Polymers for intracellular delivery of polynucleotides

Abstract

The present invention provides ionizable polymers comprising a structural unit according to formula (I). Wherein R1 and R2 are as defined in the specification. Ionizable polymers of the present invention are useful for intracellular delivery of polynucleotides in vitro and in vivo. The invention also provides compositions comprising the ionizable polymers of the invention and polynucleotides. The compositions disclosed herein are useful as medicaments, in particular as medicaments for the prevention or treatment of infectious diseases, cancer or protein deficient diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention provides an ionizable polymer comprising a structural unit according to formula (I). The present invention also provides a composition comprising an ionizable polymer of the present invention and a polynucleotide. The compositions disclosed herein can be used as a medicament, in particular as a medicament for preventing or treating infectious diseases, cancer or protein deficiency diseases. Background Art

[0002] Delivering mRNA to target cells is an important approach for upregulating target protein expression and holds broad application prospects in mRNA vaccines, protein replacement therapies, genome editing, and cell reprogramming. While mRNA therapies offer significant advantages over traditional approaches, their application still faces numerous challenges. One of the most critical challenges is the development of efficient and safe mRNA delivery systems.

[0003] At present, lipid nanoparticles (LNPs) are one of the most commonly used mRNA delivery systems. LNPs are usually composed of four components, including (i) ionizable lipids for electrostatically complexing polynucleotides, (ii) auxiliary phospholipids for supporting structure, (iii) cholesterol for regulating membrane fluidity, and (iv) polyethylene glycol (PEG)-lipids for improving colloidal stability and extending circulation time. The requirement of four components can be loaded down with trivial details. It is also known that LNPs cause undesirable side effects.

[0004] Therefore, there is a need for improved and / or alternative delivery vehicles for polynucleotides (eg, mRNA molecules) that enable safe and effective intracellular delivery. Summary of the Invention

[0005] Aspects and embodiments of the invention are described in the following numbered clauses.

[0006] 1. An ionizable polymer comprising structural units according to formula (I):

[0007]

[0008] in

[0009] R1 represents a covalent bond or a linking moiety derived from a polyalkylene glycol; and

[0010] R2 represents a linear or branched aliphatic hydrocarbon group or a linear or branched fluorinated aliphatic hydrocarbon group.

[0011] 2. The ionizable polymer of item 1, wherein the polyalkylene glycol is polyethylene glycol.

[0012] 3. The ionizable polymer of clause 1 or 2, wherein the polyalkylene glycol has a molecular weight of from about 200 to about 1000, optionally from about 300 to about 800, such as from about 400 to about 600, for example about 500.

[0013] 4. The ionizable polymer of any preceding clause, wherein R2 is a linear or branched alkyl or fluorinated alkyl group.

[0014] 5. The ionizable polymer of any preceding clause, wherein R2 has 1 to 25 carbon atoms.

[0015] 6. The ionizable polymer of any preceding clause, wherein R2 is selected from the group consisting of butyl, hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl and octadecyl.

[0016] 7. The ionizable polymer of any one of clauses 1 to 5, wherein R2 is selected from the group consisting of difluoroethyl, pentafluoropropyl, heptafluorobutyl, nonafluoropentyl and pentafluorooctyl.

[0017] 8. The ionizable polymer of any preceding clause, wherein n is an integer from 1 to 10,000.

[0018] 9. The ionizable polymer of any of the preceding clauses, wherein R1 is according to formula (II)

[0019]

[0020] in,

[0021] Each Ra is independently a covalent bond or a linear -C 1-6 Alkylene-;

[0022] Rb is -C 1-6 Alkylene-, said alkylene being a straight chain alkylene and optionally substituted by one or more groups selected from OH, NH2, C 1-4 Alkyl and halogen radical substitution;

[0023] p is an integer from 1 to 10.

[0024] 10. The ionizable polymer of any preceding clause, wherein each Ra is -C1 alkylene-, and Rb is a linear and unsubstituted -C2 alkylene-.

[0025] 11. The ionizable polymer of clause 1, wherein the ionizable polymer comprises structural units selected from the group consisting of:

[0026] i)

[0027] ii)

[0028] iii)

[0029] iv)

[0030] v)

[0031] vi) and

[0032] vii)

[0033] 12. The ionizable polymer of clause 1, wherein the ionizable polymer comprises structural units selected from the group consisting of:

[0034] viii) ix)

[0035] x) xi)

[0036] xii) xiii)

[0037] xiv) and

[0038] xvi)

[0039] 13. A composition comprising the ionizable polymer of any one of clauses 1 to 12 and a polynucleotide, wherein the polynucleotide is complexed with polymer nanoparticles (PNPs) formed from the ionizable polymer.

[0040] 14. The composition of clause 13, wherein the polynucleotide is selected from the group consisting of RNA and DNA, optionally wherein the polynucleotide is mRNA or siRNA.

[0041] 15. The composition of clause 13 or 14, wherein:

[0042] a) R1 is derived from a polyalkylene glycol and the polynucleotide-PNP complex is formed by mixing the polynucleotide, cholesterol and an ionizable polymer, optionally wherein the polyalkylene glycol is polyethylene glycol; or

[0043] b) R1 is a covalent bond and the polynucleotide-PNP complex is formed by mixing the polynucleotide and the ionizable polymer.

[0044] 16. The composition of any one of clauses 13 to 15, wherein the polynucleotide-PNP complex is capable of delivering the polynucleotide into a human or non-human animal cell, optionally wherein the polynucleotide is mRNA and / or wherein the human cell is a cancer cell.

[0045] 17. The composition of any one of clauses 13 to 16, wherein the polynucleotide is mRNA or DNA, and wherein the mRNA or DNA encodes a cancer-specific antigen, an infectious disease-specific antigen, or a therapeutic protein.

[0046] 18. A pharmaceutical composition comprising the composition of any one of clauses 13 to 17.

[0047] 19. The pharmaceutical composition of clause 18, comprising polymeric nanoparticles (PNPs) formed from a polynucleotide and an ionizable polymer, wherein:

[0048] a) R1 is a linking moiety derived from polyethylene glycol and R2 is octadecyl, or

[0049] b) R1 is a covalent bond, and R2 is a decyl group.

[0050] 20. The pharmaceutical composition of clause 18 or 19 for use in treating cancer, wherein the mRNA or DNA encodes a cancer-specific antigen.

[0051] 21. The pharmaceutical composition of clause 18 or 19 for use in preventing or treating an infectious disease, wherein the mRNA or DNA encodes an infectious disease-specific antigen.

[0052] 22. The pharmaceutical composition of clause 21, wherein the infectious disease is a virus-related disease and the mRNA or DNA encodes a virus-specific antigen.

[0053] 23. The pharmaceutical composition of clause 18 or 19 for use in treating a protein deficiency disease, wherein the mRNA or DNA encodes a protein or peptide that is absent or non-functional in the subject to be treated.

[0054] 24. A method for preventing or treating a subject, comprising administering to the subject an effective amount of:

[0055] The composition of any one of clauses 13 to 17, wherein the polynucleotide is mRNA or DNA, or

[0056] The pharmaceutical composition of any one of clauses 18 to 22.

[0057] 25. The method of clause 24, wherein the treatment is treatment of cancer in the subject and the mRNA or DNA encodes a cancer-specific antigen or a therapeutic protein.

[0058] 26. The method of clause 24, wherein the prevention is prevention of a virus-related infection in the subject and the mRNA or DNA encodes a virus-specific antigen.

[0059] 27. The method of clause 24, wherein the treatment or prevention is the treatment or prevention of a protein deficiency in a subject, wherein the mRNA or DNA encodes a protein or peptide that is absent or non-functional in the subject.

[0060] 28. Use of the composition of any one of clauses 13 to 17 or the pharmaceutical composition of any one of clauses 18 to 23 in the manufacture of a medicament for preventing or treating a disease selected from the group consisting of cancer and virus-related diseases.

[0061] 29. Use of the composition of any one of clauses 13 to 17 or the pharmaceutical composition of any one of clauses 18 to 23 in the manufacture of a medicament for preventing or treating a protein deficiency.

