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WO2025149492A1 - Rna encoding an immune inhibitory il-1 family member - Google Patents

Rna encoding an immune inhibitory il-1 family member

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Publication number
WO2025149492A1
WO2025149492A1 PCT/EP2025/050272 EP2025050272W WO2025149492A1 WO 2025149492 A1 WO2025149492 A1 WO 2025149492A1 EP 2025050272 W EP2025050272 W EP 2025050272W WO 2025149492 A1 WO2025149492 A1 WO 2025149492A1
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WO
WIPO (PCT)
Prior art keywords
rna
lipid
family
cells
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/050272
Other languages
French (fr)
Inventor
Roman Fischer
Ugur Sahin
Carine THALMAN
Micaela DOMINGUES
Elena STEHLE
Nancy GUTIERREZ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Resano GmbH
Biontech SE
Original Assignee
Resano GmbH
Biontech SE
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Filing date
Publication date
Application filed by Resano GmbH, Biontech SE filed Critical Resano GmbH
Publication of WO2025149492A1 publication Critical patent/WO2025149492A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/545—IL-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/335—Modified T or U

Definitions

  • the present invention provides an isolated RNA encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof.
  • the invention also relates to a nucleic acid particle or a pharmaceutical composition comprising the RNA of the invention. Therapeutic uses of the RNA, nucleic acid particle and pharmaceutical composition of the invention are also provided.
  • BACKGROUND TO THE INVENTION A large number of diseases are significantly exacerbated by ongoing inflammatory processes. These include atherosclerosis.
  • the interleukin (IL) 1 (IL-1) family of cytokines plays a central role in the regulation of both innate and adaptive immunity, and particularly in inflammation.
  • IL-1 family members Tight regulation of the pro-inflammatory effects of IL-1 family members by receptor antagonists and decoy receptors provides a balance between enhancement of immunity and uncontrolled inflammation, which is the cause of a variety of diseases and contributes to their development and maintenance.
  • the different pro-inflammatory IL-1 family members have been associated with a variety of diseases.
  • the pro-inflammatory influence of the IL-1 family increases the risk and severity of cardiovascular disease (CVD) and atherosclerosis.
  • CVD cardiovascular disease
  • IL-1 ⁇ One of the most important inflammatory mediators within this family is IL-1 ⁇ , which is a secreted cytokine that triggers the production of further inflammatory mediators and also activates other immune cells.
  • IL-1 ⁇ is a secreted cytokine that triggers the production of further inflammatory mediators and also activates other immune cells.
  • the polynucleotide is RNA. Where further aspects of the invention described herein refer to polynucleotide, the polynucleotide is preferably an RNA. In a further aspect, the invention provides an RNA encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof. In some embodiments, the polynucleotide may be an isolated polynucleotide.
  • IL-1 interleukin-1
  • the immune inhibitory protein of the IL-1 family is selected from the group consisting of: IL-1 receptor antagonist (IL-1RA), interleukin-18 binding protein (IL- 18BP), interleukin 36 receptor antagonist (IL-36RA), interleukin 37 (IL-37), and interleukin 38 (IL-38).
  • the immune inhibitory protein of the IL-1 family is IL-1RA, IL-18BP or IL-36RA.
  • the immune inhibitory protein of the interleukin-1 (IL-1) family further comprises the cognate of a binding moiety.
  • the cognate of a binding moiety is an antigen, an epitope or a polypeptide tag.
  • the RNA comprises more than one type of modified nucleoside, wherein the modified nucleosides are independently selected from pseudouridine ( ⁇ ), N1- methyl-pseudouridine (m1 ⁇ ), and 5-methyl-uridine (m5U).
  • the modified nucleoside is pseudouridine ( ⁇ ) or N1-methyl- pseudouridine (m1 ⁇ ).
  • the RNA comprises a 5’ cap analog.
  • the RNA comprises the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG or 3 ⁇ - O-Me-m 7 G(5')ppp(5')G.
