside- modified RNA can be administered using a single dose to induce an effective adaptive immune response.

[0267] In another embodiment, the nucleoside-modified RNA of the invention exhibits significantly less innate immunogenicity than an unmodified in vitro-synthesized RNA molecule with the same sequence. In another embodiment, the modified RNA molecule exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In another embodiment, innate immunogenicity is reduced by a 3-fold factor. In another embodiment, innate immunogenicity is reduced by a 5-fold factor. In another embodiment, innate immunogenicity is reduced by a 7-fold factor. In another embodiment, innate immunogenicity is reduced by a 10-fold factor. In another embodiment, innate immunogenicity is reduced by a 15-fold factor. In another embodiment, innate immunogenicity is reduced by a 20-fold factor. In another embodiment, innate immunogenicity is reduced by a 50-fold factor. In another embodiment, innate immunogenicity is reduced by a 100-fold factor. In another embodiment, innate immunogenicity is reduced by a 200-fold factor. In another embodiment, innate immunogenicity is reduced by a 500-fold factor. In another embodiment, innate immunogenicity is reduced by a 1000-fold factor. In another embodiment, innate immunogenicity is reduced by a 2000-fold factor. In another embodiment, innate immunogenicity is reduced by another fold difference.

[0268] In another embodiment, “exhibits significantly less innate immunogenicity” refers to adetectable decrease in innate immunogenicity. In another embodiment, the term refers to a fold decrease in innate immunogenicity (e.g., 1 of the fold decreases enumerated above). In another embodiment, the term refers to a decrease such that an effective amount of the nucleoside-modified RNA can be administered without triggering a detectable innate immune response. In another embodiment, the term refers to a decrease such that the nucleoside-modified RNA can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the recombinant protein. In another embodiment, the decrease is such that the nucleoside-modified RNA can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the recombinant protein.

[0269]

[0270] Polypeptide Therapeutic Agents

[0271] In other related aspects, the therapeutic agent includes an isolated peptide that modulates a target. For example, in one embodiment, the peptide of the invention inhibits or activates a target directly by binding to the target thereby modulating the normal functional activity of the target. In one embodiment, the peptide of the invention modulates the target by competing with endogenous proteins. In one embodiment, the peptide of the invention modulates the activity of the target by acting as a transdominant negative mutant.

[0272] The variants of the polypeptide therapeutic agents may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of the invention, (iv) fragments of the polypeptides and / or (v) one in which the polypeptide is fused with another polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.

[0273] In various embodiments, the nanoparticle is a two-component nanoparticle, three- component nanoparticle, or four-component nanoparticle. Thus, the nanoparticles may further comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least threeclasses: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids. In one embodiment, the nanoparticles do not further comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. In one embodiment, the nanoparticles do not further comprise any or all of a simple lipid, a compound lipid, or a derived lipid.

[0274] In one embodiment, the nanoparticle comprises a cationic lipid. As used herein, the term “cationic lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease. In one embodiment, the nanoparticle does not comprise a cationic lipid.

[0275] In certain embodiments, the cationic lipid which is optionally present or not present in the nanoparticles comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N- dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)- N,N,N-trimethylammonium chloride (DOTAP); 3-(N—(N′,N′-dimethylaminoethane)- carbamoyl)cholesterol (DC-Chol), N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)- N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). Additionally, a number of commercial preparations of cationic lipids are available which can be used in the invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1- (2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2- dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0276] In one embodiment, the cationic lipid is an amino lipid. Such amino lipids include those described in WO 2012 / 016184, incorporated herein by reference in its entirety. Representative aminolipids include, but are not limited to, 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin- TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N- dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0277] In various embodiments, the nanoparticles further comprise a steroid or steroid analogue. A “steroid” is a compound comprising the following carbon skeleton:.

[0278] In certain embodiments, the steroid or steroid analogue is cholesterol. In one embodiment, the nanoparticles do not comprise a steroid or steroid analogue. In one embodiment, the nanoparticles do not comprise cholesterol.

[0279] In one embodiment, the nanoparticles further comprise a stabilizer. In one embodiment, the stabilizer comprises oligooxyetylenes. In one embodiment, the stabilizer comprises a water soluble macromolecule. In one embodiment the stabilizer comprises a water soluble oligomer. In one embodiment, the stabilizer comprises a carbohydrate.

[0280] In certain embodiments, the nanoparticle comprises one or more targeting moieties which are capable of targeting the nanoparticle to a cell or cell population. For example, in one embodiment, the targeting moiety is a ligand which directs the nanoparticle to a receptor found on a cell surface.

[0281] In certain embodiments, the nanoparticle comprises one or more internalization domains. For example, in one embodiment, the nanoparticle comprises one or more domains which bind to a cell to induce the internalization of the nanoparticle. For example, in one embodiment, the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the nanoparticle. In certain embodiments, the nanoparticle is capable of binding a biomolecule in vivo, where the nanoparticle-bound biomolecule can then be recognized by a cell-surface receptor to induce internalization. For example, in one embodiment, the nanoparticle binds systemic ApoE, which leads to the uptake of the nanoparticle and associated cargo. Compositions

[0282] In one aspect, the present invention relates to compositions comprising at least one ionizable amphiphilic Janus dendrimer of the present invention and / or nanoparticle thereof. In some embodiments, the composition further comprises at least one agent described herein.

[0283] The invention also relates to compositions comprising at least one compound of Formula (I) or Formula (II), or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or derivative thereof, and methods of use thereof for delivering an encapsulated agent to a site of interest. Exemplary agents that can be encapsulated in the compositions of the invention include, but are not limited to, diagnostic agents, detectable agents, and therapeutic agents.

[0284] In one embodiment, the composition comprises nanoparticles comprising a compound of Formula (I) or Formula (II), or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or derivative thereof, and at least one agent encapsulated by the nanoparticle. In some embodiments, the encapsulated agent comprises an agent for inducing an immune response in a subject. In certain embodiments, the invention provides a composition comprising a nanoparticle encapsulating a nucleic acid molecule encoding an agent for inducing an immune response in a subject. For example, in certain embodiments, the composition comprises a vaccine comprising a nucleic acid molecule encoding an antigen.

[0285] In some embodiments, the composition comprises at least one amine has a pKa of between about 5.0 to about 7.0. In some embodiments, the composition comprises at least one amine has a pKa of between about 5.0 to about 6.5. In some embodiments, the composition comprises at least one amine has a pKa of between about 5.0 to about 6.05. In some embodiments, the composition comprises at least one amine has a pKa of between about 5.8 to about 6.05.

[0286] In some embodiments, the composition comprises at least one amine has a pKa of between about 5.0 to about 7.0 to enable delivery of the agent to a target of interest (e.g., mucosal surface, gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, spleen, or any combination thereof). In some embodiments, the composition comprises at least one amine has a pKa of between about 5.0 to about 6.5 to enable delivery of the agent to a target of interest (e.g., mucosal surface, gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, spleen, or any combination thereof). In some embodiments, the composition comprises at least one amine has a pKa of between about 5.0 to about 6.05 to enable delivery of the agent to a target of interest (e.g., mucosal surface, gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, spleen, or any combination thereof). In some embodiments, the composition comprises at least one amine has a pKa of between about 5.8 to about 6.05 to enable delivery of the agent to a target of interest (e.g., mucosal surface, gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, spleen,or any combination thereof).

[0287] In some embodiments, the composition has a pH of about 4.0. In some embodiments, the composition has a pH above about 4.0. In some embodiments, the composition has a pH of about 5.0. In some embodiments, the composition has a pH above about 5.0. In some embodiments, the composition has a pH of about 6.0. In some embodiments, the composition has a pH above about 6.0. In some embodiments, the composition has a pH of about 7.0. In some embodiments, the composition has a pH above about 7.0. In some embodiments, the composition has a pH of about 8.0. In some embodiments, the composition has a pH above about 8.0. In some embodiments, the composition has a pH of about 9.0. In some embodiments, the composition has a pH above about 9.0. In some embodiments, the composition has a pH of about 10.0. In some embodiments, the composition has a pH above about 10.0. In some embodiments, the composition has a pH of about 11.0. In some embodiments, the composition has a pH above about 11.0. In some embodiments, the composition has a pH of about 12.0. In some embodiments, the composition has a pH above about 12.0. In some embodiments, the composition has a pH of about 13.0. In some embodiments, the composition has a pH above about 13.0. In some embodiments, the composition has a pH of about 14.0. In some embodiments, the composition has a pH of between about 7.0 to about 8.0. For example, in one embodiment, the composition has a pH of about 7.4.

[0288] In some embodiments, the composition comprises between about 1 mg / mL to about 1,000 mg / mL, about 1 mg / mL to about 900 mg / mL, about 1 mg / mL to about 800 mg / mL, about 1 mg / mL to about 700 mg / mL, about 1 mg / mL to about 600 mg / mL, about 1 mg / mL to about 500 mg / mL, about 1 mg / mL to about 400 mg / mL, about 1 mg / mL to about 300 mg / mL, about 1 mg / mL to about 200 mg / mL, about 1 mg / mL to about 100 mg / mL, about 10 mg / mL to about 100 mg / mL, or about 10 mg / mL to about 80 mg / mL of at least one ionizable amphiphilic Janus dendrimer or a nanoparticle comprising thereof. For example, in some embodiments, the composition comprises about 10 mg / mL, about 18 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, or about 100 mg / mL of at least one ionizable amphiphilic Janus dendrimer or a nanoparticle comprising thereof.

[0289] In some embodiments, the composition comprises between about 0.01 µg to about 1,000 µg, about 0.05 µg to about 1,000 µg, about 0.1 µg to about 1,000 µg, about 0.25 µg to about 1,000 µg, about 0.5 µg to about 1,000 µg, about 1 µg to about 1,000 µg, about 1 µg to about 900 µg, about 1 µg to about 800 µg, about 1 µg to about 700 µg, about 1 µg to about 600 µg, about 1 µg to about 500 µg, about 1 µg to about 250 µg, about 1 µg to about 200 µg, about 1 µg to about 100 µg, or about 1 µg to about 50 µg of the agent. For example, in some embodiments, the composition comprises about 1.25 µg, about 1.5 µg, about 2.5 µg, about 5 µg, about 10 µg, or about 50 µg of the agent.

[0290] In some embodiments, the composition comprises an N / P ratio of between about 1 toabout 100, between about 1 to about 90, between about 1 to about 80, between about 1 to about 70, between about 1 to about 60, between about 1 to about 50, between about 1 to about 40, between about 5 to about 40, between about 1 to about 30, between about 1 to about 20, or between about 1 to about 10. For example, in some embodiments, the composition comprises an N / P ratio of about 5, about 10, about 20, about 30, or about 40.

[0291] In some embodiments, the composition maintains stability and activity at between about - 10 °C to about 30 °C, about 0 °C to about 20 °C, or about 0 °C to about 10 °C. For example, in one embodiment, the composition maintains stability and activity at about 4 °C, 0 °C, 1 °C, 2 °C, 3 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, or 30 °C.

[0292] In some embodiments, the composition is a one-component delivery system composition, two-component delivery system composition, three-component delivery system, four-component delivery system composition, or five-component delivery system composition.

[0293] In one embodiment, the composition is a vaccine.

[0294] In one embodiment, the composition comprises a nanoparticle and one or more nucleic acid molecules described herein. For example, in one embodiment, the composition comprises a nanoparticle and one or more nucleoside-modified RNA molecules encoding one or more antigens, adjuvants, or a combination thereof.

[0295] In one embodiment, the composition may be prepared by injection of a mixture comprising a compound described herein into a suitable solution, such as a solution comprising the agent to be encapsulated. In one embodiment, microfluidic techniques such as those required for the formation of lipid nanoparticles (LNPs) are not required for the production of the inventive nanoparticles.

[0296] In one embodiment, the composition of the invention comprises in vitro transcribed (IVT) RNA molecule. For example, in certain embodiments, the composition of the invention comprises IVT RNA molecule which encodes an agent. In certain embodiments, the IVT RNA molecule of the present composition is a nucleoside-modified mRNA molecule. In certain embodiments, the agent is at least one of a viral antigen, bacterial antigen, fungal antigen, parasitic antigen, tumor-specific antigen, or tumor-associated antigen. However, the invention is not limited to any particular agent or combination of agents. In certain embodiments, the composition comprises an adjuvant. In certain embodiments, the composition comprises a nucleic acid molecule encoding an adjuvant. In one embodiment, the composition comprises a nucleoside-modified RNA encoding an adjuvant.

[0297] In one embodiment, the composition comprises at least one nucleoside-modified RNA molecule encoding a combination of at least two agents. In one embodiment, the composition comprises a combination of two or more nucleoside-modified RNA molecules encoding a combination of two or moreagents.

[0298] In one embodiment, the invention provides a method for inducing an immune response in a subject. For example, the method can be used to provide immunity in the subject against a virus, bacteria, fungus, parasite, cancer, or the like. In some embodiments, the method comprises administering to the subject a composition comprising one or more nanoparticles comprising one or more nucleoside- modified RNA encoding at least one antigen, an adjuvant, or a combination thereof.

[0299] In one embodiment, the method comprises the systemic administration of the composition into the subject, including for example intradermal administration. In certain embodiments, the method comprises administering a plurality of doses to the subject. In another embodiment, the method comprises administering a single dose of the composition, where the single dose is effective in inducing a therapeutic response.

[0300] In some embodiments, the composition further comprises at least one second agent, buffer, adjuvant, stabilizer, solvent, polymer, or any combination thereof. Exemplary second agents include, but are not limited to, a diagnostic agent, detectable agent, therapeutic agent, nucleic acid molecule, or any combination thereof.

[0301] In some embodiments, the buffer, adjuvant, stabilizer, solvent, and polymer is any buffer, adjuvant, stabilizer, solvent, and polymer known in the art. Exemplary buffers include, but are not limited to, acetate buffer, phosphate-buffered saline, sodium phosphate buffer, or any combination thereof.

[0302] In some embodiments, the buffer has a concentration between about 1 mM to about 100 mM. In some embodiments, the buffer has a concentration between about 1 mM to about 50 mM. In some embodiments, the buffer has a concentration between about 1 mM to about 20 mM. In some embodiments, the buffer has a concentration between about 5 mM to about 20 mM. In some embodiments, the buffer has a concentration between about 10 mM to about 15 mM. For example, in some embodiments, the buffer has a concentration of about 10 mM. In some embodiments, the buffer has a concentration of about 11 mM. In some embodiments, the buffer has a concentration of about 12 mM. In some embodiments, the buffer has a concentration of about 13 mM. In some embodiments, the buffer has a concentration of about 14 mM. In some embodiments, the buffer has a concentration of about 15 mM. In some embodiments, the buffer has a concentration of about 16 mM. In some embodiments, the buffer has a concentration of about 17 mM. In some embodiments, the buffer has a concentration of about 18 mM. In some embodiments, the buffer has a concentration of about 19 mM. In some embodiments, the buffer has a concentration of about 20 mM.

[0303] Exemplary polymers include, but are not limited to, PEG.