[0062] 30. A method of producing a composition according to clauses 13-17, wherein the polynucleotide is mRNA or DNA, the method comprising the steps of:

[0063] i) combining the monomer of formula (III) and the monomer of formula (IV) by amino-epoxy ring-opening polymerization to form

[0064] an ionizable polymer comprising structural units according to formula (I); and

[0065] ii) combining the ionizable polymer with mRNA or mRNA encoding an antigenic polypeptide or therapeutic protein

[0066] DNA mixing,

[0067] in,

[0068]

[0069] in,

[0070] R1′ represents a covalent bond or a polyalkylene glycol moiety; and

[0071] R2' represents a linear or branched aliphatic hydrocarbon group or a linear or branched fluorinated aliphatic hydrocarbon group. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1The chemical structures of three series of polymers of the present invention (referred to herein as PHTA polymers) are shown: (a) PHTA-Cn, (b) PHTA-BCn, and (c) PHTA-BFn.

[0073] Figure 2 shows the PHTA-Cn polymer 1 H NMR spectra (CDCl3, 400 MHz, 298 K). (a) PHTA-C8; (b) PHTA-C10; (c) PHTA-C12; (d) PHTA-C14; (e) PHTA-C16; and (f) PHTA-C18.

[0074] Figure 3 Shown are the size distributions of PHTA-Cn / mOVA nanovaccines before and after mOVA loading.

[0075] Figure 4 Shown are the zeta potentials of PHTA-Cn / mOVA nanovaccines before and after mOVA loading.

[0076] Figure 5 Shown are representative TEM images of PHTA-based polymer nanoparticles (PNPs).

[0077] Figure 6 Shown are agarose gel electrophoresis images of PHTA-Cn / mOVA on day 1 and day 8 after mOVA loading.

[0078] Figure 7 Shown are green fluorescent protein (GFP) expression in DC 2.4 cells transfected with the indicated formulations PHTA-Cn / mGFP (1 μg / mL mGFP), examined by confocal microscopy images.

[0079] Figure 8 Shown are (a) representative flow cytometry graphs showing the expression level of GFP in DC 2.4 cells transfected with the indicated formulations of PHTA-Cn / mGFP (1 μg / mL mGFP); and (b) GFP high-expressing cells (GFP high The corresponding quantitative percentages of 347 cells (n=3) were analyzed.

[0080] Figure 9 (a) In vivo bioluminescence images after subcutaneous injection of LNP / mFluc, PHTA-C8 / mFluc, and PHTA-C18 / mFluc into mice via footpad, respectively. (b) Quantitative analysis of bioluminescence signals (n=5 / group).

[0081] Figure 10 Shown is a treatment schedule illustrating that B16-OVA tumor-bearing mice received vaccinations on days 4, 7, and 10 after tumor inoculation.

[0082] Figure 11 Shown are the mean tumor growth curves of mice (n=5 / group) receiving the indicated treatments.

[0083] Figure 12 Shown are the tumor inhibition efficiencies calculated based on the tumor volume at the end point compared with the PBS group (n=5 / group).

[0084] Figure 13 Shown are body weight curves of mice receiving indicated treatments (n=5 / group).

[0085] Figure 14 shows the PHTA-BCn polymer 1 H NMR spectrum (CDCl3, 400MHz, 298K). (a) PHTA-BC4; (b) PHTA-BC6; (c) PHTA-BC8; (d) PHTA-BC10.

[0086] Figure 15 Shown is an agarose gel electrophoresis image of the PHTA-BCn / mGFP complex loaded with mRNA encoding green fluorescent protein (mGFP).

[0087] Figure 16 shows the construction and characterization of the PHTA-BC10 / mOVA nanovaccine. (a) Scheme illustrating the construction of the PHTA-BC10 / mOVA nanovaccine by microfluidic mixing of PHTA-BC10 polymer and mRNA encoding the model antigen ovalbumin (mOVA), (b) Size of the PHTA-BC10 / mOVA nanovaccine before and after mOVA loading, (c) Zeta potential of the PHTA-BC10 / mOVA nanovaccine before and after mOVA loading.

[0088] Figure 17 Shown are semi-quantitative analyses of mean fluorescence intensity (MFI) of green fluorescent protein (GFP) expression in DC 2.4 cells transfected with the indicated formulations PHTA-BCn / mGFP (1 μg / mL mGFP).

[0089] Figure 18 Figure 3. Tumor inhibition efficacy of PHTA-BC10 / mOVA as a therapeutic cancer vaccine. (a) Average tumor growth curves of mice receiving the indicated treatments (n = 5 / group). (b) Tumor inhibition efficiency calculated based on tumor volume at endpoint compared to the PBS group (n = 5 / group).

[0090] Figure 19 shows MALDI-TOF-MS of PHTA-BFn polymers: (a) PHTA-BF2; (b) PHTA-BF5; (c) PHTA-BF7; (d) PHTA-BF9; and (e) PHTA-BF15.

[0091] Figure 20 Figure 5 shows the characterization of PHTA-BF7 / mRNA complexes. (a) Agarose gel electrophoresis image of PHTA-BF7 / mRNA complexes. (b) Size distribution of PHTA-BF7 / mRNA complexes before and after mRNA loading.

[0092] Figure 21 Shown are semi-quantitative analyses of mean fluorescence intensity (MFI) of green fluorescent protein (GFP) expression in DC 2.4, RAW 264.7, HEK 293T, and PC3 cells transfected with PHTA-BF7 / mGFP (1 μg / mL mGFP).

[0093] Figure 22 Shown are in vivo bioluminescence images of mice 8 hours and 24 hours after subcutaneous injection of PHTA-BF7 / mFluc into the mice. DETAILED DESCRIPTION

[0094] The present inventors have found that ionizable polymers comprising structural units according to formula (I) are surprisingly effective in delivering polynucleotides to cells, promoting the endosomal escape of polynucleotides, and allowing the translation of proteins or peptides encoded by polynucleotides in vitro and in vivo. Using a cancer mouse model, the present inventors have also shown that ionizable polymers can deliver mRNA encoding model cancer antigens (mOVA) in vivo, thereby producing a strong tumor inhibitory effect in mice. It was found that ionizable polymers were surprisingly well tolerated and had no significant toxicity in mice. Therefore, the ionizable polymers of the present invention show great potential as delivery vectors in therapeutic agents based on polynucleotides, such as those based on mRNA.

[0095] Thus, the present invention provides ionizable polymers for intracellular delivery of polynucleotides in vitro and in vivo, the ionizable polymers comprising structural units of formula (I):

[0096]

[0097] In formula (I), R1 represents a covalent bond or a linking portion derived from polyalkylene glycol.

[0098] As used herein, the term "polyalkylene glycol" refers to a glycol having the general formula HO-[RO] n -H polymers, where R is an alkylene group. Hydroxyl groups are terminal groups. Common examples of polyalkylene glycols are polyethylene glycols (where R = linear C2 alkylene). The alkylene group may be unsubstituted or substituted, for example, selected from OH, NH2, C 1-4The term "derived from polyalkylene glycol" as used herein refers to a group within a structural unit of formula (I) that can be obtained from polyalkylene glycol or a compound containing polyalkylene glycol. The term encompasses groups that contain a polyalkylene glycol portion.

[0099] In formula (I), n can be an integer from 1 to 10000, for example, from 1 to 5000, from 1 to 4000, from 1 to 3000, from 1 to 2000 and from 1 to 1000. As will be appreciated, ionizable polymers of the present invention can be formed in a variety of ways, including the chemical modification of one or more repeating units of monomeric polymerization and / or polymer precursors. Typically, it is formed by monomeric polymerization. Therefore, the quantity of the structural units in ionizable polymers depends on degree of polymerization, which may be inconsistent between each polymer molecule in the sample. Therefore, except the value of the integer n in formula (I), the size of ionizable polymers of the present invention can be limited by the number-average molecular weight (Mn) of ionizable polymers in a given sample.

[0100] Typically, the ionizable polymers of the present invention have a number average molecular weight (Mn) of about 1,000 to about 10,000, such as about 2,000 to about 8,000, or about 3,000 to about 7,000.

[0101] In certain embodiments, the ionizable polymers of the present invention have a number average molecular weight (Mn) of about 4100, about 4700, about 4900, about 5100, about 5600, or about 6200.