  • the RNA comprises the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG.
  • the RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12 and 31, or a nucleotide sequence having at least 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12 and 31.
  • the invention provides the polynucleotide, preferably the RNA, according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention for use in the prevention and/or treatment of a disease or condition selected from the group consisting of: cancer, a cardiovascular disease, atherosclerosis, Alzheimer's disease, an autoimmune inflammatory disease, a liver disease, or an inflammatory skin condition.
  • a disease is atherosclerosis or a cardiovascular disease.
  • the cardiovascular disease is heart failure or pericarditis.
  • the autoimmune inflammatory disease is multiple sclerosis or rheumatoid arthritis.
  • the inflammatory skin condition is psoriasis.
  • the liver disease is non-alcoholic fatty liver disease.
  • Figure 3 Receptor expression on HEK-Blue TM IL-36 cells.
  • HEK-BlueTM IL-1R (A), IL-18 (B) and IL-36 (C) cells were stimulated with increasing concentrations of recombinant human IL-1 ⁇ , IL-1 ⁇ , IL-18, IL-36 ⁇ , IL-36 ⁇ and IL-36 ⁇ .
  • the NF ⁇ B and AP-1 response was determined by adding QUANTI-BlueTM Solution, a SEAP detection reagent.
  • the absorbance was measured at 620 nm.
  • IFN ⁇ was used as a negative control.
  • the activity in % of the different reporter cell lines was calculated. The highest response of either IL-1 ⁇ , IL-18 or IL-36 ⁇ was set at 100% activity.
  • FIG. 5 Inhibitory effect of anti-inflammatory members of the IL-1 family on stimulated HEK-Blue TM reporter cell lines.
  • HEK-Blue TM IL-1R (A), IL-18 (B) and IL-36 (C) cells were stimulated with recombinant human IL-1 ⁇ (8 pg/ml), IL-18 (20 pg/ml) or IL-36 ⁇ (500 pg/ml) in the presence of serially diluted IL-1RA, IL-18BP, IL-36RA, IL-37 and IL-38.
  • IL-1RA- or IL-18BP-encoding mRNA was lipofected into HEK 293T/17 cells and the IL-1RA or IL-18BP concentration in the supernatant was quantified by ELISA.
  • HEK-Blue TM IL-1R (A) and IL-18 (B) cells were stimulated with IL- 1 ⁇ (8 pg/ml) or IL-18 (20 pg/ml) in the presence of serially diluted mRNA-encoded IL-1RA or IL-18BP supernatant or recombinant IL-1RA (purchased, Anakinra) or IL-18BP (purchased) as a reference.
  • Figure 7 IL-1 receptor family gene expression. THP-1 cells were incubated for 72 h in the presence of 100 nM PMA and then in RPMI medium for further 24 h.
  • Figure 8 IL-1 receptor family expression of THP-1 cells.
  • THP-1 cells were incubated for 72 h in the presence of 100 nM PMA and then in RPMI medium for further 24 h. Macrophages were stimulated in presence of 100 ⁇ g/ml oxLDL for 24 h, then detached with a cell scraper, collected, and washed. Expression was quantified by staining with the corresponding antibodies against the receptors IL-1R1, IL-18R1 and IL-36R and co-receptors IL 1RAP and IL-18R2 by flow cytometric analysis.
  • Figure 9 Comparison of receptor expression on the surface of unstimulated and stimulated HEK-Blue TM IL-1R cells.
  • the receptor expression was quantified by flow cytometry staining with the corresponding antibodies against the receptors IL-1R1, IL-18R ⁇ and IL-36R and the co-receptors IL-1RAcP and IL-18R ⁇ . Shown is the quantified receptor expression on HEK-Blue TM IL-1R cells.
  • the cells were also stained with the corresponding isotype of IL-1R1, IL-18R ⁇ , IL-1RAcP and IL-18R ⁇ (1:100).