[0304] In some embodiments, the composition comprises about 0.01 mol% to about 50 mol% polymer. In some embodiments, the composition comprises about 0.05 mol% to about 50 mol% polymer.In some embodiments, the composition comprises about 0.25 mol% to about 50 mol% polymer. In some embodiments, the composition comprises about 0.5 mol% to about 50 mol% polymer. In some embodiments, the composition comprises about 0.01 mol% to about 40 mol% polymer. In some embodiments, the composition comprises about 0.01 mol% to about 30 mol% polymer. In some embodiments, the composition comprises about 0.01 mol% to about 20 mol% polymer. In some embodiments, the composition comprises about 0.01 mol% to about 10 mol% polymer. In some embodiments, the composition comprises about 0.01 mol% to about 5 mol% polymer. In some embodiments, the composition comprises about 0.25 mol% to about 10 mol% polymer. In some embodiments, the composition comprises about 0.5 mol% to about 10 mol% polymer. In some embodiments, the composition comprises about 1 mol% to about 10 mol% polymer. In some embodiments, the composition comprises about 1.5 mol% to about 10 mol% polymer. In some embodiments, the composition comprises about 2 mol% to about 10 mol% polymer. In some embodiments, the composition comprises about 4 mol% to about 10 mol% polymer. For example, in some embodiments, the composition comprises about 0.25 mol% to polymer. In some embodiments, the composition comprises about 0.5 mol% polymer. In some embodiments, the composition comprises about 1 mol% polymer. In some embodiments, the composition comprises about 1.5 mol% polymer. In some embodiments, the composition comprises about 2 mol% polymer. In some embodiments, the composition comprises about 3 mol% polymer. In some embodiments, the composition comprises about 4 mol% polymer. In some embodiments, the composition comprises about 5 mol% polymer. Vaccine

[0305] In one embodiment, the invention provides an immunogenic composition for inducing an immune response in a subject. For example, in one embodiment, the immunogenic composition is a vaccine. As used herein, an “immunogenic composition” may comprise an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding an antigen, a cell expressing or presenting an antigen or cellular component, or a combination thereof. In particular embodiments the composition comprises or encodes all or part of any peptide antigen, or an immunogenically functional equivalent thereof. In other embodiments, the composition comprises a mixture of mRNA molecules that encodes one or more additional immunostimulatory agent. Immunostimulatory agents include, but are not limited to, an additional antigen, an immunomodulator, or an adjuvant. In the context of the invention, the term “vaccine” refers to a substance that induces immunity upon inoculation into animals.

[0306] A vaccine of the invention may vary in its composition of nucleic acid components. In a non-limiting example, a nucleic acid encoding an antigen might also be formulated with an adjuvant. Of course, it will be understood that various compositions described herein may further comprise additionalcomponents. A vaccine of the invention, and its various components, may be prepared and / or administered by any method disclosed herein or as would be known to one of ordinary skill in the art, in light of the present disclosure.

[0307] The induction of the immunity by the expression of the antigen can be detected by observing in vivo or in vitro the response of all or any part of the immune system in the host against the antigen.

[0308] For example, a method for detecting the induction of cytotoxic T lymphocytes is well known. A foreign substance that enters the living body is presented to T cells and B cells by the action of APCs. T cells that respond to the antigen presented by APC in an antigen specific manner differentiate into cytotoxic T cells (also referred to as cytotoxic T lymphocytes or CTLs) due to stimulation by the antigen. These antigen stimulated cells then proliferate. This process is referred to herein as “activation” of T cells. Therefore, CTL induction by an epitope of a polypeptide or peptide or combinations thereof can be evaluated by presenting an epitope of a polypeptide or peptide or combinations thereof to a T cell by APC, and detecting the induction of CTL. Furthermore, APCs have the effect of activating B cells, CD4+ T cells, CD8+ T cells, macrophages, eosinophils and NK cells.

[0309] A method for evaluating the inducing action of CTL using dendritic cells (DCs) as APC is well known in the art. DC is a representative APC having a robust CTL inducing action among APCs. In the methods of the invention, the epitope of a polypeptide or peptide or combinations thereof is initially expressed by the DC and then this DC is contacted with T cells. Detection of T cells having cytotoxic effects against the cells of interest after the contact with DC shows that the an epitope of a polypeptide or peptide or combinations thereof has an activity of inducing the cytotoxic T cells. Furthermore, the induced immune response can be also examined by measuring IFN-gamma produced and released by CTL in the presence of antigen-presenting cells that carry immobilized peptide or combination of peptides by visualizing using anti-IFN-gamma antibodies, such as an ELISPOT assay.

[0310] Apart from DC, peripheral blood mononuclear cells (PBMCs) may also be used as the APC. The induction of CTL is reported to be enhanced by culturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL has been shown to be induced by culturing PBMC in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

[0311] The antigens confirmed to possess CTL-inducing activity by these methods are antigens having DC activation effect and subsequent CTL-inducing activity. Furthermore, CTLs that have acquired cytotoxicity due to presentation of the antigen by APC can be also used as vaccines against antigen- associated disorders.

[0312] The induction of immunity by expression of the antigen can be further confirmed by observing the induction of antibody production against the antigen. For example, when antibodies againstan antigen are induced in a laboratory animal immunized with the composition encoding the antigen, and when antigen-associated pathology is suppressed by those antibodies, the composition is determined to induce immunity.

[0313] The induction of immunity by expression of the antigen can be further confirmed by observing the induction of CD4+ T cells. CD4+ T cells can also lyse target cells, but mainly supply help in the induction of other types of immune responses, including CTL and antibody generation. The type of CD4+ T cell help can be characterized, as Th1, Th2, Th9, Th17, Tregulatory, or T follicular helper (Tfh) cells. Each subtype of CD4+ T cell supplies help to certain types of immune responses. Of particular interest to this invention, the Tfhsubtype provides help in the generation of high affinity antibodies.

[0314] In some embodiments, the therapeutic compounds or compositions of the invention may be administered prophylactically (i.e., to prevent disease or disorder) or therapeutically (i.e., to treat disease or disorder) to subjects suffering from or at risk of (or susceptible to) developing the disease or disorder. Such subjects may be identified using standard clinical methods. In the context of the invention, prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or alternatively delayed in its progression. In the context of the field of medicine, the term “prevent” encompasses any activity which reduces the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Targeting Domain

[0315] In one embodiment, the composition comprises a targeting domain that directs the delivery vehicle to a site. In one embodiment, the site is a site in need of the agent comprised within the delivery vehicle. The targeting domain may comprise a nucleic acid, peptide, antibody, small molecule, organic molecule, inorganic molecule, glycan, sugar, hormone, and the like that targets the particle to a site in particular need of the therapeutic agent. In certain embodiments, the particle comprises multivalent targeting, wherein the particle comprises multiple targeting mechanisms described herein. In certain embodiments, the targeting domain of the delivery vehicle specifically binds to a target associated with a site in need of an agent comprised within the delivery vehicle. For example, the targeting domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Such a target can be a protein, protein fragment, antigen, or other biomolecule that is associated with the targeted site. In some embodiments, the targeting domain is an affinity ligand whichspecifically binds to a target. In certain embodiments, the target (e.g. antigen) associated with a site in need of a treatment with an agent. In some embodiments, the targeting domain may be co-polymerized with the composition comprising the delivery vehicle. In some embodiments, the targeting domain may be covalently attached to the composition comprising the delivery vehicle, such as through a chemical reaction between the targeting domain and the composition comprising the delivery vehicle. In some embodiments, the targeting domain is an additive in the delivery vehicle. Targeting domains of the instant invention include, but are not limited to, antibodies, antibody fragments, proteins, peptides, and nucleic acids.

[0316] In various embodiments, the targeting domain binds to a cell surface molecule of a cell of interest. For example, in various embodiments, the targeting domain binds to a cell surface molecule of an endothelial cell, a stem cell, or an immune cell. Peptides

[0317] In one embodiment, the targeting domain of the invention comprises a peptide. In certain embodiments, the peptide targeting domain specifically binds to a target of interest. The peptide of the invention may be made using chemical methods. For example, peptides can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. The peptide may alternatively be made by recombinant means or by cleavage from a longer polypeptide. The composition of a peptide may be confirmed by amino acid analysis or sequencing.

[0318] The variants of the peptides according to the invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the peptide is an alternative splice variant of the peptide of the invention, (iv) fragments of the peptides and / or (v) one in which the peptide is fused with another peptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.

[0319] As known in the art the “similarity” between two peptides is determined by comparingthe amino acid sequence and its conserved amino acid substitutes of one peptide to a sequence of a second peptide. Variants are defined to include peptide sequences different from the original sequence, preferably different from the original sequence in less than 40% of residues per segment of interest, more preferably different from the original sequence in less than 25% of residues per segment of interest, more preferably different by less than 10% of residues per segment of interest, most preferably different from the original protein sequence in just a few residues per segment of interest and at the same time sufficiently homologous to the original sequence to preserve the functionality of the original sequence. The invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.20894, Altschul, S., et al., J. Mol. Biol.215: 403-410 (1990)].

[0320] The peptides of the invention can be post-translationally modified. For example, post- translational modifications that fall within the scope of the invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No.6,103,489) to a standard translation reaction.

[0321] The peptides of the invention may include unnatural amino acids formed by post- translational modification or by introducing unnatural amino acids during translation. Nucleic Acids

[0322] In one embodiment, the targeting domain of the invention comprises an isolated nucleic acid, including for example a DNA oligonucleotide and a RNA oligonucleotide. In certain embodiments, the nucleic acid targeting domain specifically binds to a target of interest. For example, in one embodiment, the nucleic acid comprises a nucleotide sequence that specifically binds to a target of interest.

[0323] The nucleotide sequences of a nucleic acid targeting domain can alternatively comprise sequence variations with respect to the original nucleotide sequences, for example, substitutions, insertions and / or deletions of one or more nucleotides, with the condition that the resulting nucleic acid functions as the original and specifically binds to the target of interest.

[0324] In the sense used in this description, a nucleotide sequence is “substantially homologous”to any of the nucleotide sequences describe herein when its nucleotide sequence has a degree of identity with respect to the nucleotide sequence of at least 60%, advantageously of at least 70%, preferably of at least 85%, and more preferably of at least 95%. Other examples of possible modifications include the insertion of one or more nucleotides in the sequence, the addition of one or more nucleotides in any of the ends of the sequence, or the deletion of one or more nucleotides in any end or inside the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTN algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.20894, Altschul, S., et al., J. Mol. Biol.215: 403-410 (1990)]. Antibodies

[0325] In one embodiment, the targeting domain of the invention comprises an antibody, or antibody fragment. In certain embodiments, the antibody targeting domain specifically binds to a target of interest. Such antibodies include polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies.

[0326] The antibodies may be intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain Fv molecule (Ladner et al, U.S. Pat. No. 4,946,778), or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.

[0327] Such antibodies may be produced in a variety of ways, including hybridoma cultures, recombinant expression in bacteria or mammalian cell cultures, and recombinant expression in transgenic animals. The choice of manufacturing methodology depends on several factors including the antibody structure desired, the importance of carbohydrate moieties on the antibodies, ease of culturing and purification, and cost. Many different antibody structures may be generated using standard expression technology, including full-length antibodies, antibody fragments, such as Fab and Fv fragments, as well as chimeric antibodies comprising components from different species. Antibody fragments of small size, such as Fab and Fv fragments, having no effector functions and limited pharmokinetic activity may be generated in a bacterial expression system. Single chain Fv fragments show low immunogenicity. Adjuvant

[0328] In one embodiment, the composition comprises an adjuvant. In one embodiment, thecomposition comprises a nucleic acid molecule encoding an adjuvant. In one embodiment, the adjuvant- encoding nucleic acid molecule is IVT RNA. In one embodiment, the adjuvant-encoding nucleic acid molecule is nucleoside-modified mRNA.

[0329] Exemplary adjuvants include, but is not limited to, alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 having the signal sequence deleted and optionally including the signal peptide from IgE. Other genes which may be useful adjuvants include those encoding: MCP-I, MIP-Ia, MIP-Ip, IL-8, RANTES, L- selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-I, VLA-I, Mac-1, pl50.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-I, Ap-I, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP 1, TAP2, anti-CTLA4-sc, anti-LAG3-Ig, anti-TIM3-Ig and functional fragments thereof. Antibody Therapeutic Agents

[0330] The invention also contemplates a delivery vehicle comprising an antibody, or antibody fragment, specific for a target. That is, the antibody can inhibit a target to provide a beneficial effect.

[0331] The antibodies may be intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain FV molecule (Ladner et al, U.S. Pat. No. 4,946,778), or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.

[0332] Antibodies can be prepared using intact polypeptides or fragments containing an immunizing antigen of interest. The polypeptide or oligopeptide used to immunize an animal may be obtained from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Suitable carriers that may be chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The coupled polypeptide may then be used toimmunize the animal (e.g., a mouse, a rat, or a rabbit). Antigen

[0333] The invention provides a composition that induces a therapeutic response in a subject. In one embodiment, the composition comprises an antigen. In one embodiment, the composition comprises a nucleic acid sequence which encodes an antigen. For example, in certain embodiments, the composition comprises a nucleoside-modified RNA encoding an antigen. The antigen may be any molecule or compound, including but not limited to a polypeptide, peptide or protein that induces a therapeutic response, such as an adaptive immune response, in a subject.

[0334] In one embodiment, the antigen comprises a polypeptide or peptide associated with a pathogen, such that the antigen induces an adaptive immune response against the antigen, and therefore the pathogen. In one embodiment, the antigen comprises a fragment of a polypeptide or peptide associated with a pathogen, such that the antigen induces an adaptive immune response against the pathogen.

[0335] In certain embodiments, the antigen comprises an amino acid sequence that is substantially homologous to the amino acid sequence of an antigen described herein and retains the immunogenic function of the original amino acid sequence. For example, in certain embodiments, the amino acid sequence of the antigen has a degree of identity with respect to the original amino acid sequence of at least 60%, advantageously of at least 70%, preferably of at least 85%, and more preferably of at least 95%.

[0336] In one embodiment, the antigen is encoded by a nucleic acid sequence of a nucleic acid molecule. In certain embodiments, the nucleic acid sequence comprises DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. In one embodiment, the nucleic acid sequence comprises a modified nucleic acid sequence. For example, in one embodiment the antigen-encoding nucleic acid sequence comprises nucleoside-modified RNA, as described in detail elsewhere herein. In certain instances, the nucleic acid sequence comprises include additional sequences that encode linker or tag sequences that are linked to the antigen by a peptide bond.

[0337] In certain embodiments, the antigen, encoded by the nucleoside-modified nucleic acid molecule, comprises a protein, peptide, a fragment thereof, or a variant thereof, or a combination thereof from any number of organisms, for example, a virus, a parasite, a bacterium, a fungus, or a mammal. For example, in certain embodiments, the antigen is associated with an autoimmune disease, allergy, or asthma. In other embodiments, the antigen is associated with cancer, herpes, influenza, hepatitis B, hepatitis C, human papilloma virus (HPV), ebola, pneumococcus, Haemophilus influenza, meningococcus, dengue, tuberculosis, malaria, norovirus or human immunodeficiency virus (HIV). Incertain embodiments, the antigen comprises a consensus sequence based on the amino acid sequence of two or more different organisms. In certain embodiments, the nucleic acid sequence encoding the antigen is optimized for effective translation in the organism in which the composition is delivered.