[0102] In certain embodiments, R1 may comprise a polyalkylene glycol moiety having a molecular weight of about 200 to about 1000. For example, the molecular weight is about 300 to about 800, such as about 400 to about 600 (e.g., about 500). For example, the polyalkylene glycol moiety may be a polyethylene glycol moiety having a molecular weight of about 200 to about 1000. For example, the molecular weight is about 300 to about 800, such as about 400 to about 600 (e.g., about 500).

[0103] In certain embodiments, R1 is according to formula (II):

[0104]

[0105] In formula (II), each Ra is independently a covalent bond or a linear -C 1-6 Alkylene-. For example, each Ra can be a covalent bond or a linear-C 1-6Alkylene-, for example, C1 alkylene, linear C2 alkylene, linear C3 alkylene, linear C4 alkylene, linear C5 alkylene or linear C6 alkylene. In certain embodiments, each Ra is C1 alkylene. For the avoidance of doubt, when R1 is according to formula (II), R1 can still be considered to be derived from a polyalkylene glycol. In this regard, the portion of formula (II) corresponding to the polyalkylene glycol portion is represented by -O-[Rb-O] p -express.

[0106] In formula (II), Rb is -C 1-6 Alkylene-. Alkylene is a straight chain alkylene and may be replaced by one or more groups selected from OH, NH2, C 1-4 Alkyl and halogen (such as fluorine, chlorine, bromine or iodine) groups are substituted.

[0107] In formula (II), p is an integer from 1 to 1000, for example, 1 to 500, 1 to 400, 1 to 300, 1 to 200, 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 to 20, and 1 to 10. In certain embodiments, p is from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In certain exemplary embodiments, p is 8.

[0108] In formula (I), R2 represents a linear or branched aliphatic hydrocarbon group or a linear or branched fluorinated aliphatic hydrocarbon group.

[0109] As used herein, the term "hydrocarbyl" refers to a monovalent substituent containing only hydrogen and carbon atoms. The term encompasses substituents of branched or unbranched, saturated or unsaturated, cyclic, polycyclic or acyclic types. Specific examples of hydrocarbyl include alkyl, cycloalkyl, alkenyl, alkadienyl, cycloalkenyl, cycloalkadienyl, aryl and alkynyl. The hydrocarbyl can be an aliphatic hydrocarbyl. The term "aliphatic hydrocarbyl" refers to a saturated straight or branched hydrocarbon chain that is completely saturated or contains one or more unsaturated units. Examples of aliphatic hydrocarbyl include substituted or unsubstituted alkyl, alkenyl, and alkynyl. In certain embodiments, R2 can be a fluorinated aliphatic hydrocarbyl. The term "fluorinated aliphatic hydrocarbyl" refers to an aliphatic hydrocarbyl as defined herein that is substituted with one or more fluorine atoms.

[0110] In certain embodiments, R2 can be a linear or branched C optionally substituted with one or more fluorine atoms. 1-25 Alkyl (e.g. C 1-18 For example, R2 can be C 4-18 Alkyl (e.g. C 4-10 Alkyl and C 8-18 Alternatively, for example, R2 may be C 2-8In some embodiments, R can be an aliphatic hydrocarbon group selected from the group consisting of butyl, hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl and octadecyl (such as octyl, decyl, dodecyl, tetradecyl, hexadecyl and octadecyl). In some other embodiments, R can be an aliphatic hydrocarbon group selected from the group consisting of ethyl, propyl, butyl, amyl and octyl, wherein the aliphatic hydrocarbon group is replaced by one or more fluorine atoms (such as, R can be difluoroethyl, pentafluoropropyl, heptafluorobutyl, nonafluoropentyl or pentafluorooctyl).

[0111] In certain embodiments, when R1 is a linking moiety derived from a polyalkylene glycol, R2 can be an aliphatic hydrocarbon group selected from the group consisting of octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl. In certain other embodiments, when R1 is a covalent bond, R2 can be an aliphatic hydrocarbon group selected from the group consisting of butyl, hexyl, octyl, decyl. In certain other embodiments, when R1 is a covalent bond, R2 can be an aliphatic hydrocarbon group selected from the group consisting of ethyl, propyl, butyl, pentyl, and octyl, wherein the aliphatic hydrocarbon group is substituted with one or more fluorine atoms (e.g., R2 can be difluoroethyl, pentafluoropropyl, heptafluorobutyl, nonafluoropentyl, or pentafluorooctyl). Typically, the aliphatic hydrocarbon group at R2 is a straight chain aliphatic hydrocarbon group (e.g., a straight chain C 1-25 alkyl).

[0112] As used herein, the term "alkyl" refers to straight and branched saturated hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, pentyl, and hexyl. Exemplary straight-chain alkyl groups include methyl, ethyl, n-propyl, isopropyl, and n-butyl. Exemplary branched-chain alkyl groups include tert-butyl, isobutyl, 1-ethylpropyl, and 1-ethylbutyl.

[0113] As used herein, the term "aryl" refers to a monocyclic or bicyclic aromatic carbocyclic group. Examples of aryl groups include phenyl and naphthyl. In bicyclic aromatic groups, one of the rings may, for example, be partially saturated. Examples of such groups include indanyl and tetrahydronaphthyl.

[0114] As used herein, the term "halogen" refers to fluorine, chlorine, bromine or iodine. Fluorine is particularly preferred.

[0115] In certain exemplary embodiments, the ionizable polymers of the present invention comprise structural units having a structure selected from the group consisting of:

[0116] i)

[0117] ii)

[0118] iii)

[0119] iv)

[0120] v)

[0121] vi) and

[0122] vii)

[0123] In certain other exemplary embodiments, the ionizable polymers of the present invention comprise structural units having a structure selected from the group consisting of:

[0124] viii) ix)

[0125] x) xi)

[0126] xii) xiii)

[0127] xiv) xv) and

[0128] xvi)

[0129] The present invention also provides an ionizable polymer produced by amino-epoxy ring-opening polymerization between a monomer according to formula (III) and a monomer according to formula (IV):

[0130]

[0131] Here, R1′ represents a covalent bond or a polyalkylene glycol moiety, and R2′ represents a linear or branched aliphatic hydrocarbon group or a linear or branched fluorinated aliphatic hydrocarbon group.

[0132] R1′ may have a structure according to formula (IIIa):

[0133]

[0134] In formula (IIIa), Rb′ is -C 1-6 Alkylene-. Alkylene is a straight chain alkylene and may be replaced by one or more groups selected from OH, NH2, C 1-4In formula (IIIa), p' is an integer from 1 to 1000, for example, from 1 to 500, from 1 to 400, from 1 to 300, from 1 to 200, from 1 to 100, from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, and from 1 to 10. In certain embodiments, p' is from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In certain exemplary embodiments, p' is 8.

[0135] R2' can be a straight or branched chain C optionally substituted by one or more fluorine atoms 1-25 Alkyl (e.g. C 1-18 For example, R2′ can be C 4-18 Alkyl (e.g. C 4-10 Alkyl and C 8-18 Alternatively, for example, R2′ may be C 2-8 In some embodiments, R2′ can be an aliphatic hydrocarbon group selected from the group consisting of butyl, hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl. In some other embodiments, R2′ can be an aliphatic hydrocarbon group selected from the group consisting of ethyl, propyl, butyl, pentyl, and octyl, wherein the aliphatic hydrocarbon group is substituted with one or more fluorine atoms (e.g., 1 to 15 fluorine atoms, such as 2, 5, 7, 9, or 15 fluorine atoms). In some embodiments, R2′ can be an aliphatic hydrocarbon group selected from the group consisting of ethyl, propyl, butyl, pentyl, and octyl, wherein the aliphatic hydrocarbon group is substituted with one or more fluorine atoms (e.g., R2′ can be a difluoroethyl, pentafluoropropyl, heptafluorobutyl, nonafluoropentyl, or pentafluorooctyl).