  • the receptor expression was quantified by staining with the corresponding antibodies against the receptors IL-1R1, IL-18R ⁇ and IL-36R and the co- receptors IL-1RAcP and IL-18R ⁇ using flow cytometry. Shown is the quantified receptor expression on HEK-Blue TM IL-18 cells. As a control, the cells were also stained with the corresponding isotype of IL-1R1, IL-18R ⁇ , IL-1RAcP and IL-18R ⁇ (1:100). For IL-36R a mixture of Aqua Zombie (1:1,000) and streptavidin FITC (1:100) was used as a control.
  • C Comparison of the normalized MFI of the surface expression of the receptors of unstimulated and IL-18 stimulated HEK-Blue TM IL-18 (n 3 ⁇ SD).
  • Figure 11 Comparison of receptor expression on the surface of unstimulated and stimulated HEK-Blue TM IL-36 cells.
  • the receptor expression was quantified by staining with the corresponding antibodies against the receptors IL-1R1, IL-18R ⁇ and IL-36R and the co- receptors IL-1RAcP and IL-18R ⁇ using flow cytometry. Shown is the quantified receptor expression on HEK-Blue TM IL-36 cells. Isotype staining was used as a control for IL-1R1 (1:8), IL-18R ⁇ , IL-1RAcP and IL-18R ⁇ (1:100). For IL-36R a mixture of Aqua Zombie (1:1,000) and streptavidin FITC (1:100) was used as a control.
  • C Comparison of the normalized MFI of the surface expression of the receptors of unstimulated and IL-36 ⁇ stimulated HEK- Blue TM IL-36 (n 3 ⁇ SD).
  • Figure 12 Responses of the HEK-Blue TM reporter cell lines after cytokine stimulation. The bioactivity of the cytokines of the IL-1 family was quantified using SEAP assay.
  • the HEK-Blue TM cells were first stimulated for 24 h at 37°C and 5% CO2 with a x/( ⁇ 10) dilution series of IL-1 ⁇ IL-1 ⁇ , IL-18, IL-36 ⁇ , IL-36 ⁇ (1 ng/ml) or IL-36 ⁇ (100 ng/ml) or IFN- ⁇ (1 ng/ml) as a control. Afterwards the supernatant was diluted 1:3 in Quanti-Blue TM solution and incubated for 1 h at 37°C and 5% CO 2 and the absorption was measured at 620 nm. A Response of HEK-Blue TM IL-1R cells normalized to IL-1 ⁇ .
  • Figure 19 Inhibitory effect of IL-1RA on monocytes. Pro-inflammatory cytokines release was analyzed in CD14+ monocytes with MSD.
  • the invention provides a polynucleotide encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof.
  • the invention provides a polynucleotide encoding an anti-inflammatory protein of the interleukin-1 (IL-1) family or functional variant thereof.
  • RNA RNA
  • RNA RNA
  • encoding or similar terms
  • terms such as “capable of expressing”, “polynucleotide (e.g. RNA) expressing” and “polynucleotide (e.g. RNA) encoding” or similar terms are used interchangeably herein and with respect to a particular peptide or polypeptide mean that the polynucleotide, if present in the appropriate environment, e.g. within a cell, can be expressed to produce said peptide or polypeptide. It will be understood by the skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code.
  • polynucleotides of the present invention are codon optimised to enable expression in a mammalian cell, in particular a cell as described herein.
  • Polynucleotides according to the invention may comprise DNA and/or RNA.
  • the polynucleotides may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides.
  • the IL-1 family members mediate their effects via different heterodimeric IL-1 receptor (IL-1R) complexes.
  • IL-1R heterodimeric IL-1 receptor
  • Many well-defined interactions within the IL-1 family are known in the art, such as the binding of IL-1 ⁇ and IL-1 ⁇ to IL-1R, IL-18 to IL-18R, and IL-36 ⁇ , IL-36 ⁇ , and IL-36 ⁇ to IL-36R.