[0338] In one embodiment, the antigen comprises a tumor-specific antigen or tumor-associated antigen, such that the antigen induces an adaptive immune response against the tumor. In one embodiment, the antigen comprises a fragment of a tumor-specific antigen or tumor-associated antigen, such that the antigen induces an adaptive immune response against the tumor. In certain embodiment, the tumor-specific antigen or tumor-associated antigen is a mutation variant of a host protein. Viral Antigens

[0339] In one embodiment, the antigen comprises a viral antigen, or fragment thereof, or variant thereof. In certain embodiments, the viral antigen is from a virus from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. In certain embodiments, the viral antigen is from papilloma viruses, for example, human papillomoa virus (HPV), human immunodeficiency virus (HIV), polio virus, hepatitis B virus, hepatitis C virus, smallpox virus (Variola major and minor), vaccinia virus, influenza virus, rhinoviruses, dengue fever virus, equine encephalitis viruses, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), California encephalitis virus, Hanta virus (hemorrhagic fever), rabies virus, Ebola fever virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex 1 (oral herpes), herpes simplex 2 (genital herpes), herpes zoster (varicella- zoster, a.k.a., chickenpox), cytomegalovirus (CMV), for example human CMV, Epstein-Barr virus (EBV), flavivirus, foot and mouth disease virus, chikungunya virus, lassa virus, arenavirus, severe acute respiratory syndrome (SARS) virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a cancer causing virus. Parasite Antigens

[0340] In certain embodiments, the antigen comprises a parasite antigen or fragment or variant thereof. In certain embodiments, the parasite is a protozoa, helminth, or ectoparasite. In certain embodiments, the helminth (i.e., worm) is a flatworm (e.g., flukes and tapeworms), a thorny-headed worm, or a round worm (e.g., pinworms). In certain embodiments, the ectoparasite is lice, fleas, ticks, and mites.

[0341] In certain embodiments, the parasite is any parasite causing the following diseases:Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinosis, and Trichuriasis.

[0342] In certain embodiments, the parasite is Acanthamoeba, Anisakis, Ascaris lumbricoides, Botfly, Balantidium coli, Bedbug, Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke, Loa loa, Paragonimus - lung fluke, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosoma, Whipworm, or Wuchereria bancrofti. Bacterial Antigens

[0343] In one embodiment, the antigen comprises a bacterial antigen or fragment or variant thereof. In certain embodiments, the bacterium is from any one of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

[0344] In certain embodiments, the bacterium is a gram positive bacterium or a gram negative bacterium. In certain embodiments, the bacterium is an aerobic bacterium or an anaerobic bacterium. In certain embodiments, the bacterium is an autotrophic bacterium or a heterotrophic bacterium. In certain embodiments, the bacterium is a mesophile, a neutrophile, an extremophile, an acidophile, an alkaliphile, a thermophile, psychrophile, halophile, or an osmophile.

[0345] In certain embodiments, the bacterium is an anthrax bacterium, an antibiotic resistant bacterium, a disease causing bacterium, a food poisoning bacterium, an infectious bacterium, Salmonella bacterium, Staphylococcus bacterium, Streptococcus bacterium, or tetanus bacterium. In certain embodiments, bacterium is a mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile. Fungal Antigens

[0346] In one embodiment, the antigen comprises a fungal antigen or fragment or variant thereof. In certain embodiments, the fungus is Aspergillus species, Blastomyces dermatitidis, Candidayeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium. Tumor Antigens

[0347] In certain embodiments, the antigen comprises a tumor antigen, including for example a tumor-associated antigen or a tumor-specific antigen. In the context of the invention, “tumor antigen” or “hyperporoliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” refer to antigens that are common to specific hyperproliferative disorders. In certain aspects, the hyperproliferative disorder antigens of the invention are derived from cancers including, but not limited to, primary or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin’s lymphoma, Hodgkins lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.

[0348] Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. In one embodiment, the tumor antigen of the invention comprises one or more antigenic cancer epitopes immunogenically recognized by tumor infiltrating lymphocytes (TIL) derived from a cancer tumor of a mammal. The selection of the antigen will depend on the particular type of cancer to be treated or prevented by way of the composition of the invention.

[0349] Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate- carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0350] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostatic acid phosphatase (PAP) and prostate- specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation- related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma the tumor-specificidiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. B-cell differentiation antigens such as CD19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0351] The type of tumor antigen referred to in the invention may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells.

[0352] Non-limiting examples of TSA or TAA antigens include the following: Differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor- specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0353] In a preferred embodiment, the antigen includes but is not limited to CD19, CD20, CD22, ROR1, Mesothelin, CD33 / IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and the like. Combinations

[0354] In one embodiment, the composition of the invention comprises a combination of agents described herein. In certain embodiments, a composition comprising a combination of agents describedherein has an additive effect, wherein the overall effect of the combination is approximately equal to the sum of the effects of each individual agent. In other embodiments, a composition comprising a combination of agents described herein has a synergistic effect, wherein the overall effect of the combination is greater than the sum of the effects of each individual agent.

[0355] A composition comprising a combination of agents comprises individual agents in any suitable ratio. For example, in one embodiment, the composition comprises a 1:1 ratio of two individual agents. However, the combination is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed. Conjugation

[0356] In various embodiments of the invention, the delivery vehicle is conjugated to a targeting domain. Exemplary methods of conjugation can include, but are not limited to, covalent bonds, electrostatic interactions, and hydrophobic (“van der Waals”) interactions. In one embodiment, the conjugation is a reversible conjugation, such that the delivery vehicle can be disassociated from the targeting domain upon exposure to certain conditions or chemical agents. In another embodiment, the conjugation is an irreversible conjugation, such that under normal conditions the delivery vehicle does not dissociate from the targeting domain.

[0357] In some embodiments, the conjugation comprises a covalent bond between an activated polymer conjugated lipid and the targeting domain. The term “activated polymer conjugated lipid” refers to a molecule comprising a lipid portion and a polymer portion that has been activated via functionalization of a polymer conjugated lipid with a first coupling group. In one embodiment, the activated polymer conjugated lipid comprises a first coupling group capable of reacting with a second coupling group. In one embodiment, the activated polymer conjugated lipid is an activated pegylated lipid. In one embodiment, the first coupling group is bound to the lipid portion of the pegylated lipid. In another embodiment, the first coupling group is bound to the polyethylene glycol portion of the pegylated lipid. In one embodiment, the second functional group is covalently attached to the targeting domain.

[0358] The first coupling group and second coupling group can be any functional groups known to those of skill in the art to together form a covalent bond, for example under mild reaction conditions or physiological conditions. In some embodiments, the first coupling group or second coupling group are selected from the group consisting of maleimides, N-hydroxysuccinimide (NHS) esters, carbodiimides, hydrazide, pentafluorophenyl (PFP) esters, phosphines, hydroxymethyl phosphines, psoralen, imidoesters, pyridyl disulfide, isocyanates, vinyl sulfones, alpha-haloacetyls, aryl azides, acyl azides, alkyl azides, diazirines, benzophenone, epoxides, carbonates, anhydrides, sulfonyl chlorides, cyclooctyne, aldehydes, and sulfhydryl groups. In some embodiments, the first coupling group or second coupling group isselected from the group consisiting of free amines (–NH2), free sulfhydryl groups (–SH), free hydroxide groups (–OH), carboxylates, hydrazides, and alkoxyamines. In some embodiments, the first coupling group is a functional group that is reactive toward sulfhydryl groups, such as maleimide, pyridyl disulfide, or a haloacetyl. In one embodiment, the first coupling group is a maleimide.

[0359] In one embodiment, the second coupling group is a sulfhydryl group. The sulfhydryl group can be installed on the targeting domain using any method known to those of skill in the art. In one embodiment, the sulfhydryl group is present on a free cysteine residue. In one embodiment, the sulfhydryl group is revealed via reduction of a disulfide on the targeting domain, such as through reaction with 2- mercaptoethylamine. In one embodiment, the sulfhydryl group is installed via a chemical reaction, such as the reaction between a free amine and 2-iminothilane or N-succinimidyl S-acetylthioacetate (SATA).

[0360] In some embodiments, the polymer conjugated lipid and targeting domain are functionalized with groups used in “click” chemistry. Bioorthogonal “click” chemistry comprises the reaction between a functional group with a 1,3-dipole, such as an azide, a nitrile oxide, a nitrone, an isocyanide, and the link, with an alkene or an alkyne dipolarophiles. Exemplary dipolarophiles include any strained cycloalkenes and cycloalkynes known to those of skill in the art, including, but not limited to, cyclooctynes, dibenzocyclooctynes, monofluorinated cyclcooctynes, difluorinated cyclooctynes, and biarylazacyclooctynone Pharmaceutical Compositions

[0361] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0362] Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0363] Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.

[0364] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0365] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0366] In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.

[0367] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[0368] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.

[0369] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared,packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0370] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0371] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent / powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0372] Low boiling propellants generally include liquid propellants having a boiling point ofbelow 65 °F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w / w) of the composition, and the active ingredient may constitute 0.1 to 20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).

[0373] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0374] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0375] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents,demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington’s Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference. Methods

[0376] The invention provides methods of delivering an agent to a cell, tissue, or organ of a subject. In one aspect, the present invention relates to methods of delivering an agent to a target of interest (e.g., cell, tissue, or organ of a subject) using at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof. In another aspect, the present invention relates to methods of delivering an agent to two or more targets of interest (e.g., cell, tissue, or organ of a subject) using at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof.

[0377] In some embodiments, the method comprises a one-component delivery system, two- component delivery system, three-component delivery system, or four-component delivery system comprising at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof.

[0378] Examples of such cells of interest include, but are not limited to, epithelial cell, connective tissue cell, muscle cell, nerve cell, endothelial cell, parenchymal cell, blood cell, T cell, macrophages, neutrophils, dendritic cell (DC), monocytes, and any combination thereof. Examples of such organs include, but are not limited to, mucosal surface, gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, spleen, or any combination thereof.

[0379] For example, in some embodiments, the method comprises delivering at least one agent to mucosal surface. In some embodiments, the method comprises delivering at least one agent to gastrointestinal tract. In some embodiments, the method comprises delivering at least one agent to stomach. In some embodiments, the method comprises delivering at least one agent to small intestine. In some embodiments, the method comprises delivering at least one agent to large intestine. In some embodiments, the method comprises delivering at least one agent to kidney. In some embodiments, the method comprises delivering at least one agent to heart. In some embodiments, the method comprises delivering at least one agent to lung. In some embodiments, the method comprises delivering at least one agent to liver. In some embodiments, the method comprises delivering at least one agent to lymph nodes. In some embodiments, the method comprises delivering at least one agent to spleen. In some embodiments, the method comprises simultaneously delivering at least one agent to mucosal surface,gastrointestinal tract, such as stomach, small intestine, large intestine, etc., kidney, heart, lung, liver, lymph nodes, and / or spleen.

[0380] In some embodiments, the method comprises delivering at least one agent to at least one cell having mucosal surface. In some embodiments, the method comprises delivering at least one agent to at least one gastrointestinal tract cell. In some embodiments, the method comprises delivering at least one agent to at least one stomach cell. In some embodiments, the method comprises delivering at least one agent to at least one small intestine cell. In some embodiments, the method comprises delivering at least one agent to at least one large intestine cell. In some embodiments, the method comprises delivering at least one agent to at least one kidney cell. In some embodiments, the method comprises delivering at least one agent to at least one heart cell. In some embodiments, the method comprises delivering at least one agent to at least one lung cell. In some embodiments, the method comprises delivering at least one agent to at least one liver cell. In some embodiments, the method comprises delivering at least one agent to at least one lymph node cell. In some embodiments, the method comprises delivering at least one agent to at least one spleen cell. In some embodiments, the method comprises simultaneously delivering at least one agent to at least one cell having mucosal surface, at least one gastrointestinal tract cell, at least one stomach cell, at least one small intestine cell, at least one large intestine cell, at least one kidney cell, at least one heart cell, at least one lung cell, at least one liver cell, at least one lymph node cell, and / or at least one spleen cell.

[0381] In some embodiments, the agent is a diagnostic agent to detect at least one marker associated with a disease or disorder. In some embodiments, the agent is a therapeutic agent for the treatment or prevention of a disease or disorder. Therefore, in some embodiments, the invention provides methods for diagnosing, treating or preventing a disease or disorder comprising administering an effective amount of a composition comprising one or more diagnostic or therapeutic agents, one or more adjuvants, or a combination thereof.

[0382] In some embodiments, the method provides immunity in the subject to an infection, disease, or disorder associated with an antigen. The invention thus provides a method of treating or preventing the infection, disease, or disorder associated with the antigen. For example, the method may be used to treat or prevent a viral infection, bacterial infection, fungal infection, parasitic infection, or cancer, depending upon the type of antigen of the administered composition. Exemplary antigens and associated infections, diseases, and tumors are described elsewhere herein.

[0383] In one embodiment, the composition is administered to a subject having an infection, disease, or cancer associated with the antigen. In one embodiment, the composition is administered to a subject at risk for developing the infection, disease, or cancer associated with the antigen. For example, the composition may be administered to a subject who is at risk for being in contact with a virus, bacteria,fungus, parasite, or the like. In one embodiment, the composition is administered to a subject who has increased likelihood, though genetic factors, environmental factors, or the like, of developing cancer.

[0384] In one embodiment, the method comprises administering a composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more antigens and one or more adjuvant. In one embodiment, the method comprises administering a composition comprising a first nucleoside-modified nucleic acid molecule encoding one or more antigens and a second nucleoside- modified nucleic acid molecule encoding one or more adjuvants. In one embodiment, the method comprises administering a first composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more antigens and administering a second composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more adjuvants.

[0385] In certain embodiments, the method comprises administering to subject a plurality of nucleoside-modified nucleic acid molecules encoding a plurality of antigens, adjuvants, or a combination thereof.

[0386] In certain embodiments, the method of the invention allows for sustained expression of the antigen or adjuvant, described herein, for at least several days following administration. However, the method, in certain embodiments, also provides for transient expression, as in certain embodiments, the nucleic acid is not integrated into the subject genome.

[0387] In certain embodiments, the method comprises administering nucleoside-modified RNA which provides stable expression of the antigen or adjuvant described herein. In some embodiments, administration of nucleoside-modified RNA results in little to no innate immune response, while inducing an effective adaptive immune response.

[0388] Administration of the compositions of the invention in a method of treatment can be achieved in a number of different ways, using methods known in the art. In one embodiment, the method of the invention comprises systemic administration of the subject, including for example enteral or parenteral administration. In certain embodiments, the method comprises intradermal delivery of the composition. In another embodiment, the method comprises intravenous delivery of the composition. In some embodiments, the method comprises intramuscular delivery of the composition. In one embodiment, the method comprises subcutaneous delivery of the composition. In one embodiment, the method comprises inhalation of the composition. In one embodiment, the method comprises intranasal delivery of the composition.

[0389] It will be appreciated that the composition of the invention may be administered to a subject either alone, or in conjunction with another agent.

[0390] The therapeutic and prophylactic methods of the invention thus encompass the use of pharmaceutical compositions encoding an antigen, adjuvant, or a combination thereof, described herein topractice the methods of the invention. The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of from ng / kg / day and 100 mg / kg / day. In one embodiment, the invention envisions administration of a dose which results in a concentration of the compound of the invention from 10nM and 10 μM in a mammal.