[0136] For example, when R1′ is a polyalkylene glycol moiety, R2′ can be an aliphatic hydrocarbon group selected from the group consisting of octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl. Alternatively, for example, when R1′ is a covalent bond, R2′ can be an aliphatic hydrocarbon group selected from the group consisting of butyl, hexyl, octyl, decyl. Alternatively, for example, when R1′ is a covalent bond, R2′ can be an aliphatic hydrocarbon group selected from the group consisting of ethyl, propyl, butyl, pentyl, and octyl, wherein the aliphatic hydrocarbon group is substituted with one or more fluorine atoms (for example, R2′ can be difluoroethyl, pentafluoropropyl, heptafluorobutyl, nonafluoropentyl, or pentafluorooctyl). Typically, the aliphatic hydrocarbon group at R2′ is a straight chain aliphatic hydrocarbon group (for example, a straight chain C 1-25 alkyl).

[0137] In certain preferred embodiments, the ionizable polymer of the present invention is produced by amino-epoxy ring-opening polymerization between poly(ethylene glycol) diglycidyl ether or 1,3-butadiene diepoxide and an amine selected from the group consisting of butylamine, hexylamine, octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, octadecylamine, and combinations of two or more thereof. In certain other preferred embodiments, the ionizable polymer of the present invention is produced by amino-epoxy ring-opening polymerization between poly(ethylene glycol) diglycidyl ether or 1,3-butadiene diepoxide and an amine selected from the group consisting of difluoroethylamine, pentafluoropropylamine, heptafluorobutylamine, nonafluoropentylamine, pentafluorooctylamine, and combinations of two or more thereof.

[0138] For example, the ionizable polymer of the present invention can be produced by amino-epoxy ring-opening polymerization between poly(ethylene glycol) diglycidyl ether and an amine selected from the group consisting of octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, octadecylamine, and combinations of two or more thereof. Alternatively, for example, the ionizable polymer of the present invention can be produced by amino-epoxy ring-opening polymerization between 1,3-butadiene diepoxide and an amine selected from the group consisting of butylamine, hexylamine, octylamine, decylamine, and combinations of two or more thereof. Alternatively, for example, the ionizable polymer of the present invention can be produced by amino-epoxy ring-opening polymerization between 1,3-butadiene diepoxide and an amine selected from the group consisting of difluoroethylamine, pentafluoropropylamine, heptafluorobutylamine, nonafluoropentylamine, pentafluorooctylamine, and combinations of two or more thereof.

[0139] Typically, the poly(ethylene glycol) portion of the poly(ethylene glycol) diglycidyl ether has a number average molecular weight (Mn) of about 100 to about 1000, e.g., about 200 to about 800. In certain exemplary embodiments, the poly(ethylene glycol) portion of the poly(ethylene glycol) diglycidyl ether has an Mn of about 500.

[0140] As used herein, when used with respect to the polymers disclosed herein, the terms "number average molecular weight" and "Mn" refer to the average molecular weight of a polymer calculated by dividing the total weight of a polymer sample by the total number of molecules in the sample. The use of the term "molecular weight" with respect to a polymer herein should be understood to refer to the number average molecular weight of a given polymer. The number average molecular weight of a polymer can be determined by techniques known in the art, such as gel permeation chromatography, viscometry, mass spectrometry, or particle number-dependent methods, such as vapor pressure osmometry, end group measurement, or proton NMR.

[0141] The ionizable polymer of the present invention can form polymer nanoparticles (PNPs) comprising an ionizable polymer and polynucleotides. Polymer nanoparticles can be formed by mixing an ionizable polymer and polynucleotides and optionally cholesterol. A specific mixing method suitable for forming polymer nanoparticles (PNPs) is microfluidic mixing. Additional or alternative methods include, but are not limited to, pipette mixing and vortex mixing. Specific methods are disclosed in the Examples section of this article.

[0142] In certain embodiments, the ionizable polymers of the present invention consist essentially of structural units according to formula (I) as described above.

[0143] Composition

[0144] The present invention also provides compositions comprising ionizable polymers of the present invention and polynucleotides, wherein polynucleotides are compounded with the ionizable polymers to form polymer nanoparticles (PNPs). The polymer nanoparticles (PNPs) formed by ionizable polymers and polynucleotides generally have an average diameter of about 300nm or less (e.g., about 200 or less, such as an average diameter of about 10nm to about 200nm, about 40nm to about 150nm or about 100nm to about 200nm). The polymer nanoparticles comprising ionizable polymers of the present invention and polynucleotides can be referred to as "polynucleotide-PNP complexes" or "polymer nano vaccines" in this article.

[0145] The polynucleotide-PNP complex disclosed herein is capable of delivering the polynucleotide present in the complex to eukaryotic cells, such as human or non-human eukaryotic cells. Typically, the polynucleotide-PNP complex disclosed herein is used to deliver the polynucleotide to human cells, such as human cancer cells.

[0146] For the avoidance of doubt, the term "polynucleotide" as used herein refers to any molecule of a polymer comprising nucleotides. A polynucleotide can be an isolated polynucleotide molecule or a polynucleotide construct (e.g., messenger RNA (mRNA) or plasmid DNA (pDNA)). A polynucleotide can be linear (e.g., mRNA or siRNA) or circular (e.g., plasmid). A polynucleotide can also be a double-stranded or single-stranded polynucleotide. Typically, a polynucleotide is an RNA molecule or a deoxyribonucleic acid (DNA) molecule. However, it can also be a derivative of RNA and / or DNA, for example, it can be a peptide nucleic acid oligomer (PNA). In certain exemplary embodiments, a polynucleotide is an mRNA molecule.

[0147] The polynucleotide encodes a protein or peptide of interest. For example, the polynucleotide may encode a protein or peptide that is a cancer-specific antigen or a protein or peptide that is an infectious disease-specific antigen, or the polynucleotide may encode a therapeutic protein or peptide (e.g., a therapeutic antibody or antibody fragment).

[0148] When the polynucleotide encodes a cancer-specific antigen, the cancer-specific antigen can be a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). TAA is an immunogenic protein or peptide abnormally expressed by cancer cells. For example, TAA can be an immunogenic protein or peptide expressed by both normal cells and cancer cells but expressed at a higher level by cancer cells. TSA is an immunogenic protein or peptide expressed by cancer cells rather than normal cells. A special example of a tumor-specific antigen is a neoantigen. Special examples of tumor-associated antigens include NY-ESO-1, tyrosinase, MAGE-A3 and TPTE, MAGE-C1, MAGE-C2, TPBG 5T4, survivin, and MUC-1.

[0149] When the polynucleotide encodes an infectious disease specific antigen, the antigen can be any immunogenic protein or peptide derived from a microorganism that can cause disease by invading a subject. For example, an infectious disease specific antigen can be an immunogenic protein or peptide derived from a pathogenic bacterium, fungus, parasite or virus. Particular examples of infectious disease specific antigens include the spike protein of SARS-CoV-2, the hemagglutinin protein of influenza virus, the membrane or envelope protein of Zika virus, the fusion protein of respiratory syncytial virus (RSV), and the surface glycoprotein of human immunodeficiency virus (HIV), Ebola virus or rabies virus.

[0150] When the polynucleotide encodes a therapeutic protein or peptide, the protein or peptide can be any protein or peptide that is beneficial for treating or preventing any hereditary or acquired disease or improving the condition of the subject. A particular example of a therapeutic protein is a therapeutic antibody or antibody fragment. Further examples of therapeutic proteins or peptides include CRISPR-associated proteins (CAS), cytokines (e.g., OX40L, IL-2, IL-12, IL-12sc, IL-15sushi, IL-23, and IL-36γ, IFNα, and GM-CSF), methylmalonyl-CoA mutase, propionyl-CoA (CoA) carboxylase, cystic fibrosis transmembrane conductance regulator, ornithine transcarbamylase (OTC), glycogen debranching enzyme; PTEN and p53.

[0151] In certain embodiments, the compositions of the present invention further comprise cholesterol. Cholesterol present in the compositions of the present invention can facilitate intracellular delivery of polynucleotides and PNPs into cells by regulating membrane fluidity.

[0152] The present invention also provides a method for producing the composition of the present invention, the method comprising the steps of:

[0153] (i) combining a monomer of formula (III) and a monomer of formula (IV) by amino-epoxy ring-opening polymerization to form an ionizable polymer comprising structural units according to formula (I); and

[0154] (ii) Mixing the ionizable polymer with mRNA or DNA encoding the antigenic polypeptide or therapeutic protein.