  • the different pro-inflammatory IL-1 family members have been associated with a variety of diseases. For example, the pro-inflammatory influence of the IL-1 family increases the risk and severity of cardiovascular disease (CVD).
  • CVD cardiovascular disease
  • IL-1 signaling by administering immune inhibitory or anti-inflammatory IL-1 family members or neutralizing antibodies has been shown to be therapeutically effective in various autoimmune inflammatory diseases and inflammatory conditions such as cardiovascular disease.
  • blocking IL-1 ⁇ is the current standard of care for some autoimmune inflammatory diseases and IL-1 antagonists such as canakinumab or anakinra are used clinically for the treatment of CVD.
  • IL-1 antagonists such as canakinumab or anakinra are used clinically for the treatment of CVD.
  • an anti-atherosclerotic effect was observed when anakinra was administrated in ApoE KO mice.
  • neutralizing anti-IL-1 ⁇ antibodies have been used to demonstrate the central role of IL-1 ⁇ in the development and pathogenesis of atherosclerosis, a chronic inflammatory vascular disease.
  • a poly(A) sequence comprises or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides, and, in particular, about 110 nucleotides.
  • a poly(A) sequence comprises or consists of from about 80 to about 150 nucleotides, optionally from about 110 to about 120 nucleotides.
  • the poly(A) sequence only consists of A nucleotides.
  • the poly(A) sequence comprises or consists of about 120 A nucleotides.
  • the poly(A) sequence essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, and U), as disclosed in WO 2016/005324 A1, hereby incorporated by reference. Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • a poly(A) cassette present in the coding strand of DNA that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g.
  • RNA comprises a poly-A tail.
  • the poly-A tail comprises at least 100 nucleotides.
  • the poly-A tail is an interrupted poly-A tail.
  • poly-A tail sequence is provided below: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 14)
  • the poly-A tail comprises the poly-A tail shown in SEQ ID NO: 14.
  • the RNA of the invention may comprise a poly-A tail shown in SEQ ID NO: 14 and the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG.
  • the RNA of the invention may comprise a poly-A tail shown in SEQ ID NO: 14, and the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG, and a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto.
  • the RNA of the invention may comprise a poly-A tail shown in SEQ ID NO: 14, and the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG, and a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the nucleotide sequence of SEQ ID NO: 31.
  • the RNA of the invention may comprise a poly-A tail shown in SEQ ID NO: 14, and the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG, and a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto, and a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
  • Illustrative polynucleotide sequences Illustrative RNA backbone sequence (5’UTR shown in bold, Kozak sequence shown in italics, 3’UTR shown underlined, and poly-A tail shown in italics and underlined): AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC – [coding sequence] - CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCC CCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGAC ACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAG UGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGU GCCAGCCACACCCUGGAGCUAGCA
  • RNA backbone sequence such as SEQ ID NO: 15.
  • the polynucleotide comprises a sequence as set forth in SEQ ID NO: 23 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto.
  • the envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic.
  • the particle may be a monolamellar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids) optionally in combination with additional substances (e.g., additional lipids) which do not have to be amphiphilic.
  • the term "particle” relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term “particle” includes nanoparticles.
  • liposomes comprise unilamellar or multilamellar phospholipid bilayers enclosing an aqueous core (also referred to herein as an aqueous lumen). They may be prepared from materials possessing polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups.
  • cationic lipids employed in formulating liposomes designed for the delivery of nucleic acids are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked to a hydrocarbon chain or cholesterol derivative via glycerol.
  • electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, in particular cationic or cationically ionizable amphiphilic lipids) and negatively charged nucleic acid (especially mRNA) results in complexation and spontaneous formation of nucleic acid lipoplex particles.
  • Positively charged liposomes may be generally synthesized using a cationic or cationically ionizable amphiphilic lipid, such as DOTMA and/or DODMA, and additional lipids, such as DOPE.