[0391] Typically, dosages which may be administered in a method of the invention to a mammal, preferably a human, range in amount from 0.01 μg to about 50 mg per kilogram of body weight of the mammal, while the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of mammal and type of disease state being treated, the age of the mammal and the route of administration. Preferably, the dosage of the compound will vary from about 0.1 μg to about 10 mg per kilogram of body weight of the mammal. More preferably, the dosage will vary from about 1 μg to about 1 mg per kilogram of body weight of the mammal.

[0392] The composition may be administered to a mammal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the mammal, etc.

[0393] In certain embodiments, administration of an immunogenic composition or vaccine of the invention may be performed by single administration or boosted by multiple administrations.

[0394] In one embodiment, the invention includes a method comprising administering one or more compositions encoding one or more antigens or adjuvants described herein. In certain embodiments, the method has an additive effect, wherein the overall effect of the administering the combination is approximately equal to the sum of the effects of administering each antigen or adjuvant. In other embodiments, the method has a synergistic effect, wherein the overall effect of administering the combination is greater than the sum of the effects of administering each antigen or adjuvant. Treatment Methods

[0395] In one embodiment, the invention provides methods of inducing an adaptive immune response against a disease or disorder of interest (e.g., a disease or disorder associated with the gastrointestinal tract, SARS-CoV-2, etc.) in a subject comprising administering an effective amount of a composition comprising an amphiphilic Janus dendrimer of Formula (I) or Formula (II), or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or derivative thereof, and one or more isolated nucleic acids encoding one or more antigens associated with the disease or disorder of interest (e.g., one or more SARS-CoV-2 antigens).

[0396] For example, in one embodiment, the method provides immunity in the subject to SARS- CoV-2, SARS-CoV-2 infection, or to a disease or disorder associated with SARS-CoV-2. The invention thus provides a method of treating or preventing the infection, disease, or disorder associated with SARS- CoV-2. In one embodiment, the disease or disorder associated with SARS-CoV-2 is COVID-19 or a comorbidity of COVID-19.

[0397] In some embodiments, the invention is a method of administering to a subject a composition comprising at least one nucleoside-modified RNA encoding at least one antigen associated with the disease or disorder of interest (e.g., at least one SARS-CoV-2 antigen).

[0398] In one embodiment, the composition is administered to a subject having an infection, disease, or disorder of interest (e.g., infection, disease, or disorder associated with SARS-CoV-2). In one embodiment, the composition is administered to a subject at risk for developing the infection, disease, or disorder of interest (e.g., infection, disease, or disorder associated with SARS-CoV-2).

[0399] For example, the composition may be administered to a subject who is at risk for being in contact with a SARS-CoV-2. In one embodiment, the composition is administered to a subject who lives in, traveled to, or is expected to travel to a geographic region in which SARS-CoV-2 is prevalent. In one embodiment, the composition is administered to a subject who is in contact with or expected to be in contact with another person who lives in, traveled to, or is expected to travel to a geographic region in which SARS-CoV-2 is prevalent. In one embodiment, the composition is administered to a subject who has knowingly been exposed to SARS-CoV-2 through their occupation or contact.

[0400] In one embodiment, the method comprises administering a composition comprising at least one dendrimer of Formula (I) or Formula (II), or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or derivative thereof, and one or more nucleoside-modified nucleic acid molecules encoding one or more antigens associated with the disease or disorder of interest (e.g., one or more SARS-CoV-2 antigens) and one or more adjuvant. In one embodiment, the method comprises administering a composition comprising a first nucleoside-modified nucleic acid molecule encoding one or more antigens associated with the disease or disorder of interest (e.g., one or more SARS-CoV-2 antigens) and a second nucleoside-modified nucleic acid molecule encoding one or more adjuvants. In one embodiment, the method comprises administering a first composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more antigens associated with the disease or disorder of interest (e.g., one or more SARS-CoV-2 antigens) and administering a second composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more adjuvants.

[0401] In certain embodiments, the method comprises administering to subject a plurality of nucleoside-modified nucleic acid molecules encoding a plurality of antigens associated with the diseaseor disorder of interest (e.g., a plurality of SARS-CoV-2 antigens), adjuvants, or a combination thereof.

[0402] In certain embodiments, the method of the invention allows for sustained expression of the antigen or adjuvant of a disease or disorder of interest (e.g., SARS-CoV-2 antigen), described herein, for at least several days following administration. In certain embodiments, the method of the invention allows for sustained expression of the antigen or adjuvant of a disease or disorder of interest (e.g., SARS- CoV-2 antigen), described herein, for at least 2 weeks following administration. In certain embodiments, the method of the invention allows for sustained expression of the antigen or adjuvant of a disease or disorder of interest (e.g., SARS-CoV-2 antigen), described herein, for at least 1 month following administration. However, the method, in certain embodiments, also provides for transient expression, as in certain embodiments, the nucleic acid is not integrated into the subject genome.

[0403] In certain embodiments, the method comprises administering nucleoside-modified RNA, which provides stable expression of the antigen or adjuvant of a disease or disorder of interest (e.g., SARS-CoV-2 antigen) described herein. In some embodiments, administration of nucleoside-modified RNA results in little to no innate immune response, while inducing an effective adaptive immune response.

[0404] In certain embodiments, the method provides sustained protection against a disease or disorder of interest (e.g., a disease or disorder associated with the gastrointestinal tract, SARS-CoV-2, etc.). For example, in certain embodiments, the method provides sustained protection against SARS-CoV- 2 for more than 2 weeks. In certain embodiments, the method provides sustained protection against SARS-CoV-2 for 1 month or more. In certain embodiments, the method provides sustained protection against SARS-CoV-2 for 2 months or more. In certain embodiments, the method provides sustained protection against SARS-CoV-2 for 3 months or more. In certain embodiments, the method provides sustained protection against SARS-CoV-2 for 4 months or more. In certain embodiments, the method provides sustained protection against SARS-CoV-2 for 5 months or more. In certain embodiments, the method provides sustained protection against SARS-CoV-2 for 6 months or more. In certain embodiments, the method provides sustained protection against SARS-CoV-2 for 1 year or more.

[0405] In one embodiment, a single immunization of the composition induces a sustained protection against a disease or disorder of interest (e.g., a disease or disorder associated with the gastrointestinal tract, SARS-CoV-2, etc.) for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, or 1 year or more.

[0406] Administration of the compositions of the invention in a method of treatment can be achieved in a number of different ways, using methods known in the art. In one embodiment, the method of the invention comprises systemic administration of the subject, including for example enteral or parenteral administration. In certain embodiments, the method comprises intradermal delivery of thecomposition. In another embodiment, the method comprises intravenous delivery of the composition. In some embodiments, the method comprises intramuscular delivery of the composition. In one embodiment, the method comprises subcutaneous delivery of the composition. In one embodiment, the method comprises inhalation of the composition. In one embodiment, the method comprises intranasal delivery of the composition.

[0407] It will be appreciated that the composition of the invention may be administered to a subject either alone, or in conjunction with another agent.

[0408] The therapeutic and prophylactic methods of the invention thus encompass the use of pharmaceutical compositions encoding an antigen of a disease or disorder of interest (e.g., a disease or disorder associated with the gastrointestinal tract, SARS-CoV-2, etc.), adjuvant of a disease or disorder of interest (e.g., a disease or disorder associated with the gastrointestinal tract, SARS-CoV-2, etc.), or a combination thereof, described herein to practice the methods of the invention. The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of from 1 ng / kg / day and 100 mg / kg / day. In one embodiment, the invention envisions administration of a dose, which results in a concentration of the compound of the invention from 10 nM and 10 μM in a mammal.

[0409] Typically, dosages which may be administered in a method of the invention to a mammal, such as a human, range in amount from 0.01 μg to about 50 mg per kilogram of body weight of the mammal, while the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of mammal and type of disease state being treated, the age of the mammal and the route of administration. In certain embodiments, the dosage of the compound will vary from about 0.1 μg to about 10 mg per kilogram of body weight of the mammal. In certain embodiments, the dosage will vary from about 1 μg to about 1 mg per kilogram of body weight of the mammal.

[0410] The composition may be administered to a mammal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the mammal, etc.

[0411] In certain embodiments, administration of an immunogenic composition or vaccine of the invention may be performed by single administration or boosted by multiple administrations.

[0412] In one embodiment, the invention includes a method comprising administering one or more compositions encoding one or more antigens or adjuvants of a disease or disorder of interest (e.g., a disease or disorder associated with the gastrointestinal tract, SARS-CoV-2, etc.) described herein. In certain embodiments, the method has an additive effect, wherein the overall effect of the administeringthe combination is approximately equal to the sum of the effects of administering each antigen or adjuvant of a disease or disorder of interest (e.g., a disease or disorder associated with the gastrointestinal tract, SARS-CoV-2, etc.). In other embodiments, the method has a synergistic effect, wherein the overall effect of administering the combination is greater than the sum of the effects of administering each antigen or adjuvant of a disease or disorder of interest (e.g., a disease or disorder associated with the gastrointestinal tract, SARS-CoV-2, etc.). EXPERIMENTAL EXAMPLES

[0413] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0414] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0415] Example 1: Towards a Complete Elucidation of the Primary Structure-Activity in Pentaerythritol-Based One-Component Ionizable Amphiphilic Janus Dendrimers for In Vivo Delivery of Luc-mRNA

[0416] During the Covid-19 pandemic, studies were conducted to eliminate some of the LNPs’ weaknesses by incorporating all 4-component activities of LNPs into a single architecture named 1- component ionizable amphiphilic Janus dendrimer (IAJD) (Zhang, et al., J. Am. Chem. Soc.2021, 143, 12315–12327; ; Zhang, et al., J. Am. Chem. Soc.2021, 143, 17975–17982; Zhang, et al., J. Am. Chem. Soc.2022, 144, 4746–4753; Lu, et al., Pharmaceutics 2023, 15, 1572; Lu, et al., J. Am. Chem. Soc.2023, 145 (34), 18760–18766; Sahoo, et al., J. Am. Chem. Soc.2024, 146 (6), 3627–3634; Zhang, et al., J. Am. Chem. Soc.2023, 145, 4311-4323; Arshad, et al., Biomacromolecules 2024, 25, 6871–6882), with targeted delivery, which requires 5-components with LNPs (Cheng, et al., Nat. Nanotechnol.2020, 15, 313–320; Han, et al., Nat. Nanotechnol.2023, 18, 1105–1114). Amphiphilic Janus dendrimers (JDs) (Percec, et al., Science 2010, 328, 1009–1014; Sherman, et al., Chem. Rev.2017, 117, 6538–6631; Peterca, et al., J. Am. Chem. Soc.2011, 133, 20507–20520; Zhang, et al., ACS Nano 2014, 8, 1554– 1565; Zhang et al., Proc. Natl. Acad. Sci. U. S. A.2014, 111, 9058–9063; Xiao, et al., Proc. Natl. Acad. Sci. U. S. A.2016, 113, E1134–E1141; Yadavalli, et al., Proc. Natl. Acad. Sci. U. S. A.2019, 116, 744–752; Xiao, et al., Proc. Natl. Acad. Sci. U. S. A.2017, 114, E7045–E7053; Torre, et al., Proc. Natl. Acad. Sci. U. S. A.2019, 116, 15378–15385; Li, et al., ACS Nano 2020, 14, 7398–7411; Kostina, et al., Soft Matter 2021, 17, 254–267; Kostina, et al., Nano Lett.2019, 19, 5732–5738; Buzzacchera, et al., Biomacromolecules 2019, 20, 712–727; Rosen, et al., Chem. Rev.2009, 109, 6275–6540; Sun, et al., Chem. Soc. Rev.2015, 44, 3900–3923; Kopitz, et al., Angew. Chem. Int. Ed. 2017, 56, 14677–14681; Ludwig, et al., Proc. Natl. Acad. Sci. U. S. A. 2019, 11, 2837–2842; Percec, et al., Bull. Chem. Soc. Jpn. 2021, 94, 900–928; Xiao, et al., Proc. Natl. Acad. Sci. U. S. A. 2018, 115, E2509–E2518; Xiao, et al., J. Am. Chem. Soc.2016, 138, 12655–12663; Zhang, et al., Angew. Chem. Int. Ed. 2014, 53, 10899– 10903; Zhang, et al., Angew. Chem. Int. Ed. 2015, 54, 4036–4040; Zhang, et al., Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 5585–5590), and Janus glycodendrimers (JGDs) (Percec, et al., J. Am. Chem. Soc. 2013, 135, 9055−9077; Zhang, et al., J. Am. Chem. Soc.2015, 137, 13334–13344; Xiao, et al., Proc. Natl. Acad. Sci. U. S. A.2016, 113, 1162–1167; Rodriguez-Emmenegger, et al., Proc. Natl. Acad. Sci. U. S. A.2019, 116, 5376–5382; Xiao, et al., Proc. Natl. Acad. Sci. U. S. A.2020, 117, 11931–11939; Kostina, et al., Angew. Chem. Int. Ed.2021, 60, 8352–8360; Klajnert-Maculewicz, et al., Chem. Commun.2023, 59 (99), 14611–14625; Sahoo, et al., Biomacromolecules 2024, 25, 1353-1370) were known to self-assemble into stable and monodisperse vesicles with predictable dimensions, known as dendrimersomes (DSs) and glycodendrimersomes (GDSs). This process is accomplished by simple injection of the JD or JGD ethanol solution into water or buffer, taking less than one minute and requiring no special equipment.

[0417] The elucidation of role of sequence and concentration of the carbohydrate part of the JGDs on the activity of their GDSs during agglutination to sugar-binding proteins was accomplished by using GDSs as models of the glycans in biological membranes. Therefore, replacing the carbohydrate part of the sequence-defined JGD with an IA provided access to the first class of IAJDs. Rapid screening of primary structure, including architecture, pKa of IAs, sequence and structure of hydrophilic and hydrophobic domains of IAJD libraries obtained via modular-orthogonal methodologies, and co-assembly of IAJDs with mRNA into DNPs, allowed rapid discovery of molecular design principles for targeted delivery to lung, liver, spleen, and lymph-nodes (LNs). One of the most active IAJDs reported is based on pentaerythritol (PE). This PE-based IAJD belongs to the simplified single-single, sSS, architecture which is synthesized in only three reaction steps, fewer than any other IAJD.

[0418] The role of the linker length interconnecting the IA to the hydrophobic fragment of IAJDs has never been investigated. Therefore, given the fact that PE-based IAJDs are made in just 3- steps, they became the target for the first series of investigations on the role of linker length interconnecting the IA to the hydrophobic domain of the IAJD. It was hypothesized that the linker length must affect the pKa of the IAJD through inductive effects and the stability of the DNPs through itsnumber of conformational isomers. While it was not possible to predict the outcome of these investigations, new principles were identified, as has been the case in all previous studies on the role of the primary structure of the IAJD on in vivo luciferase activity. The preparation of a library of 12 sSS PE- based IAJDs to be discussed in this report is outlined in Figure 1.

[0419] First, the experimental details will be described.