[0155] For the avoidance of doubt, formula (III) and (IV) in step i) are according to formula (III) and formula (IV) described above for the ionizable polymers of the present invention. In certain embodiments, in step i), the monomer according to formula (III) is poly(ethylene glycol) diglycidyl ether or 1,3-butadiene diepoxide, and the monomer according to formula (IV) is an amine selected from the group consisting of butylamine, hexylamine, octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, octadecylamine, and combinations of two or more thereof. In certain other embodiments, in step i), the monomer according to formula (III) is poly(ethylene glycol) diglycidyl ether or 1,3-butadiene diepoxide, and the monomer according to formula (IV) is an amine selected from the group consisting of difluoroethylamine, pentafluoropropylamine, heptafluorobutylamine, nonafluoropentylamine, pentafluorooctylamine, and combinations of two or more thereof.

[0156] The composition comprising the ionizable polymer and polynucleotide of the present invention can be provided as a pharmaceutical composition, optionally comprising one or more pharmaceutically acceptable excipients. The pharmaceutical composition of the present invention can be provided as any composition suitable for parenteral administration (including subcutaneous, intradermal, intralymphatic, intraosseous infusion, intramuscular, intravascular (bolus or infusion) and intramedullary), intratumoral, intracerebral, intraperitoneal, nasal or oral inhalation administration. Typically, the pharmaceutical composition is a pharmaceutical composition suitable for intradermal, intranasal or intravenous administration. The most suitable route of administration and therefore the most suitable form of the composition may depend, for example, on the condition of the recipient.

[0157] The pharmaceutical compositions of the present invention may include aqueous or non-aqueous sterile injection solutions that may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. Pharmaceutical compositions suitable for inhalation administration include solutions in saline, which may contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters that enhance bioavailability, and / or other solubilizers or dispersants, such as those known in the art. The pharmaceutical compositions may be present in unit dose or multi-dose containers, such as sealed ampoules and vials, and may be stored under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier, such as saline or water for injection, immediately before use. Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets. Various pharmaceutically acceptable carriers and their formulations are described in standard formulation papers, such as Remington's Pharmaceutical Sciences by EW Martin. See also Wang, YJ and Hanson, MA, Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S, 1988.

[0158] In embodiments where the polynucleotide in the pharmaceutical composition encodes an antigenic protein or peptide (e.g., a protein or peptide that is a cancer antigen or an infectious disease specific antigen), the pharmaceutical composition may further comprise an adjuvant. As used herein, the term "adjuvant" is understood to mean any substance that enhances a subject's immune response to an antigenic protein or peptide. Examples of adjuvants include, but are not limited to, polyinosinic:polycytidylic acid, Freund's incomplete adjuvant (IFA), cytokines (e.g., interleukins), CD40, keyhole limpet hemocyanin, Toll-like receptor liposomes, CpG oligodeoxynucleotides, saponin, colloidal alum, and lipopolysaccharide lipid A analogs.

[0159] It should be understood that, in addition to the ingredients specifically mentioned above, the compositions used in the present invention may include other agents conventional in the art with respect to the type of composition in question. Otherwise, the preparation of suitable formulations can be routinely achieved by the skilled artisan using conventional techniques and / or in accordance with standard and / or generally accepted pharmaceutical practices.

[0160] treat

[0161] The composition of the present invention can be used as a medicine. In particular, the composition of the present invention can be used to treat or prevent diseases.

[0162] The type of disease that can be treated or prevented by the composition of the present invention depends on the type of protein or peptide encoded by the polynucleotide present in the composition. For example, when the polynucleotide present in the composition encodes a cancer-specific antigen, the composition can be used to prevent or treat cancer in a subject. In this case, the composition of the present invention can be considered a therapeutic vaccine. Examples of cancers that can be prevented or treated using the composition of the present invention include, but are not limited to, melanoma, colorectal cancer, head and neck squamous cell carcinoma, and non-small cell lung cancer (NSCLC).

[0163] When the polynucleotide encoding infectious disease specific antigens present in the composition, the composition can be used to prevent or treat the infectious disease in the subject. In this case, the composition of the present invention can be considered as a preventive or therapeutic vaccine. When the infectious disease specific antigen is a virus-specific antigen, the composition can be used to prevent or treat virus-related diseases, such as those caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus, Zika virus, respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), Ebola virus, rabies virus, cytomegalovirus (CMV). The composition of the present invention can also be used to treat diseases caused by bacteria, fungi or parasite infections (such as Plasmodium infection).

[0164] When the polynucleotide present in the composition encodes a therapeutic protein or peptide, the composition can be used to treat a genetic or acquired disease that can be treated by a therapeutic protein or peptide. For example, when the polynucleotide encodes a therapeutic antibody that exhibits anti-cancer activity, the composition can be used to treat cancer in a subject.

[0165] The compositions of the present invention can also be used to prevent or treat protein deficiency diseases. As used herein, the term "protein deficiency disease" refers to a disease associated with a specific protein or protein group that is not present or non-functional in a patient. In this case, the compositions of the present invention can be considered to be protein replacement therapy. Examples of protein deficiency diseases include methylmalonyl-CoA mutase deficiency, propionyl-CoA carboxylase deficiency, cystic fibrosis, ornithine transcarbamylase deficiency, glycogen storage disease type III (GSD III), PTEN hamartoma syndrome, and cancers associated with p53. Therefore, in certain embodiments, when the polynucleotide encoding present in the composition does not exist in the subject or when a non-functional protein or peptide, the composition can be used to treat protein deficiency diseases. In certain other embodiments, the compositions of the present invention may include polynucleotides encoding CRISPR-associated proteins (CAS) and / or guide RNA (gRNA), in which case the compositions can be used for genome editing of the subject, for example, for genome editing of the protein deficiency disease for treating or preventing the subject.

[0166] The amount of the composition of the present invention administered to a subject will depend on various factors, such as the severity of the condition to be treated, the specific patient to be treated, the immunogenicity of the antigenic polypeptides encoded by the polynucleotides in the composition, or the efficacy of the therapeutic polypeptides encoded by the polynucleotides in the composition. In any case, a medical practitioner or other technician will be able to routinely determine the actual dosage that is most suitable for an individual patient. However, in general, the dosage administered is what can be considered a "therapeutic amount" or "effective amount."

[0167] As used herein, the term "therapeutic amount" or "effective amount" may refer to an amount of the composition of the present invention that is capable of preventing a disease or improving the condition of a subject suffering from a disease. For example, when the polynucleotides in the composition encode antigenic proteins or peptides, a "therapeutic amount" or "effective amount" may be considered to be the amount of the composition that is capable of eliciting an immune response in a subject against the antigenic polypeptides. For example, a primary and / or secondary immune response that prevents a subject from acquiring an infectious disease or cancer and / or improves the condition of a subject suffering from an infectious disease or cancer. When the polynucleotides in the composition encode therapeutic proteins or peptides (e.g., antibodies or antibody fragments), a "therapeutic amount" or "effective amount" may refer to the amount of the composition that is sufficient to induce expression of the therapeutic protein or peptide in a subject at a level that results in an improvement in the condition of a subject suffering from a disease.

[0168] In certain embodiments, the composition can be administered to a subject by intradermal, intranasal, or intravenous administration.

[0169] Also disclosed herein is a kit comprising a composition of the present invention. That is, the kit comprises a composition comprising an ionizable polymer as disclosed herein and a polynucleotide as disclosed herein, wherein the polynucleotide is complexed with polymer nanoparticles (PNPs) formed from the ionizable polymer.

[0170] Also disclosed herein are kits comprising a compound according to formula (III) and a compound according to formula (IV) as disclosed herein, and a polynucleotide encoding a cancer-specific antigen, an infectious disease-specific antigen, or a therapeutic protein or peptide as disclosed herein. Such kits of the present invention can be used to prepare the compositions of the present invention.

[0171] For the avoidance of doubt, the kit according to the invention is in a form and amount suitable for use according to the invention. A person skilled in the art can readily determine the amount of each component (i.e., ionizable polymer, compound of formula (III) and (IIV) and / or polynucleotide) suitable for inclusion in the kit of the invention and for use according to the invention.