  • a nucleic acid (such as RNA and/or DNA, especially mRNA) lipoplex particle is a nanoparticle.
  • a lipid nanoparticle is obtainable from direct mixing of nucleic acid (such as RNA and/or DNA) in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol.
  • nucleic acid such as RNA and/or DNA
  • lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.
  • LNPs comprise or consist of a cationic/ionisable lipid and helper lipids such as phospholipids, cholesterol, and/or polymer-conjugated lipids (i.e., stealth lipids), such as polyethylene glycol (PEG) lipids.
  • PEG polyethylene glycol
  • nucleic acid LNPs such as RNA/DNA LNPs
  • the nucleic acid is bound by ionisable lipid that occupies the central core of the LNP.
  • PEG lipid forms the surface of the LNP, along with phospholipids.
  • the surface comprises a bilayer.
  • cholesterol and ionisable lipid in charged and uncharged forms can be distributed throughout the LNP.
  • nucleic acid such as RNA and/or DNA, e.g., mRNA
  • nucleic acid may be noncovalently associated with a particle as described herein.
  • the nucleic acid (such as RNA and/or DNA, especially mRNA) may be adhered to the outer surface of the particle (surface nucleic acid) and/or may be contained in the particle (encapsulated nucleic acid (such as encapsulated DNA or mRNA).
  • the particles (e.g., LNPs and LPXs) described herein have a size (such as a diameter) in the range of about 10 to about 2000 nm, such as at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and/or at most 1900 nm (e.g., at most about 1900 nm, at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm) and/
  • the particles described herein are nanoparticles.
  • nanoparticle relates to a nano-sized particle comprising nucleic acid (especially RNA and/or DNA) as described herein and at least one cationic or cationically ionisable lipid, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least about 1 nm and below about 1000 nm.
  • the size of a particle is its diameter.
  • a nucleic acid particle of the disclosure may be a particle comprising at least one lipid.
  • the particles formed from the polynucleotides (e.g., RNA) of the disclosure and at least one lipid are lipid nanoparticles (LNP), lipoplexes (LPX) or liposomes.
  • the particles are LPX particles.
  • the polynucleotide encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof of the disclosure is an RNA or mRNA molecule.
  • an LPX particle comprises an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, and RNA (especially mRNA) as described herein.
  • electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, in particular cationic or cationically ionizable amphiphilic lipids) and negatively charged nucleic acid (especially mRNA) results in complexation and spontaneous formation of nucleic acid lipoplex particles.
  • Positively charged liposomes may be generally synthesized using a cationic or cationically ionizable amphiphilic lipid, such as DOTMA and/or DODMA, and additional lipids, such as DOPE.
  • an RNA (especially mRNA) lipoplex particle is a nanoparticle.
  • the at least one lipid comprises at least one cationic or cationically ionizable lipid, preferably is a cationic lipid.
  • a "cationic lipid” refers to a lipid or lipid-like material having a net positive charge. Cationic lipids bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic or cationically ionizable lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.
  • the particles according to the present disclosure comprises RNA in the format of a lipoplex.
  • lipoplex refers to a complex of lipids and nucleic acids such as RNA.
  • Lipoplexes can be formed of cationic (positively charged) liposomes and the anionic (negatively charged) nucleic acid.
  • the cationic liposomes can also include a neutral “helper” lipid.
  • the lipoplexes form spontaneously by mixing the nucleic acid with the liposomes with a certain mixing protocol, however various other protocols may be applied. It is understood that electrostatic interactions between positively charged liposomes and negatively charged nucleic acid are the driving force for the lipoplex formation (WO 2013/143555 A1).
  • the net charge of the RNA lipoplex particles is close to zero or negative. It is known that electro- neutral or negatively charged lipoplexes of RNA and liposomes lead to substantial RNA expression in spleen dendritic cells (DCs) after systemic administration and are not associated with the elevated toxicity that has been reported for positively charged liposomes and lipoplexes (cf. WO 2013/143555 A1).