[0420] Materials. Pentaerythritol (Aldrich, 98%), tetrabutylammonium bromide (TBAB) (Acros Organics, 99%), 2-ethylhexyl bromide, (Acros, Organics, 95%), 1-bromooctane (Aldrich, 99%), 3- bromopropanoic acid (Aldrich, 97%), 4-bromobutyric acid (Acros, 98%), 5-bromovaleric acid (TGI, 97%), 4-dimethylaminopyridine (DMAP) (TCI, 99%), N,N-dimethylformamide (Fisher Scientific), sodium hydroxide (Fisher Scientific), thionyl chloride (Aldrich, 99%), triethylamine (TCI, 99%), potassium carbonate (Fisher Scientific), 1-(2-hydroxyethyl)piperazine (Acros, 99%), and 1-2-(2- hydroxyethoxy)ethyl piperazine (TCI, 98%) were used as received. The decision for the selection of these two ionizable amines and of the hydrophobic fragment will be discussed in a different publication. All other reagents and solvents, such as dichloromethane and acetonitrile, were sourced from commercial suppliers and used without further purification. Dichloromethane (DCM) and triethylamine (NEt3) were dried over CaH2and were distilled under N2just before being used. A 10 mM acetate buffer was made by dissolving 2.3 mM sodium acetate and 7.7 mM acetic acid in ultra-pure water. The final pH was rearranged by using 0.1 M HCl or 0.1 M NaOH solution. Nucleoside-modified messenger RNA encoding firefly luciferase (Luc-mRNA) was synthesized as previously described (Pardi, et al., Methods Mol. Biol. 2013, 969, 29–42). DPBS (Corning), OptiMEM (Gibco), UltraPure DNase / RNase-Free Distilled Water (Invitrogen), Trypsin-EDTA (0.25%, Gibco), Trypan Blue (Sigma-Aldrich), Cell Culture Lysis 5X Reagent (Promega), Luciferase Assay System (Promega) and D-luciferin sodium salt (Regis Technologies) were used as received.

[0421] Methods and Techniques. The assessment of purity and structural characteristics of intermediates and and end compounds was carried out using a range of analytical methods, which consists of a combination of thin-layer chromatography (TLC), proton and carbon nuclear magnetic resonance spectroscopy (¹H and ¹³C NMR), high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0422] 1H and13C NMR. NMR were recorded on a 400 MHz Bruker NEO NMR spectrometer with autosampling capability (¹H at 400 MHz, ¹³C at 101 MHz). Measurements were conducted at 23°C using the solvent CDCl₃. Chemical shifts (δ, ppm), with resonance multiplicities named singlet (s), doublet (d), triplet (t), multiplet (m), or broad resonance (br). Internal referencing was made with residual CHCl3from CDCl₃ (¹H: δ 7.26 ppm; ¹³C: δ 77.16 ppm) and tetramethylsilane (TMS: δ, 0 ppm). Analysis was performed with MNova 14 software.

[0423] Thin-Layer Chromatography (TLC). Reaction progress and compound purity were monitored using silica gel 60 F₂₅₄ TLC plates (E. Merck). Aromatic molecules were detected by UV light (λ = 254 nm), while non-aromatic compounds by staining with iodine vapors. Purification was achieved through flash column chromatography or column chromatography, employing silica gel (60 Å, 40–63 μm) from Silicycle and solvents specified in the experimental procedure for each compound.

[0424] High-Pressure Liquid Chromatography (HPLC). HPLC analysis was conducted to determine compound purity using a Shimadzu LC-20AD pump, a PE Nelson Analytical 900 Series integration station, a Shimadzu SPD-10A VP UV-vis detector (λ = 254 nm) and a Shimadzu RID-10A refractive index (RI) detector. A setup of two AM gel columns (two 500 Å, 10 μm columns) was employed, with tetrahydrofuran (THF) containing 5% NEt₃ as the solvent at 23°C. Compounds were identified using a refractive index (RI) detector. HPLC traces of IAJDs 313, 314, 93, 288, 325, 326, 333, 334, 317, 318, 249, 312, 319, 320, 331, and 332 are presented in Figure 2 through Figure 5.

[0425] Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry. Molecular masses were measured with a PerSeptive Biosystem Voyager-DE mass spectrometer (Framingham, MA) containing a nitrogen laser (337 nm) functioning in linear mode. Calibration was done with angiotensin II and bombesin standards. Samples for analysios were obtained by dissolving compounds in THF (5–10 mg / mL) and mixing with a matrix solution of 2,5- dihydroxybenzoic acid in THF (10 mg / mL) at a 1:5 ratio. After drying on a MALDI plate, samples were analyzed at 23°C with instrument parameters tailored to the compound's molar mass. MALDI-TOF plots of IAJDs 313, 314, 93, 288, 325, 326, 333, 334, 317, 318, 249, 312, 319, 320, 331, and 332 are presented in Figure 6 through Figure 9.

[0426] Dynamic Light Scattering (DLS). Particle size and polydispersities of DNPs were determined using a Malvern Zetasizer Nano S DLS nachine with a 4 mW He-Ne laser (633 nm) and a photodiode at 175° to the beam. Measurements were performed at 23°C in semi-micro cuvettes (10 × 10 × 45 mm, Greiner Bio-One) containing approximately 0.2 mL sample. DLS data of DNPs assembled from IAJDs 313, 93, 325, 314, 288, 326, 317, 249, 319318, 312, 320, 331, 332, 333 and 334 are presented in Figure 10 through Figure 12.

[0427] pKa Measurements of Individual IAJDs. The pKa values of IAJDs were determined by titration curve at the half-equivalence point. Solutions of IAJDs in ethanol saturated with NaCl (3 mL at 1.5 mg / mL) were incrementally titrated against 0.1 M HCl (increment of 7.5 mL), and the pH was determined with a Thermo Scientific Orion Star A121 meter and Thermo Scientific Orion 8220BNWP pH probe. Titration curves showing changes in solution pH in response to addition of a strong acid and calculated pKa for IAJDs 93, 249, 288, 312, 313, 314, 317, 318, 319, 320, 325 ,326, 331, 332, 333 and 334 are presented in Figures 13 through Figure 16. pKa values of the studied compounds are presented inTable 1 below:

[0428] Table 1. pKa of Individual IAJDs 93, 249, 288, 313, 314, 317, 318, 93, 288, 249, 312, 325, 326, 319, 320, 331, 332, 333, and 334. The * marked results are from previously published data

[0429] Production of Nucleoside-Modified Luc-mRNA. mRNA was synthesized as reported using T7 RNA polymerase on linearized DNA encoding codon-optimized Firefly Luciferase and a 101-nt poly(A) tail (Weissman, et al., Methods Mol. Biol. 2013, 969, 43–54).1-Methylpseudouridine-5’- triphosphate was employed in place of UTP. A trinucleotide cap1 analog was added cotranscriptionally. Purification followed previously reported procedures (Baiersdörfer, et al., Mol. Ther. Nucleic Acids 2019, 15, 26–35). mRNA was analyzed for RNase, dsRNA, endotoxin, and other contaminants, then frozen and maintained at -20°C.

[0430] Formulation of DNPs Co-Assembled from IAJDs and Luc-mRNA. A solution of nucleoside-modified mRNA encoding firefly luciferase (Luc-mRNA) in DNase / RNase-free distilledwater was prepared at 4.0 mg / mL. IAJDs were dissolved in ethanol at 80 mg / mL. To prepare the mixture, 12.5 μL of the Luc-mRNA solution was placed into a clean, RNase-free 1.5 mL Eppendorf tube, followed by addition of 463 μL of 10 mM acetate buffer (pH 4.0). Subsequently, 25 μL of the IAJD stock solution in ethanol was injected into the Luc-mRNA solution in acetate buffer, and the mixture was vortexed for 5 sec. Rapid screening experiments demonstrated that bimodal distribution as well as polydispersity have no influence on in vivo activity. A quantitative discussion will be reported in an independent publication.

[0431] In Vivo mRNA Delivery in Mice with DNPs. All mice used were handled in agreement with the guidelines and approval of the University of Pennsylvania Institutional Animal Care and Use Committee. Female BALB / c mice (6–8 weeks old, from Charles River Laboratories) were anesthetized with 3% isoflurane (Piramal Healthcare Limited) and injected via the retro-orbital sinus with 100 μL of DNP solution containing 10 μg of Luc-mRNA. After 4– 6 hours post injection, mice underwent luminescence characterization

[0432] Luminescence Characterization for In Vivo Transfection Experiments. Bioluminescence visualization was carried out using an IVIS Spectrum visualization system (PerkinElmer, Waltham, MA). Mice were anesthetized with 3% isoflurane (Piramal Healthcare Limited) and administered D-luciferin (Regis Technologies) intraperitoneally (i.p.) (150 mg / kg of body weight). Ten minutes after administering D-luciferin, the mice were placed on the imaging platform, maintained on isoflurane via a nose cone, and visualized at exposure times of 60, 30, or 15 sec with medium binning (binning = 8) to ensure the signal was within the operative detection range. For imaging organs, the mice were sacrificed, and the heart, lungs, liver, and spleen were collected for bioluminescence visualization. Image analysis was performed using Living Image software (PerkinElmer), and bioluminescence values were determined by measuring photon flux (photons / second) in the region of interest (ROI).

[0433] Molecular Modelling. Molecular models of the IAJDs were built using DS ViewerPro (version 5.0). Energy minimizations of the built models on the supramolecular structures were carried out with Material Studio (version 3.1) software from Accelrys. Display style and coloring were applied using BIOVIA Discovery Studio Visualizer (version 2019). The color codes used matched those in the ChemDraw structure: blue for the hydrophilic part, light and dark green for the hydrophobic part, pink for oxygen and OH groups, gray for carbons in the pentaerythritol core, light pink for the linker, and white for H groups on the aromatic ring.

[0434] General Procedure for the Synthesis of PE-IAJDs with Two-, Three-, Four- and Five-Carbon Linkers. Synthesis of IAJDs involves the alkylation of PE under phase transfer catalyzed conditions (Nouguier, et al., J. Org. Chem.1985, 50, 3296-3298; ; Nouguier, et al., Tetrahedron 1988, 44, 2477-2481; Nouguier, et al., J. Org. Chem.1987, 52, 2995-2997 ; Dumoulin, et al., Chem. Eur. J.2007, 13, 5585-5600; May, et al., Bioconjugate Chem.2016, 27, 226-237) with n-octylbromide (n = 8) and 2-ethylhexyl bromide (EH) in the presence of 50% NaOH at 80oC for 5 h. Following the addition of water, the mixture containing tetra-alkylated, tri-alkylated, and di-alkylated PE, along with the ether of the alkyl bromide, was extracted with DCM. The trialkyl compounds, n.PE (where n = 8 and EH), were then isolated by column chromatography (silica gel, hexane / ethyl acetate 100 / 1 to 60 / 1) with yields of 22% and 18%, respectively. Compounds n.PE were esterified with 2-bromoacetyl, 3-bromopropyl, 4- bromobutyl, and 5-bromopentyl chloride, which were produced in situ from the corresponding acids in dry DCM with SOCl₂ in the presence of one drop of dry DMF for 1 hour. This was followed by distillation of DCM and excess SOCl₂ using a rotary evaporator. The esterification of n.PE with bromoalkyl chloride was carried out at 0–23°C for 2 hours in dry DCM with NEt₃ base and DMAP as a supernucleophilic catalyst. This process generated compounds n.2 to n.5 with yields ranging from 60% to 92% after purification by column chromatography (silica gel, hexane / ethyl acetate 100 / 1 to 60 / 1). Compounds n.2 to n.5 were reacted with the ionizable amines N-(2-hydroxyethyl)piperazine, and N-(2- hydroxyethoxyethyl)piperazine in MeCN in the presence of K2CO3for 3h at 95oC to generate IAJDs 331, 333, 332, 334, 317, 313, 318, 314, 249, 93, 312, 288, 319, 325, 320 and 326 in isolated yields (after two purifications by column chromatography, silicagel, DCM / MeOH 50 / 1 to 20 / 1) reported after their name in Figure 1. The purity of all IAJDs, as determined by a combination of techniques including TLC,1HNMR,13C-NMR, HPLC, and MALDI-TOF, was over 99%. All IAJDs from Figure 1 were co- assembled with Luc-mRNA using the ethanol injection method into an acetate buffer containing Luc- mRNA (pH 4.0).

[0435] 3-((2-Ethylhexyl)oxy)-2,2-bis(2-Ethylhexyl)oxy)methyl)propan-1-ol (EH.PE). Pentaerythritol (12.00 g, 88.1 mmol, 1 equiv) was added to a freshly made NaOH solution (140.8 g NaOH and 120 mL water) and the mixture was stirred for 1 h at 80 °C.3-(Bromomethyl)heptane (51.07 g, 264.4 mmol, 3 equiv) and tetrabutylammonium bromide (TBAB) (3.55 g, 11.01 mmol, 0.125 equiv) were then added to the mixture and heating was continued for 5 h. After 5 hours, the reaction was halted and allowed to cool to room temperature. The resulting mixture was then diluted with 400 mL of water. The organic layer was extracted three times with 40 mL of DCM. The combined organic extracts were dried over anhydrous MgSO₄, filtered, and concentrated. The residue was purified by column chromatography (SiO₂) using hexane / ethyl acetate (100:1 to 60:1) as the eluent, yielding the title compound as a colorless oil (7.53 g, 18.1%).1H NMR (400 MHz, CDCl3) δ 3.71 (d, 2 H, CH2OH), 3.41(s, 6H, -CH2OCH2CH(CH2CH3)(CH2)3CH3), 3.27 (d, 6 H, -CH2OCH2CH(CH2CH3)(CH2)3CH3), 3.14 (s, 1H, CH2OH), 1.47 (m, 3 H, -CH2OCH2CH(CH2CH3)(CH2)3CH3), 1.40 – 1.20 (br, 24 H, - CH2OCH2CH(CH2CH3)(CH2)3CH3), 0.88 (m, 18 H, -CH2OCH2CH(CH2CH3)(CH2)3CH3).13C NMR (101 MHz, CDCl3) δ 74.4, 71.8, 70.0, 67.0, 45.0, 41.1, 39.7, 39.6, 39.1, 34.7, 34.5, 31.9, 31.6, 30.7, 29.1, 28.8, 25.3, 25.2, 24.0, 23.1, 22.8, 22.7, 20.7, 14.1, 14.1, 11.1.

[0436] 3-((2-Ethylhexyl)oxy)-2,2-bis(((2-ethylhexyl)oxy)methyl)propyl 2-bromoacetate (EH.2).2-Bromoacetic acid (176 mg, 1.27 mmol, 2.0 equiv) was dissolved in 10 mL dry DCM and 1 drop of DMF was added. SOCl2(91 mg, 0.762 mmol, 1.2 equiv) was added and the mixture was stirred at 23 °C for 1 h. Afterwards, DCM and excess SOCl₂ were removed under reduced pressure, yielding 2- bromoacetyl chloride. Compound EH.PE (300 mg, 0.635 mmol, 1.0 equiv), NEt3(77 mg, 0.762 mmol, 1.2 equiv) were dissolved in 10 mL anhydrous DCM. To this mixture was added the DCM solution (10 mL) and the above 2-bromoacetyl chloride dropwise at 0 °C. The mixture was allowed to warm to 23°C with stirring for 2 hours. Subsequently, 20 mL of water was added, and the mixture was extracted three times with 20 mL of DCM. The organic phase was collected, dried over anhydrous MgSO₄, and filtered. The filtrate was concentrated and purified by column chromatography (SiO₂) using hexane / ethyl acetate (40:1) as the eluent, yielding the title compound as a colorless oil (350 mg, 92%).1H NMR (400 MHz, CDCl3) δ 4.20 (s, 2 H, -CH2COO), 3.82 (s, 2 H, -OCOCH2Br), 3.36 – 3.24 (m, 12 H, - CH2OCH2(CH2)6CH3), 1.45 (m, 6 H, -OCH2CH2(CH2)5CH3), 1.28 (br, 30 H, -OCH2CH2(CH2)5CH3), 0.88 (m, 18 H, -OCH2CH(CH2CH3)(CH2CH2CH2CH3).13C NMR (101 MHz, CDCl3) δ 166.9, 77.2, 74.2, 69.6, 69.5, 66.0, 44.9, 39.7, 30.7, 29.7, 29.1, 29.1, 24.0, 23.1, 14.1, 11.1.