[0172] In the embodiments herein, the word "comprising" can be interpreted as requiring the features mentioned, but does not limit the presence of other features. Alternatively, the word "comprising" can also refer to situations in which only the components / features listed are intended to be present (e.g., the word "comprising" can be replaced by the phrase "consisting of..." or "consisting essentially of..."). It is clearly intended that both wider and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word "comprising" and its synonyms can be replaced by the phrase "consisting of..." or the phrase "consisting essentially of..." or their synonyms, and vice versa.

[0173] The phrase "consisting essentially of" and its pseudonyms may be interpreted herein to refer to a material in which small amounts of impurities may be present. For example, the purity of the material may be greater than or equal to 90%, such as greater than 95%, such as greater than 97%, such as greater than 99%, such as greater than 99.9%, such as greater than 99.99%, such as greater than 99.999%, or 100%.

[0174] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes a mixture of two or more such compositions, and the like.

[0175] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

[0176] Example

[0177] Materials and methods:

[0178] An ionizable alternating copolymer having hydroxy tertiary amine (HTA) repeating units (herein referred to as PHTA, ie, "polymer having hydroxy tertiary amine") was synthesized by amino-epoxy polymerization of diepoxide monomer 1 and amine monomer 2.

[0179] Diepoxide monomer 1 can be poly (ethylene glycol) diglycidyl ether, 1,3-butadiene diepoxide (BDE) or other diepoxide monomers. Amine monomer 2 can be an alkylamine, such as butylamine, hexylamine, octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine or octadecylamine. Amine monomer 2 can also be a fluorine-substituted alkylamine, such as difluoroethylamine, pentafluoropropylamine, heptafluorobutylamine, nonafluoropentylamine or pentafluorooctylamine. By changing the type of diepoxide monomer 1 and amine monomer 2, three representative series of PHTA polymers (i.e., PHTA-Cn, PHTA-BCn and PHTA-BFn) were synthesized, and the mRNA delivery efficiency was studied. The synthesis methods of these three series of PHTA polymers are described as follows:

[0180] Synthesis of PHTA-Cn series

[0181] Ionizable alternating copolymers PHTA-Cn were synthesized by amino-epoxy polymerization of poly(ethylene glycol) diglycidyl ether monomer 1 and different alkylamine monomers 2 (octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, and octadecylamine), where “n” represents the number of carbon atoms in the polymer side chain ( Figure 1 a). Specifically, poly(ethylene glycol) diglycidyl ether and octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, or octadecylamine were dissolved in propylene glycol methyl ether. The mixture was heated under reflux at 120° C. under a nitrogen atmosphere, and the reaction was stirred for 24 hours. The crude product was purified by ethanol dialysis for 3 days to remove unreacted monomers, and the final product was obtained by vacuum drying.

[0182] Synthesis of PHTA-BCn series

[0183] Ionizable alternating copolymers PHTA-BCn were synthesized by amino-epoxy polymerization of 1,3-butadiene diepoxide (BDE) monomer 1 and different alkylamine monomers 2 (butylamine, hexylamine, octylamine, decylamine), where “n” represents the number of carbon atoms on the polymer side chain ( Figure 1 b). Specifically, 1,3-butadiene diepoxide (BDE) and butylamine, hexylamine, octylamine, or decylamine were dissolved in ethanol and prepolymerized at room temperature for 3 hours. The reaction was then heated to 80°C under an argon atmosphere and stirred for 24 hours. The crude product was purified by ethanol dialysis for 3 days to remove unreacted monomers, and the final product was obtained by vacuum drying.

[0184] Synthesis of PHTA-BFn series

[0185] Ionizable alternating copolymers PHTA-BFn were synthesized by aminoepoxy polymerization of 1,3-butadiene diepoxide (BDE) monomer 1 and different fluorinated alkylamine monomers 2 (difluoroethylamine, pentafluoropropylamine, heptafluorobutylamine, nonafluoropentylamine, pentafluorooctylamine), where “n” represents the number of fluorine atoms on the polymer side chain ( Figure 1 c). Specifically, 1,3-butadiene diepoxide (BDE) and difluoroethylamine, pentafluoropropylamine, heptafluorobutylamine, nonafluoropentylamine, or pentafluorooctylamine were dissolved in ethanol and prepolymerized at room temperature for 3 hours. The reaction was then heated to 80°C and stirred for 24 hours under an argon atmosphere. The crude product was purified by ethanol dialysis for 3 days to remove unreacted monomers, and the final product was obtained by vacuum drying.

[0186] mRNA loading and characterization

[0187] mRNA was loaded into PHTA-based polymer nanocarriers using microfluidic mixing technology. Specifically, mRNA was loaded into PHTA-Cn-based polymer nanocarriers by microfluidic mixing of PHTA-Cn polymer, cholesterol, and mRNA, and mRNA was loaded into PHTA-BCn- / PHTA-BFn-based polymer nanocarriers by microfluidic mixing of PHTA-BCn / PHTA-BFn polymer and mRNA (without cholesterol).

[0188] In more detail, PHTA-based polymer nanocarriers (without mRNA) are prepared using a microfluidic mixing method. PHTA ionizable polymer is dissolved in ethanol (if the polymer is PHTA-Cn, cholesterol is also included). The polymer-ethanol solution is rapidly mixed with citrate buffer via a microfluidic chip, and the mixture is then dialyzed with PBS to provide ionizable polymer nanoparticles (in the form of nanoparticles without mRNA). An ionizable polymer / mRNA composition is prepared using a microfluidic mixing method. PHTA ionizable polymer is dissolved in ethanol (if the polymer is PHTA-Cn, cholesterol is also included). mRNA is diluted in citrate buffer. The polymer-ethanol solution is rapidly mixed with an mRNA citrate buffer solution via a microfluidic chip, and the mixture is then dialyzed with PBS to provide an ionizable polymer / mRNA composition (in the form of nanoparticles).

[0189] The physicochemical properties of polymer nanocarriers (without mRNA) and ionizable polymer / mRNA compositions were characterized by dynamic light scattering (DLS), zeta potential, and transmission electron microscopy (TEM). The mRNA loading capacity was evaluated by agarose gel electrophoresis.

[0190] Evaluation of mRNA delivery efficiency

[0191] To evaluate the efficiency of in vitro mRNA delivery, mRNA encoding green fluorescent protein (mGFP) was loaded into PHTA-based polymer nanocarriers, and their in vitro transfection efficiency in cells was characterized by confocal microscopy images or flow cytometry. To evaluate the efficiency of in vivo mRNA delivery, mRNA encoding firefly luciferase (mFluc) was loaded into PHTA-based polymer nanocarriers, and their in vivo transfection efficiency in mice was characterized by an IVIS optical imaging system.

[0192] Proof-of-concept study of mRNA therapeutics delivered via PHTA-based polymer nanocarriers

[0193] To evaluate the efficacy of mRNA therapy delivered via PHTA-based polymer nanocarriers, mRNA encoding the model antigen ovalbumin (mOVA) was loaded into PHTA-based polymer nanocarriers to prepare a polymer mRNA vaccine. The polymer mRNA vaccine was subcutaneously injected into B16-OVA tumor-bearing mice via the footpad to evaluate its antitumor efficacy.

[0194] Example 1-PHTA-Cn series

[0195] pass 1 The synthesis of PHTA-Cn polymer was examined by H NMR spectroscopy. The proton signals and integral ratios of the repeating units were compared with those of 1 The agreement with the theoretical values in the HNMR spectrum indicated the integrity of the chemical structure and the successful synthesis of the PHTA-Cn polymer (Figure 2).

[0196] Characterization of PHTA-Cn / mOVA nanovaccine

[0197] PHTA-Cn / mOVA nanovaccines were constructed by mixing OVA-encoding mRNA with PHTA-Cn polymers and cholesterol using microfluidics. The physicochemical properties of PHTA-Cn / mOVA nanovaccines were characterized by DLS, zeta potential, and TEM. DLS results showed that all PHTA-Cn / mOVA nanovaccines were nanoparticles with a diameter of <200 nm and a narrow distribution (<0.3). Figure 3 The decreased zeta potential of the PHTA-Cn / mOVA complex compared to PHTA-Cn polymer nanoparticles (PNPs) indicated the successful loading of mOVA ( Figure 4 Representative TEM images showing the nanosphere morphology of PHTA-based PNPs ( Figure 5 The mRNA binding ability of the PHTA-Cn polymer nanocarriers was verified by agarose gel electrophoresis on the 1st and 8th day after mRNA loading. The fixed complexes retained in the starting area of the gel and the bands without free mRNA indicated that the mRNA was effectively loaded into the polymer nanocarriers ( Figure 6 These findings collectively indicate that the PHTA-Cn / mOVA nanovaccine was successfully constructed.

[0198] In vitro mRNA delivery efficiency of PHTA-Cn / mGFP complex

[0199] To evaluate the in vitro mRNA delivery efficiency, different PHTA-Cn / mGFP complexes were prepared and their in vitro transfection efficiency in DC 2.4 cells was characterized by confocal microscopy images and flow cytometry. The fluorescence signals in the confocal microscopy images showed that all polymer nanocarriers exhibited different degrees of mRNA delivery ability and were able to successfully express GFP ( Figure 7 GFP expression analysis by flow cytometry showed that PHTA-C18 / mGFP exhibited the highest GFP expression level ( Figure 8 It can be speculated that the stronger hydrophobicity imparted by the longer alkyl chain of PHTA-C18 polymer promotes polymer self-assembly and improves the stability of PNP through hydrophobic interaction, thereby leading to higher mRNA transfection efficiency.

[0200] In vivo mRNA delivery efficiency of PHTA-Cn / mFluc complex

[0201] To evaluate the in vivo mRNA delivery efficiency, PHTA-C18 / mFluc and PHTA-C8 / mFluc complexes were prepared and injected subcutaneously into C57BL / 6 mice via the footpad and analyzed by IVIS optical imaging system. Bioluminescence images and corresponding quantitative results showed that PHTA-C18 / mFluc treatment resulted in much stronger luminescence intensity than the PHTA-C8 / mFluc treatment group due to the expression of luciferase protein ( Figure 9 These results demonstrate that PHTA-C18 PNP can promote efficient mRNA migration to lymph nodes (LNs) and allow for sustained and rapid protein translation in vivo.

[0202] Proof-of-concept study of PHTA-Cn / mOVA nanovaccine as a therapeutic cancer vaccine

[0203] The antitumor effect of PHTA-C18 / mOVA nanovaccine was evaluated in a subcutaneous B16-OVA melanoma tumor model. PHTA-C18 / mOVA and PHTA-C8 / mOVA as control formulations and PBS were subcutaneously injected into mice on days 4, 7, and 10 after vaccination. Figure 10 The antitumor efficacy was evaluated by measuring the tumor volume growth in the different treatment groups. Mice vaccinated with PHTA-C8 / mOVA showed a slight degree of tumor inhibition compared with the PBS-treated group, while PHTA-C18 / mOVA treatment produced sustained tumor inhibition ( Figure 11 ). Accordingly, the tumor inhibition efficiency of PHTA-C18 / mOVA was calculated to be 87%, which was higher than that of PHTA-C8 / mOVA (36%) ( Figure 12 The essentially unchanged body weights of all groups indicated that the PHTA-based polymer mRNA vaccine was well tolerated and had no significant toxicity ( Figure 13 ).

[0204] Example 2 - PHTA-BCn series

[0205] Characterization of PHTA-BCn polymer

[0206] PHTA-BCn polymers were synthesized by amino-epoxy polymerization of 1,3-butadiene diepoxide (BDE) and various alkylamines, where "B" represents the BDE monomer and "n" represents the number of carbon atoms in the polymer side chain. 1 The synthesis of PHTA-BCn polymer was examined by H NMR spectroscopy. The proton signals and integral ratios of the repeating units were compared with those of 1 The agreement with the theoretical value in the H NMR spectrum indicated that the PHTA-BCn polymer was successfully synthesized ( FIG14 ).

[0207] Characterization of PHTA-BCn / mRNA complexes

[0208] The PHTA-BCn / mGFP complex was constructed by simple microfluidic mixing of PHTA-BCn polymer and mGFP. The mRNA loading capacity of the PHTA-BCn polymer nanocarrier was verified by agarose gel electrophoresis. As shown in the agarose gel electrophoresis image ( Figure 15 ), all PHTA-BCn / mGFP complexes were retained in the starting area of the gel, and no free mRNA band was observed, indicating that PHTA-BCn-based polymer nanocarriers can successfully load mGFP.

[0209] A representative PHTA-BC10 / mOVA nanovaccine was constructed by microfluidic mixing of PHTA-BC10 polymer and mOVA ( Figure 16a ). The physicochemical properties of the PHTA-BC10 / mOVA nanovaccine were characterized by DLS and zeta potential. DLS results showed that the PHTA-BC10 / mOVA nanovaccine was a nanoparticle with a diameter of <200 nm (Figure 16b). The zeta potential of the PHTA-BC10 / mOVA nanovaccine was lower than that of the PHTA-BC10 PNPs without mRNA loading, further indicating the successful loading of mOVA (Figure 16c). These results confirmed the successful loading of mOVA and the construction of the PHTA-BC10 / mOVA nanovaccine.

[0210] In vitro mRNA delivery efficiency of PHTA-BCn / mGFP complex

[0211] To evaluate the in vitro mRNA delivery efficiency of PHTA-BCn-based polymeric nanocarriers, PHTA-BCn / mGFP complexes were prepared by loading mGFP and their transfection efficiency in DC 2.4 cells was studied. As indicated by the semi-quantitative results of the mean fluorescence intensity (MFI) of green fluorescent protein (GFP), DC 2.4 cells treated with different PHTA-BCn / mGFP complexes showed varying degrees of GFP expression, confirming the successful in vitro mRNA delivery capability of PHTA-BCn-based polymeric nanocarriers ( Figure 17 ).

[0212] Proof-of-concept study of PHTA-BCn / mOVA nanovaccine as a therapeutic cancer vaccine

[0213] The anti-tumor effect of a representative PHTA-BC10 / mOVA nanovaccine was evaluated in a subcutaneous B16-OVA melanoma tumor model. PHTA-BC10 / mOVA nanovaccine and PBS as a control formulation were subcutaneously injected into mice via the footpad on days 4, 7, and 10 after vaccination. Anti-tumor efficacy was evaluated by measuring tumor volume growth in different treatment groups. The results showed that mice vaccinated with the PHTA-BC10 / mOVA nanovaccine produced effective and sustained tumor suppression compared to mice treated with PBS ( Figure 18 a). The tumor inhibition efficiency of PHTA-BC10 / mOVA nanovaccine was calculated to be 89% ( Figure 18 b) These results demonstrate the potential of PHTA-BCn-based polymeric nanocarriers for the delivery of mRNA therapeutics.

[0214] Example 3-PHTA-BFn series

[0215] Characterization of PHTA-BFn polymer

[0216] PHTA-BFn polymers were synthesized via aminoepoxy polymerization of 1,3-butadiene diepoxide (BDE) and various fluorine-substituted alkylamines, where "B" represents the BDE monomer and "n" represents the number of fluorine atoms in the polymer side chain. The synthesis of the PHTA-BFn polymers was characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). As shown in the MALDI-TOF-MS spectra ( FIG19 ), three series of peaks with the same m / z interval consistent with the molar mass of the PHTA-BFn polymer repeating units were present in each MALDI-TOF-MS spectrum, indicating the successful synthesis of the alternating copolymer PHTA-BFn.

[0217] Characterization of PHTA-BF7 / mRNA complexes

[0218] Representative PHTA-BF7 / mRNA complexes were constructed by microfluidic mixing of PHTA-BF7 polymer and mRNA. The mRNA loading capacity of PHTA-BF7 polymer nanocarriers was verified by agarose gel electrophoresis. As shown in the agarose gel electrophoresis image ( Figure 20As shown in a), the PHTA-BF7 / mRNA complex was retained in the starting area of the gel, and no free mRNA band was observed, indicating that the PHTA-BF7 polymer nanocarrier can successfully load mRNA. DLS results showed that the PHTA-BF7 / mRNA complex was a nanoparticle with a diameter of <200 nm and a narrow distribution ( Figure 20 b). These results indicate that the PHTA-BF7 / mRNA complex was successfully constructed.