[0437] 3-((2-Ethylhexyl)oxy)-2,2-bis(((2-ethylhexyl)oxy)methyl)propyl 3-bromopropanoate (EH.3).3-Bromopropanoic acid (1.05 g, 6.87 mmol, 1.3 equiv) was dissolved in 15 mL anhydrous DCM and 1 drop of DMF was added. SOCl2(1.63 g, 13.75 mmol, 2.6 equiv) was added and the mixture was stirred at 23 °C for 1 h. Afterwards, DCM and excess SOCl2were removed under vacuum to give the 3- bromopropanoyl chloride. Compound EH.PE (2.5 g, 5.29 mmol, 1.0 equiv), dry NEt3(0.69 g, 6.87 mmol, 1.3 equiv), and 4-dimethylaminopyridine (1.05 mg, 1.05 mmol, 0.2 equiv) were dissolved in 10 mL anhydrous DCM. To this mixture was added the dry DCM solution (10 mL) of the above 3- bromopropanoyl chloride dropwise at 0 °C. The mixture was allowed to warm to 23°C while stirring for 2 hours. Subsequently, 20 mL of water was added, and the mixture was extracted three times with 20 mL of DCM. The organic phase was collected, dried over anhydrous MgSO₄, and filtered. The filtrate was then concentrated and purified by column chromatography (SiO₂) using hexane / ethyl acetate (40:1) as the eluent, yielding the title compound as a colorless oil (2.93 g, 91.2%).1H NMR (400 MHz, CDCl3) δ 4.12 (s, 2 H, -CH2COO), 3.45 (t, 2 H, -OCOCH2CH2CH2Br), 3.40 – 3.31 (m, 12 H, -CH2OCH2(CH2)4CH3), 2.48 (t, 2 H, -OCOCH2CH2CH2Br), 2.24-2.06 (m, 2 H, -CH2OCOCH2CH2CH2Br), 1.54 – 1.46 (m, 12 H, 3 × -OCH2CH2(CH2)3CH3), 1.28 (br, 18 H, 3 × -OCH2CH2(CH2)3CH3), 0.90-0.84 (m, 9 H, 3 × - O(CH2)5CH3).13C NMR (101 MHz, CDCl3) δ 172.3, 71.7, 69.6, 64.5, 53.5, 44.6, 32.8, 32.7, 31.8, 29.7, 28.0, 26.0, 22.8, 14.2. 3-((2-Ethylhexyl)oxy)-2,2-bis(((2-ethylhexyl)oxy)methyl)propyl 4-bromobutanoate (EH.4).4-Bromobutyric acid (0.96 g, 5.77 mmol, 1.3 equiv) was dissolved in 15 mL dry DCM and 1drop of DMF was added. SOCl2(1.37 g, 11.54 mmol, 2.6 equiv) was added and the mixture was stirred at 23 °C for 1 h. Afterwards, DCM and excess SOCl2were removed under vacuum to give the 4- bromopropanoyl chloride. Compound EH.PE (2.1 g, 4.44 mmol, 1.0 equiv), dry NEt3(584 mg, 5.77 mmol, 1.3 equiv) and 4-dimethylaminopyridine (108 mg, 0.8 mmol, 0.2 equiv) were dissolved in 10 mL anhydrous DCM. To this mixture was added the dry DCM solution (10 mL) of the above 4- bromopropanoyl chloride dropwise at 0 °C. The mixture was allowed to warm to 23 °C with stirring during a 2 h period. Afterwards, water (20 mL) was added, and the mixture was extracted three times with DCM (20 mL). The organic phase was collected, dried over anhydrous MgSO₄, and filtered. The filtrate was concentrated and purified by column chromatography (SiO₂) using hexane / ethyl acetate (40:1) as the eluent, yielding the title compound as a colorless oil (2.32 g, 84%).1H NMR (400 MHz, CDCl3) δ 4.09 (s, 2 H, -CH2COO), 3.45 (t, 2 H, -OCOCH2CH2CH2Br), 3.34 (s, 6H, -CH2OCH2CH(CH2CH3)(CH2)3CH3), 3.23 (d, 6 H, -CH2OCH2CH(CH2CH3)(CH2)3CH3), 2.48 (t, 2 H, -OCOCH2CH2CH2Br), 2.16 (m, 2H, - OCOCH2CH2CH2Br), 1.45 (m, 3 H, -CH2OCH2CH(CH2CH3)(CH2)3CH3), 1.39 – 1.14 (br, 24 H, - CH2OCH2CH(CH2CH3)(CH2)3CH3), 0.88 (m, 18 H, -CH2OCH2CH(CH2CH3)(CH2)3CH3).13C NMR (101 MHz, CDCl3) δ 174.5, 172.2, 74.2, 74.1, 69.8, 69.7, 69.3, 64.4, 64.2, 44.7, 44.6, 39.7, 32.7, 32.5, 31.6, 30.7, 29.2, 27.9, 24.0, 23.1, 22.7, 14.1, 11.1.

[0438] 3-((2-Ethylhexyl)oxy)-2,2-bis(((2-ethylhexyl)oxy)methyl)propyl 5-bromopentanoate (EH.5).5-Bromovaleric acid (2.49 g, 13.7 mmol, 1.3 equiv) was dissolved in 15 mL anhydrous DCM and 1 drop of DMF was added. SOCl2(3.27 g, 27.5 mmol, 2.6 equiv) was added and the mixture was stirred at 23 °C for 1 h. Afterwards, DCM and excess SOCl2were removed under vacuum to give the 5- bromovaleryl chloride. Compound EH.PE (5.0 g, 10.06 mmol, 1.0 equiv), dry NEt3(1.39 g, 13.7 mmol, 1.3 equiv) and 4-dimethylaminopyridine (258 mg, 2.1 mmol, 0.2 eq.) were dissolved in 10 mL anhydrous DCM. To this mixture was added the dry DCM solution (10 mL) of the above 5-bromovaleryl chloride dropwise at 0 °C. The mixture was allowed to warm to 23°C while stirring for 2 hours. Afterward, 20 mL of water was added, and the mixture was extracted three times with 20 mL of DCM. The combined organic phases were dried over anhydrous MgSO₄ and filtered. The filtrate was then concentrated and purified by column chromatography (SiO₂) using hexane / ethyl acetate (40:1) as the eluent, yielding the title compound as a colorless oil (5.3 g, 78.8%).1H NMR (400 MHz, CDCl3) δ 4.10 (s, 2 H, -CH2COO), 3.54 (t, 2 H, -OCOCH2CH2CH2Br), 3.25 (d, 6 H, -CH2OCH2CH(CH2CH3)-), 2.53 (t, 2 H, - OCOCH2CH2CH2Br), 2.17 (m, 4 H, -CH2OCOCH2CH2CH2CH2Br), 1.51 (m, 6 H, -OCH2CH2(CH2)5CH3), 1.45–1.41 (m, 3H, CH2OCH2CH(CH2CH3)-), 1.34–1.23 (br, 24 H, - OCH2CH(CH2CH3)(CH2CH2CH2CH3), 0.91–0.85 (m, 18 H, -OCH2CH(CH2CH3)(CH2CH2CH2CH3).13C NMR (101 MHz, CDCl3) δ 173.4, 71.8, 69.7, 64.3, 59.3, 57.8, 57.7, 53.2, 53.0, 44.6, 32.4, 32.0, 29.8, 29.8, 29.3, 26.3, 22.8, 22.3, 14.2.

[0439] 3-((2-Ethylhexyl)oxy)-2,2-bis(((2-ethylhexyl)oxy)methyl)propyl 2-(4-(2- hydroxyethyl)piperazin-1-yl)acetate (EH.2.EO, IAJD 333). A mixture of compound EH.2 (200 mg, 0.337 mmol, 1.0 equiv), 2-(piperazin-1-yl)ethan-1-ol (66 mg, 0.506 mmol, 1.5 equiv), K₂CO₃ (65 mg, 0.506 mmol, 1.5 equiv), and MeCN (10 mL) was heated to 95°C and refluxed for 3 hours.89After cooling to 23°C, the MeCN was removed under vacuum. Subsequently, 20 mL of water was added, and the mixture was extracted three times with 20 mL of DCM. The combined organic phases were dried over anhydrous MgSO₄ and filtered. The filtrate was concentrated to approximately 3 mL and purified by column chromatography (SiO₂) using DCM / MeOH (50:1 to 20:1) as the eluent. The purified product was then dried over anhydrous MgSO₄, and after filtration and solvent evaporation, the title compound wasobtained as a light-yellow oil (200 mg, 92%).1H NMR(400 MHz, CDCl3) δ 4.14 (s, 2 H, -CH2OCO-),3.60 (t, 2H, -NCH2CH2OH), 3.33 (s, 6 H, -CH2OCH2CH(CH2CH3)-), 3.26–3.16 (d, 8 H, - CH2OCH2CH(CH2CH3)- and -COOCH2), 2.68–2.50 (br, 10 H, - N(CH2CH2)2NCH2CH2OH), 1.45 (m, 3H, CH2OCH2CH(CH2CH3)-), 1.38–1.16 (br, 24 H, -OCH2CH(CH2CH3)(CH2CH2CH2CH3), 0.95–0.79 (m, 18 H, -OCH2CH(CH2CH3)(CH2CH2CH2CH3).13C NMR (101 MHz, CDCl3) δ 170.1, 77.2, 74.2, 69.6, 64.4, 59.2, 59.1, 57.7, 52.9, 52.6, 44.7, 39.7, 30.7, 29.1, 24.0, 23.1, 14.1, 11.1. Purity by HPLC: 99+%. MALDI-TOF MS m / z of [M + H]+calculated for C37H75N2O6: 644.0; Found: 645.0.

[0440] 3-((2-Ethylhexyl)oxy)-2,2-bis(((2-ethylhexyl)oxy)methyl)propyl 2-(4-(2-(2- hydroxyethoxy)ethyl)piperazin-1-yl)acetate (EH.2.2EO, IAJD 334). A mixture of compound EH.2 (200 mg, 0.337 mmol, 1.0 equiv), 2-(2-(piperazin-1-yl)ethoxy)ethan-1-ol (88 mg, 0.506 mmol, 1.5 equiv), K₂CO₃ (65 mg, 0.506 mmol, 1.5 equiv), and MeCN (10 mL) was heated to 95°C and refluxed for 3 hours. The reaction mixture was then cooled to 23°C, and the MeCN was removed under vacuum. Subsequently, 20 mL of water was added, and the mixture was extracted three times with 20 mL of DCM. The combined organic phases were dried over anhydrous MgSO₄ and filtered. The filtrate was concentrated to approximately 3 mL and purified by column chromatography (SiO₂) using DCM / MeOH (50:1 to 20:1) as the eluent. Finally, the product was dried over anhydrous MgSO₄, and after filtration and solventevaporation, the title compound was obtained as a light-yellow oil (210 mg, 91%).1H NMR(400 MHz,CDCl3) δ 4.13 (s, 2 H, -CH2OCO-), 3.75–3.55 (m, 6 H, -NCH2CH2OCH2CH2OH), 3.32 (s, 6 H, - CH2OCH2CH(CH2CH3)-), 3.27–3.11 (m, 8 H, -CH2OCH2CH(CH2CH3)- and -COOCH2), 2.60 (br, 10 H, - N(CH2CH2)2NCH2CH2OH), 1.44 (m, 3H, CH2OCH2CH(CH2CH3)-) and -COOCH2CH2CH2CH2), 1.34– 1.15 (br, 24 H, -OCH2CH(CH2CH3)(CH2CH2CH2CH3), 0.93–0.75 (m, 18 H, - OCH2CH(CH2CH3)(CH2CH2CH2CH3).13C NMR (101 MHz, CDCl3) δ 174.3, 77.2, 74.2, 72.4, 69.6, 67.4, 64.4, 62.1, 57.8, 53.0, 52.8, 44.7, 39.6, 30.7, 29.1, 24.0, 23.1, 14.1, 11.1. Purity by HPLC: 99+%. MALDI-TOF MS m / z of Purity by HPLC: 99+%. MALDI-TOF MS m / z of [M + H]+calculated for C39H79N2O7: 687.6; Found: 688.7; [M + Na]+calculated for C39H78N2O7Na: 709.6; Found: 710.3.

[0441] 3-(Pentadecyloxy)-2,2-bis((pentadecyloxy)methyl)propan-1-ol (8.PE). Pentaerythritol (12.00 g, 88.1 mmol, 1 equiv) was added to a freshly made NaOH solution (140.8 g NaOH and 120 mL water) and the mixture was stirred for 1 h at 80 °C.1-Bromooctane (51.07 g, 264.4 mmol, 3 equiv), and tetrabutylammonium bromide (TBAB) (3.55 g, 11.01 mmol, 0.125 equiv) were then added to the mixture and heating was continued for 5 h. After 5 hours, the reaction was halted and allowed to cool to room temperature. The resultant mixture was diluted with 400 mL of water and extracted three times with 40 mL of DCM. The combined organic phases were dried over anhydrous MgSO₄ and filtered. The filtrate was concentrated and purified by column chromatography (SiO₂) using hexane / ethyl acetate (100:1 to60:1) as the eluent, yielding the title compound as a colorless oil (9.14 g, 21.9%).1H NMR(400 MHz,CDCl3) δ 3.70 (d, 2 H, CH2OH), 3.43(s, 6H, -CH2OCH2(CH2)6CH3), 3.38 (t, 6 H, -CH2OCH2(CH2)6CH3), 3.14(br, 1 H, CH2OH), 1.63 – 1.45 (m, 6H, -OCH2CH2(CH2)5CH3), 1.28 (br, 30 H, - OCH2CH2(CH2)5CH3), 0.88 (m, 9 H, -O(CH2)7CH3).13C NMR (101 MHz, CDCl3) δ 71.8, 71.5, 66.7, 44.7, 31.9, 31.6, 29.6, 29.4, 29.3, 26.2, 22.7, 14.1.

[0442] 3-(Octyloxy)-2,2-bis((octyloxy)methyl)propyl 2-bromoacetate (8.2).2-Bromoacetic acid (177 mg, 1.27 mmol, 1.2 equiv) was dissolved in 10 mL of dry DCM, and 1 drop of DMF was added. After that, SOCl₂ (252 mg, 2.12 mmol, 2 equiv) was then added to the mixture, and allowed to stir at 23°C for 1 hour. Afterwards, DCM and excess SOCl2were removed under vacuum to give the 5- bromovaleric chloride. Compound 8.PE (500 mg, 1.06 mmol, 1.0 equiv), dry NEt3(177 mg, 1.27 mmol, 1.2 equiv) were dissolved in 10 mL anhydrous DCM. To this mixture was added the dry DCM solution (10 mL) of the above 2-bromoacetic chloride dropwise at 0 °C. The mixture was stirred and allowed to warm to 23°C over 2 hours. Then, 20 mL of water was added, and the mixture was extracted three times with 20 mL of DCM. The combined organic phases were dried over anhydrous MgSO₄ and filtered. The filtrate was then concentrated and purified by column chromatography (SiO₂) using hexane / ethyl acetate(40:1) as the eluent, yielding the title compound as a colorless oil (380 mg, 60%).1H NMR(400 MHz,CDCl3) δ 4.24 (s, 2 H, -CH2COO), 4.04 (s, 2 H, -OCOOCH2Br), 3.44 – 3.26 (m, 12 H, - CH2OCH2(CH2)6CH3), 1.51 (m, 6 H, -OCH2CH2(CH2)5CH3), 1.29 (br, 30 H, -OCH2CH2(CH2)5CH3), 0.88 (m, 9 H, -O(CH2)7CH3).13C NMR (101 MHz, CDCl3) δ 167.1, 77.2, 71.6, 69.4, 69.3, 66.0, 44.6, 41.0, 31.9, 29.6, 29.5, 29.3, 26.2, 22.7, 14.1.

[0443] 3-(Octyloxy)-2,2-bis((octyloxy)methyl)propyl 3-bromopropanoate (8.3).3- Bromopropanoic acid (2.10 g, 13.7 mmol, 1.3 equiv) was dissolved in 15 mL dry DCM and 1 drop of DMF was added. SOCl2(1.99 g, 27.5 mmol, 2.6 equiv) was added and the mixture was stirred at 23 °C for 1 h. Afterwards, DCM and excess SOCl2were removed under reduced pressure to give the 3- bromopropanoyl chloride. Compound 8.PE (5.0 g, 10.5 mmol, 1.0 equiv), dry NEt3(1.39 g, 13.7 mmol, 1.3 equiv) and 4-dimethylaminopyridine (0.258 g, 2.1 mmol, 0.2 equiv) were dissolved in 10 mL dryDCM. To this mixture was added the dry DCM solution (10 mL) of the above 3-bromopropanoyl chloride dropwise at 0 °C. The mixture was stirred and allowed to warm to 23°C over 2 hours. Subsequently, 20 mL of water was added, and the mixture was extracted three times with 20 mL of DCM. The combined organic phases were dried over anhydrous MgSO₄ and filtered. The filtrate was concentrated and purified by column chromatography (SiO₂) using hexane / ethyl acetate (40:1) as the eluent, yielding the titlecompound as a colorless oil (4.9 g, 76.2%).1H NMR(400 MHz, CDCl3) δ 4.12 (s, 2 H, -CH2COO), 3.43(t, 2 H, -OCOCH2CH2CH2Br), 3.38–3.30 (m, 12 H, -CH2OCH2(CH2)6CH3), 2.37–2.31 (m, 2 H, - CH2OCOCH2CH2CH2Br), 1.67–1.57 (m, 2 H, -CH2OCOCH2CH2CH2Br), 1.51 (m, 6 H, - OCH2CH2(CH2)5CH3), 1.29 (br, 30 H, -OCH2CH2(CH2)5CH3), 0.89 (m, 9 H, -O(CH2)7CH3).13C NMR (101 MHz, CDCl3) δ 173.0, 77.4, 71.7, 68.5, 68.5, 64.5, 44.5, 32.5, 32.5, 31.9, 29.5, 29.4, 29.5, 28.0, 26.1, 22.5, 14.0.

[0444] 3-(Octyloxy)-2,2-bis((octyloxy)methyl)propyl 4-bromobutanoate (8.4).4- Bromobutyric acid (2.29 g, 13.74 mmol, 1.3 equiv) was dissolved in 15 mL dry DCM and 1 drop of DMF was added. SOCl2(3.27 g, 27.48 mmol, 2.6 equiv) was added and the vacuum to give the 4- bromopropanoyl chloride. Compound 8.PE (5.0 g, 10.5 mmol, 1.0 equiv), dry NEt3(1.39 g, 13.74 mmol, 1.0 equiv) and 4-dimethylaminopyridine (258 mg, 2.1 mmol, 0.2 equiv) were dissolved in 10 mL dry DCM. To this mixture was added the dry DCM solution (10 mL) and the above 4-bromopropanoyl chloride dropwise at 0 °C. The mixture was stirred and allowed to warm to 23°C over a 2-hour period. Afterward, 20 mL of water was added, and the mixture was extracted three times with 20 mL of DCM. The combined organic phases were dried over anhydrous MgSO₄ and filtered. The filtrate was then concentrated and purified by column chromatography (SiO₂) using hexane / ethyl acetate (40:1) as theeluent, yielding the title compound as a colorless oil (5.3 g, 80.6%).1H NMR(400 MHz, CDCl3) δ 4.13(s, 2 H, -CH2COO), 3.46 (t, 2 H, -OCOCH2CH2CH2Br), 3.39 – 3.31 (m, 12 H, -CH2OCH2(CH2)6CH3), 2.49 (t, 2 H, -OCOCH2CH2CH2Br), 2.18 (m, 2 H, -CH2OCOCH2CH2CH2Br), 1.51 (m, 6 H, - OCH2CH2(CH2)5CH3), 1.29 (br, 30 H, -OCH2CH2(CH2)5CH3), 0.88 (m, 9 H, -O(CH2)7CH3).13C NMR (101 MHz, CDCl3) δ 172.3, 77.2, 71.6, 69.5, 69.5, 64.4, 44.4, 32.7, 32.6, 31.9, 29.6, 29.5, 29.3, 27.9, 26.2, 22.7, 14.1.

[0445] 3-(Octyloxy)-2,2-bis((octyloxy)methyl)propyl 5-bromopentanoate (8.5).5- Bromovaleric acid (2.74 g, 13.75 mmol, 1.3 equiv) was dissolved in 15 mL dry DCM and 1 drop of DMF was added. SOCl2(3.27 g, 27.5 mmol, 2.6 equiv) was added and the mixture was stirred at 23 °C for 1 h. Afterwards, DCM and excess SOCl2were removed under vacuum to give the 5-bromovaleric chloride. Compound 8.PE (5.0 g, 10.57 mmol, 1.0 equiv), dry NEt3(1.39 g, 13.75 mmol, 1.3 equiv) and 4- dimethylaminopyridine (0.258 g, 2.1 mmol, 0.2 equiv) were dissolved in 10 mL dry DCM. To this mixture was added the dry DCM solution (10 mL) and the above 5-bromovaleric chloride dropwise at 0°C. The mixture was stirred and allowed to warm to 23°C over a 2-hour duration. Subsequently, 20 mL of water was added, and the mixture was extracted three times with 20 mL of DCM. The combined organic phases were dried over anhydrous MgSO₄ and filtered. The filtrate was then concentrated and purified by column chromatography (SiO₂) using hexane / ethyl acetate (40:1) as the eluent, yielding the titlecompound as a colorless oil (5.3 g, 78.8%).1H NMR(400 MHz, CDCl3) δ 4.15 (s, 2 H, -CH2COO), 3.53(t, 2 H, -OCOCH2CH2CH2Br), 3.39 – 3.33 (m, 12 H, -CH2OCH2(CH2)6CH3), 2.47 (t, 2 H, - OCOCH2CH2CH2Br), 2.18 (m, 4 H, -CH2OCOCH2CH2CH2CH2Br), 1.53 (m, 6 H, -OCH2CH2(CH2)5CH3), 1.30 (br, 30 H, -OCH2CH2(CH2)5CH3), 0.89 (m, 9 H, -O(CH2)7CH3).13C NMR (101 MHz, CDCl3) δ 173.7, 71.7, 69.3, 64.1, 59.0, 57.3, 57.2, 53.1, 53.0, 45.0, 32.1, 32.3, 29.7, 29.7, 29.7, 26.1, 22.5, 22.1, 14.0.

[0446] 3-(Octyloxy)-2,2-bis((octyloxy)methyl)propyl 2-(4-(2-hydroxyethyl)piperazin-1-yl) acetate (8.2.EO, IAJD 331). A mixture of compound 8.2 (500 mg, 0.84 mmol, 1.0 equiv), 2-(piperazin- 1-yl)ethan-1-ol (131 mg, 1.01 mmol, 1.2 equiv), K2CO3(139 mg, 1.01 mmol, 1.2 equiv), and MeCN (15 mL) was heated to 95 °C and refluxed for 3 h. The reaction mixture was cooled to 23°C, and the MeCN was removed under vacuum. Subsequently, 20 mL of water was added, and the mixture was extracted three times with 20 mL of DCM. The combined organic phases were dried over anhydrous MgSO₄ and filtered. The filtrate was concentrated to approximately 3 mL and purified by column chromatography (SiO₂) using DCM / MeOH (50:1 to 20:1) as the eluent. The product was dried over anhydrous MgSO₄, and after filtration and solvent evaporation, the title compound was obtained as a light-yellow oil (500mg, 92%).1H NMR(400 MHz, CDCl3) δ 4.16 (s, 2 H, -CH2OCO-), 3.61 (t, 2 H, -NCH2CH2OH), 3.39 –3.30 (m, 12 H, -CH2OCH2(CH2)6CH3), 3.21 (s, 2H, -COOCH2-), 2.70–2.51 (br, 10 H, - N(CH2CH2)2NCH2CH2OH); 1.561 (m, 6 H, -OCH2CH2(CH2)5CH3), 1.37–1.19 (br, 30 H, - OCH2CH2(CH2)5CH3), 0.92–0.81 (m, 9 H, -O(CH2)7CH3).13C NMR (101 MHz, CDCl3) δ 170.1, 77.2, 71.6, 69.5, 64.4, 59.2, 59.2, 57.6, 52.9, 52.6, 44.5, 31.9, 29.6, 29.5, 29.3, 26.2, 22.7, 14.1. Purity by HPLC: 99+%. MALDI-TOF MS m / z of [M + H]+calculated for C37H75N2O6: 643.6; Found: 643.0.

[0447] 3-(Octyloxy)-2,2-bis((octyloxy)methyl)propyl 2-(4-(2-(2- hydroxyethoxy)ethyl)piperazin-1-yl)acetate (8.2.EO, IAJD 332). A mixture of compound 8.2 (630 mg, 1.06 mmol, 1 equiv), 2-(2-(piperazin-1-yl)ethoxy)ethan-1-ol (221 mg, 1.27 mmol, 1.2 equiv), K2CO3(176 mg, 1.27 mmol, 1.2 equiv) and MeCN (15 mL) was heated to 95 °C and refluxed for 3 h. The reaction mixture was cooled to 23°C, and the MeCN was removed under reduced pressure. Subsequently, 20 mL of water was added, and the mixture was extracted three times with 20 mL of DCM. The combined organic phases were dried over anhydrous MgSO₄ and filtered. The filtrate was concentrated to approximately 3 mL and purified by column chromatography (SiO₂) using DCM / MeOH (50:1 to 20:1) as the eluent. The product was then dried over anhydrous MgSO₄. Finally, filtration and evaporation of thesolvent yielded the title compound as a light-yellow oil (550 mg, 76%).1H NMR(400 MHz, CDCl3) δ4.15 (s, 2 H, -CH2OCO-), 3.71–3.58 (m, 6 H, -NCH2CH2OCH2CH2OH), 3.40 – 3.28 (m, 12 H, - CH2OCH2(CH2)6CH3), 3.18 (s, 2H, -COOCH2-), 2.60 (br, 10 H, - N(CH2CH2)2NCH2CH2O-), 1.50 (m, 6H, -OCH2CH2(CH2)5CH3), 1.27 (br, 30 H, -OCH2CH2(CH2)5CH3), 0.93 – 0.76 (m, 9 H, -O(CH2)7CH3).13C NMR (101 MHz, CDCl3) δ 170.1, 77.2, 72.4, 71.6, 69.4, 67.4, 64.3, 62.1, 59.2, 57.8, 53.0, 52.8, 44.5, 31.9, 29.6, 29.5, 29.3, 26.2, 22.7, 14.1. Purity by HPLC: 99+%. MALDI-TOF MS m / z of [M + H]+calculated for C39H79N2O7: 687.6; Found: 687.5.

[0448] 3-(Octyloxy)-2,2-bis((octyloxy)methyl)propyl 2-(4-(2-hydroxyethyl)piperazin-1-yl) acetate (8.2.EO, IAJD 331). A mixture of compound 8.2 (500 mg, 0.84 mmol, 1.0 equiv), 2-(piperazin- 1-yl)ethan-1-ol (131 mg, 1.01 mmol, 1.2 equiv), K2CO3(139 mg, 1.01 mmol, 1.2 equiv), and MeCN (15 mL) was heated to 95 °C and refluxed for 3 h. After cooling the reaction mixture to 23°C, the MeCN was removed under reduced pressure. The resulting mixture was then extracted three times with 20 mL of DCM after adding 20 mL of water. The combined organic phases were dried over anhydrous MgSO₄ and filtered. The filtrate was concentrated to approximately 3 mL and purified by column chromatography (SiO₂) using DCM / MeOH (50:1 to 20:1) as the eluent. The product was dried over anhydrous MgSO₄. Finally, filtration and evaporation of the solvent yielded the title compound as a light-yellow oil (500 mg,92%).1H NMR(400 MHz, CDCl3) δ 4.16 (s, 2 H, -CH2OCO-), 3.61 (t, 2 H, -NCH2CH2OH), 3.39 – 3.30(m, 12 H, -CH2OCH2(CH2)6CH3), 3.21 (s, 2H, -COOCH2-), 2.70–2.51 (br, 10 H, - N(CH2CH2)2NCH2CH2OH); 1.561 (m, 6 H, -OCH2CH2(CH2)5CH3), 1.37–1.19 (br, 30 H, - OCH2CH2(CH2)5CH3), 0.92–0.81 (m, 9 H, -O(CH2)7CH3).13C NMR (101 MHz, CDCl3) δ 170.1, 77.2, 71.6, 69.5, 64.4, 59.2, 59.2, 57.6, 52.9, 52.6, 44.5, 31.9, 29.6, 29.5, 29.3, 26.2, 22.7, 14.1. Purity by HPLC: 99+%. MALDI-TOF MS m / z of [M + H]+calculated for C37H75N2O6: 643.6; Found: 643.0.

[0449] 3-(Octyloxy)-2,2-bis((octyloxy)methyl)propyl 2-(4-(2-(2- hydroxyethoxy)ethyl)piperazin-1-yl)acetate (8.2.EO, IAJD 332). A mixture of compound 8.2 (630 mg, 1.06 mmol, 1 equiv), 2-(2-(piperazin-1-yl)ethoxy)ethan-1-ol (221 mg, 1.27 mmol, 1.2 equiv), K2CO3(176 mg, 1.27 mmol, 1.2 equiv) and MeCN (15 mL) was heated to 95 °C and refluxed for 3 h. The reaction mixture was cooled to 23°C, and the MeCN was removed under reduced pressure. Then, 20 mL of water was added, and the mixture was extracted three times with 20 mL of DCM. The combined organic phases were dried over anhydrous MgSO₄ and filtered. The filtrate was concentrated to approximately 3 mL and purified by column chromatography (SiO₂) using DCM / MeOH (50:1 to 20:1) as the eluent. The product was further dried over anhydrous MgSO₄. Filtration and evaporation of the solvent yielded the titlecompound as a light-yellow oil (550 mg, 76%).1H NMR(400 MHz, CDCl3) δ 4.15 (s, 2 H, -CH2OCO-),3.71–3.58 (m, 6 H, -NCH2CH2OCH2CH2OH), 3.40 – 3.28 (m, 12 H, -CH2OCH2(CH2)6CH3), 3.18 (s, 2H, - COOCH2-), 2.60 (br, 10 H, - N(CH2CH2)2NCH2CH2O-), 1.50 (m, 6H, -OCH2CH2(CH2)5CH3), 1.27 (br,30 H, -OCH2CH2(CH2)5CH3), 0.93 – 0.76 (m, 9 H, -O(CH2)7CH3).13C NMR (101 MHz, CDCl3) δ 170.1, 77.2, 72.4, 71.6, 69.4, 67.4, 64.3, 62.1, 59.2, 57.8, 53.0, 52.8, 44.5, 31.9, 29.6, 29.5, 29.3, 26.2, 22.7, 14.1. Purity by HPLC: 99+%. MALDI-TOF MS m / z of [M + H]+calculated for C39H79N2O7: 687.6; Found: 687.5.