[0219] In vitro mRNA delivery efficiency of PHTA-BF7 / mGFP complex

[0220] The in vitro mRNA delivery efficiency of PHTA-BF7 / mGFP complex was studied in antigen-presenting cells DC 2.4, macrophages RAW 264.7, and tumor cells HEK 293T and PC3. The semi-quantitative results of the mean fluorescence intensity (MFI) of green fluorescent protein (GFP) were shown ( Figure 21 ), all four cell lines showed effective GFP protein expression after treatment with PHTA-BF7 / mGFP complexes. These results indicate that PHTA-BF7-based polymer nanocarriers can successfully deliver mRNA in vitro.

[0221] In vivo mRNA delivery efficiency of PHTA-BF7 / mFluc complex

[0222] To further investigate the in vivo mRNA delivery efficiency of the PHTA-BF7 / mFluc complex, the PHTA-BF7 / mFluc complex was subcutaneously injected into mice and characterized by an IVIS optical imaging system at 8 and 24 hours after injection. Figure 22 ), PHTA-BF7 / mFluc treatment induced efficient luciferase protein expression in mice, demonstrating the in vivo mRNA delivery ability of PHTA-BFn-based polymer nanocarriers.

[0223] in conclusion

[0224] In summary, a new ionizable alternating copolymer (PHTA) with hydroxyl tertiary amine (HTA) repeating units has been developed for polynucleotide delivery. PHTA polymers were synthesized by amino-epoxy polymerization of diepoxide monomer 1 and amine monomer 2. By varying the types of diepoxide monomer 1 and amine monomer 2, three representative series of PHTA polymers (i.e., PHTA-Cn, PHTA-BCn, and PHTA-BFn) were synthesized and their polynucleotide delivery efficiency was studied. The results showed that all three series of PHTA polymers could successfully deliver polynucleotides into cells in vitro and in vivo.

Claims

1. An ionizable polymer comprising structural units according to formula (I): in R1 represents a covalent bond or a linking moiety derived from a polyalkylene glycol; and R2 represents a linear or branched aliphatic hydrocarbon group or a linear or branched fluorinated aliphatic hydrocarbon group.

2. The ionizable polymer according to claim 1, wherein the polyalkylene glycol is polyethylene glycol.

3. The ionizable polymer according to claim 1 or 2, wherein the polyalkylene glycol has a molecular weight of about 200 to about 1000, optionally about 300 to about 800, such as about 400 to about 600, such as about 500.

4. An ionizable polymer according to any one of the preceding claims, wherein R2 is a linear or branched alkyl group or a fluorinated alkyl group.

5. An ionizable polymer according to any one of the preceding claims, wherein R2 has 1 to 25 carbon atoms.

6. An ionizable polymer according to any one of the preceding claims, wherein R2 is selected from the group consisting of butyl, hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl and octadecyl.

7. The ionizable polymer according to any one of clauses 1 to 5, wherein R2 is selected from the group consisting of difluoroethyl, pentafluoropropyl, heptafluorobutyl, nonafluoropentyl and pentafluorooctyl.

8. An ionizable polymer according to any one of the preceding claims, wherein n is an integer from 1 to 10,000.

9. An ionizable polymer according to any one of the preceding claims, wherein R1 is according to formula (II) in, Each Ra is independently a covalent bond or a linear -C 1-6 Alkylene-; Rb is -C 1-6 Alkylene-, said alkylene being a straight chain alkylene and optionally substituted by one or more groups selected from OH, NH2, C 1-4 Alkyl and halogen radical substitution; p is an integer from 1 to 10.

10. An ionizable polymer according to any preceding claim, wherein each Ra is -C1 alkylene- and Rb is a linear and unsubstituted -C2 alkylene-.

11. The ionizable polymer according to claim 1 , wherein the ionizable polymer comprises structural units selected from the group consisting of: i) ii) iii) iv) v) vi) and vii) 12. The ionizable polymer of claim 1 , wherein the ionizable polymer comprises structural units selected from the group consisting of: viii) ix) x) xi) 12) xiii) xiv) xv) and xvi) 13. A composition comprising the ionizable polymer according to any one of claims 1 to 12, and a polynucleotide, wherein the polynucleotide is complexed with polymer nanoparticles (PNPs) formed from the ionizable polymer.

14. The composition of claim 13, wherein the polynucleotide is selected from the group consisting of RNA and DNA, optionally wherein the polynucleotide is mRNA or siRNA.

15. The composition according to claim 13 or 14, wherein: c) R1 is derived from a polyalkylene glycol, and the polynucleotide-PNP complex is formed by mixing the polynucleotide, cholesterol, and an ionizable polymer, optionally wherein the polyalkylene glycol is polyethylene glycol; or d) R1 is a covalent bond and the polynucleotide-PNP complex is formed by mixing the polynucleotide and the ionizable polymer.

16. The composition of any one of claims 13 to 15, wherein the polynucleotide-PNP complex is capable of delivering the polynucleotide into a human or non-human animal cell, optionally wherein the polynucleotide is mRNA and / or wherein the human cell is a cancer cell.

17. The composition of any one of claims 13 to 16, wherein the polynucleotide is mRNA or DNA, and wherein the mRNA or DNA encodes a cancer-specific antigen, an infectious disease-specific antigen, or a therapeutic protein.

18. A pharmaceutical composition comprising the composition according to any one of claims 12 to 16.

19. The pharmaceutical composition of claim 18, comprising polymer nanoparticles (PNPs) formed from a polynucleotide and an ionizable polymer, wherein: a) R1 is a linking moiety derived from polyethylene glycol, and R2 is octadecyl, or b) R1 is a covalent bond, and R2 is a decyl group.

20. The pharmaceutical composition according to claim 18 or 19, for use in treating cancer, wherein the mRNA or DNA encodes a cancer-specific antigen.

21. The pharmaceutical composition according to claim 18 or 19, for preventing or treating an infectious disease, wherein the mRNA or DNA encodes an infectious disease-specific antigen.

22. The pharmaceutical composition according to claim 21, wherein the infectious disease is a virus-related disease, and the mRNA or DNA encodes a virus-specific antigen.

23. The pharmaceutical composition according to claim 18 or 19, for use in treating a protein deficiency disease, wherein the mRNA or DNA encodes a protein or peptide that is absent or non-functional in the subject to be treated.

24. A method for preventing or treating a subject, comprising administering to the subject an effective amount of: The composition according to any one of claims 13 to 17, wherein the polynucleotide is mRNA or DNA, or The pharmaceutical composition according to any one of claims 18 to 22.

25. The method of claim 24, wherein the treatment is treatment of cancer in the subject and the mRNA or DNA encodes a cancer-specific antigen or a therapeutic protein.

26. The method of claim 24, wherein the prevention is prevention of a virus-related infection in a subject and the mRNA or DNA encodes a virus-specific antigen.

27. The method of claim 24, wherein the treatment or prevention is the treatment or prevention of a protein deficiency in a subject, wherein the mRNA or DNA encodes a protein or peptide that is absent or non-functional in the subject.

28. Use of the composition according to any one of claims 13 to 17 or the pharmaceutical composition according to any one of claims 17 to 22 in the manufacture of a medicament for preventing or treating a disease selected from the group consisting of cancer and virus-related diseases.

29. Use of the composition according to any one of claims 13 to 17 or the pharmaceutical composition according to any one of claims 17 to 22 in the manufacture of a medicament for preventing or treating protein deficiency.

30. A method of producing a composition according to claims 13-17, wherein the polynucleotide is mRNA or DNA, comprising the steps of: i) combining a monomer of formula (III) and a monomer of formula (IV) by amino-epoxy ring-opening polymerization to form an ionizable polymer comprising structural units according to formula (I); and ii) mixing the ionizable polymer with mRNA or DNA encoding an antigenic polypeptide or therapeutic protein, in, in, R1′ represents a covalent bond or a polyalkylene glycol moiety; and R2' represents a linear or branched aliphatic hydrocarbon group or a linear or branched fluorinated aliphatic hydrocarbon group.

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