[0450] 3-((2-Ethylhexyl)oxy)-2,2-bis(((2-ethylhexyl)oxy)methyl)propyl 3-(4-(2- hydroxyethyl)piperazin-1- yl)propanoate (EH.3.EO, IAJD 313) A mixture of compound EH.3 (0.40 g,0.65 mmol, 1.0 equiv), 2-(piperazin-1-yl)ethan-1-ol (102 mg, 0.78mmol, 1.2 equiv), K2CO3(109 mg,0.78 mmol, 1.2 equiv) and MeCN (25 mL) was heated to 82 °C andrefluxed for 3 h. The reaction mixturewas cooled to 23 °C and MeCN was removed under reduced pressure. Then water (20 mL) was added,and the resulting mixture was extracted with DCM (20 mL) forthree times. The organic phases werecollected and dried over anhydrous MgSO4. After filtration, the obtained filtrate was concentrated to around 3 mL and purified by column chromatography (SiO2) with DCM / MeOH = 50 / 1 to 20 / 1 as theeluent. Then the obtained product was dried over anhydrous MgSO4.Filtration and evaporation of thesolvent yielded the title compound as a light-yellow oil (310 mg, 71.7%).1H NMR (400 MHz, CDCl3) δ4.11 (s, 2 H, -CH2OCO-), 3.61 (t, 2H, -NCH2CH2OH), 3.33 (s, 6 H, - CH2OCH2CH(CH2CH3)-),3.23 (d, 6 H, -CH2OCH2CH(CH2CH3)-), 2.69 (t, 2H, -COOCH2CH2N), 2.54–2.47 (br, 12 H, -N(CH2CH2)2NCH2CH2OH) and (-COOCH2CH2N), 1.48–1.34 (m, 3 H, -CH2OCH2CH(CH2CH3)-), 1.32–1.20 (br, 24 H, -OCH2CH(CH2CH3)(CH2CH2CH2CH3), 0.90–0.83 (m,18 H, -OCH2CH(CH2CH3)(CH2CH2CH2CH3). 13C NMR(101 MHz, CDCl3) δ 172.1, 74.2, 69.7, 64.2,59.2,57.7, 53.6, 53.0, 52.8, 44.7, 39.7, 32.4, 30.7, 24.0, 23.1, 14.1, 11.1. Purity by HPLC: 99+%. MALDI-TOFMS m / z of Purity by HPLC: 99+%. MALDI-TOF MS m / z of [M + H]+ calculated forC38H76N2O6: 658.6;Found: 659.0.

[0451] 3-((2-Ethylhexyl)oxy)-2,2-bis(((2-ethylhexyl)oxy)methyl)propyl-3-(4-(2-(2- hydroxyethoxy)ethyl)piperazin-1-yl)propanoate (EH.3.2EO, IAJD 314): A mixture of compound EH.3(1.19 g, 195 mmol, 1.0 equiv), 2-(2-(piperazin-1-yl)ethoxy)ethan-1-ol (409mg, 234 mmol, 1.2 equiv),K2CO3(325 mg, 234 mmol, 1.2 equiv) and MeCN (35 mL) was heated to 82°C and refluxed for 3 h.The reaction mixture was cooled to 23 °C and MeCN was removed under reduced pressure. Then water(20 mL) was added, and the resulting mixture was extracted with DCM (20mL) for three times. Theorganic phases were collected and dried over anhydrous MgSO4. After filtration, the obtained filtrate was concentrated to around 3 mL and purified by column chromatography (SiO2) with DCM / MeOH = 50 / 1 to20 / 1 as the eluent. Then the obtained product was dried over anhydrous MgSO4.Filtration andevaporation of the solvent yielded the title compound as a light-yellow oil (1.03 g, 75.1%).1H NMR (400MHz, CDCl3) δ 4.09 (s, 2 H, -CH2OCO-), 3.69–3.58 (m, 6 H, -NCH2CH2OCH2CH2OH), 3.32 (s, 6 H,-CH2OCH2CH(CH2CH3)-), 3.21 (d, 6 H, -CH2OCH2CH(CH2CH3)-), 2.68 (t, 2H, - COOCH2CH2N), 2.58–2.45 (br, 12 H, - N(CH2CH2)2NCH2CH2OH) and (-COOCH2CH2N), 1.46–1.37 (m, 3 H, - CH2OCH2CH(CH2CH3)-), 1.33–1.21 (br, 24 H, -OCH2CH(CH2CH3)(CH2CH2CH2CH3), 0.90– 0.82 (m, 18 H, -OCH2CH(CH2CH3)(CH2CH2CH2CH3).13C NMR(101 MHz, CDCl3) δ 172.0, 74.2, 72.4, 69.7, 67.5, 64.2, 62.1, 57.9, 53.5, 53.2, 52.6, 44.7, 39.7, 32.4, 30.7, 29.1, 24.0, 23.1, 14.1, 11.1. Purity by HPLC: 99+%. MALDI-TOF MS m / z of of [M + H]+ calculated for C40H81N2O7: 701.6; Found: 701.5; [M+ Na]+ calculated for C40H80N2O7Na: 723.6; Found: 723.6; [M + K]+ calculated for C40H80N2O7K:739.6;Found: 735.6.

[0452] 3-((2-Ethylhexyl)oxy)-2,2-bis(((2-ethylhexyl)oxy)methyl)propyl 4-(4-(2- hydroxyethyl)piperazin-1- yl)butanoate (EH.4.EO, IAJD 93): A mixture of compound EH.4 (700 mg, 1.12 mmol, 1.0 equiv), 2-(piperazin-1-yl)ethan-1-ol (17.58 mg, 1.35 mmol, 1.2 equiv), K2CO3 (18.67 mg, 1.35 mmol, 1.2 equiv), and MeCN (25 mL) was heated to 82 °C and refluxed for 3 h. The reaction mixture was cooled to 23 °C and MeCN was removed under reduced pressure. Then water (20 mL) was added, and the resulting mixture was extracted with DCM (20 mL) for three times. The organic phases were collected and dried over anhydrous MgSO4. After filtration, the obtained filtrate was concentrated to around 3 mL and purified by column chromatography (SiO2) with DCM / MeOH = 50 / 1 to 20 / 1 as the eluent. Then the obtained product was dried over anhydrous MgSO4. Filtration and evaporation of the solvent yielded the title compound as a light-yellow oil (530 mg, 70.2%).(1H NMR 400 MHz, CDCl3) δ 4.09 (s, 2 H, -CH2OCO-), 3.62 (t, 2H, -NCH2CH2OH), 3.34 (s, 6 H, - CH2OCH2CH(CH2CH3)-), 3.23 (d, 6 H, -CH2OCH2CH(CH2CH3)-), 2.57–2.49 (br, 10 H, - N(CH2CH2)2NCH2CH2OH), 2.39–2.31 (m, 4 H, -COOCH2CH2CH2), 1.84–1.77 (m, 2 H, - OCOCH2CH2CH2), 1.51–1.40 (m, 3 H, - CH2OCH2CH(CH2CH3)-), 1.39–1.21 (br, 24 H, - OCH2CH(CH2CH3)(CH2CH2CH2CH3), 0.90–0.83 (m, 18 H, -OCH2CH(CH2CH3)(CH2CH2CH2CH3).13C NMR (101 MHz, CDCl3) δ 173.2, 74.2, 69.7, 64.2, 59.2, 57.6, 57.6, 53.1, 52.8, 44.7, 39.7, 32.2, 30.7, 29.1, 24.0, 23.1, 22.2, 14.1, 11.1. Purity by HPLC: 99+%. MALDI-TOF MS m / z of Purity by HPLC: 99+%. MALDI-TOF MS m / z of [M + H]+ calculated for C39H79N2O6: 671.6; Found: 671.6.

[0453] 3-((2-ethylhexyl)oxy)-2,2-bis(((2-ethylhexyl)oxy)methyl)propyl 4-(4-(2-(2- hydroxyethoxy)ethyl)piperazin-1-yl)butanoate (EH.4.2EO, IAJD 288): A mixture of compound EH.4 (2.7 g, 4.34 mmol, 1equiv), 2-(2-(piperazin-1-yl)ethoxy)ethan-1-ol (907.9 mg, 5.21 mmol, 1.2 equiv), K2CO3 (720 mg, 5.21 mmol, 1.2 equiv) and MeCN (35 mL) was heated to 82 °C and refluxed for 3 h. The reaction mixture was cooled to 23 °C and MeCN was removed under reduced pressure. Then water (20 mL) was added, and the resulting mixture was extracted with DCM (20 mL) for three times. The organic phases were collected and dried over anhydrous MgSO4. After filtration, the obtained filtrate was concentrated to around 3 mL and purified by column chromatography (SiO2) with DCM / MeOH = 50 / 1 to20 / 1 as the eluent. Then the obtained product was dried over anhydrous MgSO4. Filtration and evaporation of the solvent yielded the title compound as a light-yellow oil (2.2 g, 70.8%).1H NMR (400 MHz, CDCl3) δ 4.09 (s, 2H, –CH2OCO–), 3.69–3.60 (m, 6 H, -NCH2CH2OCH2CH2OH),3.33 (s, 6 H, - CH2OCH2CH(CH2CH3), 3.22 (d, 6 H, -CH2OCH2CH(CH2CH3)-), 2.60–2.51 (br, 10 H, - N(CH2CH...

Claims

CLAIMS What is claimed is:

1. An ionizable amphiphilic Janus dendrimer represented by Formula (I):or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or derivative thereof, wherein X is a hydrophilic group comprising at least one amine; Y1, Y2, and Y3each independently represent C1-C30-alkyl; and m is an integer having the value of 1, 2, 4, or 5.

2. The ionizable amphiphilic Janus dendrimer of claim 1, wherein m is 2.

3. The ionizable amphiphilic Janus dendrimer of claim 1, wherein X comprises at least two tertiary amines.

4. The ionizable amphiphilic Janus dendrimer of claim 1, wherein X is selected from the group consisting ofwherein dashed lines indicate the connection to Formula (I); each occurrence of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10is independently selected from the group consisting of hydrogen, deuterium, halide, alkyl, aryl, cycloalkyl, amine, heterocycloalkyl, carbonyl, and combinations thereof; wherein any two of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10optionally together form a ring; and each occurrence of Rx, Ry, and Rzis independently selected from the group consisting of hydrogen, deuterium, halide, alkyl, alkoxy, hydroxy, ester, ether, and combinations thereof.

5. The ionizable amphiphilic Janus dendrimer of claim 4, wherein at least one of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, Rx, Ry, and Rzrepresents the following structure:wherein n is 0, 1, 2, or 3.

6. The ionizable amphiphilic Janus dendrimer of claim 1, wherein X is selected from the group consisting of:wherein dashed lines indicate the connection to Formula (I); and each occurrence of Rx, Ry, and Rzis independently selected from the group consisting of hydrogen, deuterium, halide, alkyl, alkoxy, hydroxy, ester, ether, and combinations thereof.

7. The ionizable amphiphilic Janus dendrimer of claim 6, wherein at least one of Rx, Ry, and Rzrepresents the following structure:wherein n is 0, 1, 2, or 3.

8. The ionizable amphiphilic Janus dendrimer of claim 1, wherein the ionizable amphiphilic Janus dendrimer has a pKa of between about 5.50 and about 6.

50.

9. The ionizable amphiphilic Janus dendrimer of claim 1, wherein Y1, Y2, and Y3independently represent a linear C6-C16-alkyl chain or branched C6-C16-alkyl chain.

10. The ionizable amphiphilic Janus dendrimer of claim 1, wherein the ionizable amphiphilic Janus dendrimer is an ionizable amphiphilic Janus dendrimer has one of the following structures:

11. A nanoparticle comprising the ionizable amphiphilic Janus dendrimer of claim 1.

12. The nanoparticle of claim 11, wherein the nanoparticle further comprises an agent encapsulated within the nanoparticle; wherein the agent is selected from the group consisting of a diagnostic agent, detectable agent, therapeutic agent, nucleic acid molecule, or a combination thereof.

13. A composition comprising the nanoparticle of claim 12, wherein the composition further comprises a second agent, a buffer, an adjuvant, a stabilizer, a solvent, or a polymer.

14. The nanoparticle of claim 12, wherein the agent is selected from the group consisting of a linear mRNA, circular mRNA, DNA, RNA, cDNA, cRNA, CirRNA, mRNA, miRNA, siRNA, microRNA, CRISPR-Cas9, sgRNA, modified RNA, tRNA, antagomir, antisense molecule, nucleic acid molecule, targeted nucleic acid, small molecule, protein, antibody, peptide, protein, and any combination thereof.

15. The nanoparticle of claim 12, wherein the agent is a nucleic acid molecule encoding at least one selected from the group consisting of an antigen, antibody, gene editing molecule, chimeric antigen receptor (CAR), and any combination thereof.

16. A method of delivering an agent to a subject in need thereof, wherein the method comprises administering to the subject the nanoparticle of claim 12.

17. A method of preventing or treating a disease or disorder in a subject in need thereof, wherein the method comprises administering to the subject at least one nanoparticle encapsulating an agent; wherein the nanoparticle comprises an ionizable amphiphilic Janus dendrimer represented by Formula (I):Formula (I), or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or derivative thereof, wherein X is a hydrophilic group comprising at least one amine; Y1, Y2, and Y3each independently represent C1-C30-alkyl; and m is an integer having the value of 1, 2, 4, or 5.

18. The method of claim 17, wherein the disease or disorder is selected from the group consisting of a viral infection, bacterial infection, fungal infection, parasitic infection, cancer, disease or disorder associated with cancer, autoimmune disease or disorder, and any combination thereof.

19. A method of inducing an immune response in a subject in need thereof, wherein the method comprises administering to the subject at least one nanoparticle encapsulating an agent; wherein the nanoparticle comprises an ionizable amphiphilic Janus dendrimer represented by Formula (I):Formula (I), or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or derivative thereof, wherein X is a hydrophilic group comprising at least one amine; Y1, Y2, and Y3each independently represent C1-C30-alkyl; and m is an integer having the value of 1, 2, 4, or 5.

20. The method of claim 19, wherein the nanoparticle targets the mucosal surface of the subject, gastrointestinal tract of the subject, the stomach of the subject, the small intestine of the subject, the large intestine of the subject, the kidney of the subject, the heart of the subject, the liver of the subject, the spleen of the subject, the lungs of the subject, the lymph nodes of the subject, or any combination thereof.

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