  • DCs spleen dendritic cells
  • the composition according to the present disclosure comprises RNA encoding an immune inhibitory protein of the IL-1 family or functional variant thereof, in the format of nanoparticles, preferably lipoplex nanoparticles, in which (i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles and/or (ii) the nanoparticles have a net negative charge and/or (iii) the charge ratio of positive charges to negative charges in the nanoparticles is 1.4:1 or less and/or (iv) the zeta potential of the nanoparticles is 0 or less.
  • zeta potential is a scientific term for electrokinetic potential in colloidal systems.
  • the zeta potential and (b) the charge ratio of the cationic or cationically ionizable lipid to the RNA in the nanoparticles can both be calculated as disclosed in WO 2013/143555 A1.
  • particles which are nanoparticulate lipoplex formulations with a defined particle size, wherein the net charge of the particles is negative, as disclosed in WO 2013/143555 A1 are preferred particles in the context of the present disclosure.
  • the lipoplexes are obtained according to a method as disclosed in WO 2019/077053 A1. According to WO 2019/077053 A1, lipoplexes can be obtained by adding liposome colloid with a solution comprising RNA.
  • the liposome colloid can be obtained by a method comprising injecting a lipid solution in ethanol into an aqueous phase to produce the liposome colloid, wherein the concentration of at least one of the lipids in the lipid solution corresponds to or is higher than the equilibrium solubility of the at least one lipid in ethanol.
  • a particularly preferred method of producing a liposome colloid comprises injecting a lipid solution comprising DOTMA and DOPE in a molar ratio of about 2:1 in ethanol into water stirred at a stirring velocity of about 150 rpm to produce the liposome colloid, wherein the concentration of DOTMA and DOPE in the lipid solution is about 330 mM.
  • the RNA particles target the spleen, in particular professional antigen presenting cells, preferably monocytes and/or macrophages or their precursors.
  • a composition comprising nanoparticles which comprise at least one cationic lipid, at least one neutral helper lipid and RNA encoding an immune inhibitory protein of the IL-1 family, or functional variant thereof, of the present disclosure, where the charge ratio of positive charges to negative charges in the nanoparticles can be between 1:1 and 1:8 (such as between 1:1 and 1:4, optionally between 1:2 and 1:1.2).
  • the positive charges are contributed by the at least one cationic lipid and the negative charges are contributed by the RNA.
  • the nanoparticles can be lipoplexes comprising DOTAP and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.
  • RNA lipoplex particles target immune cells, preferably monocytes and/or macrophages.
  • the composition comprises RNA lipoplex particles as described in WO2022069632A1 or WO2019077053A1.
  • a LNP comprises about 40 mol% to about 50 mol% (e.g., about 47.5 mol%) (relative to the total amount of lipids in a LNP) of a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ; BODD-C2C2-1Me-Pyr; ALC-0315; ALC-0366; SM-102; HY-501; EA-405; HY-405; DODMA; and Dlin-MC3-DMA.
  • a LNP comprises about 40 mol% to about 50 mol% (e.g., about 47.5 mol%) (relative to the total amount of lipids in a LNP) of a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ; BODD-C2C2-1Me-Pyr; ALC-315, SM- 102; HY-501; and DODMA.
  • Helper lipids As described herein, lipid nanoparticles of the present disclosure comprise a helper lipid.
  • a helper lipid is or comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), sphingomyelins, N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), 1,2-diacylglyceryl-3-O-4’-(N,N,
  • a helper lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DSPE, and SM.
  • the helper lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.
  • the helper lipid is DSPC.
  • Helper lipids may be synthetic or naturally derived. Other helper lipids suitable for use in a lipid nanoparticle are described in WO 2021/026358, WO 2017/075531, and WO 2018/081480, the entire contents of each of which are incorporated herein by reference.
  • a lipid nanoparticle comprises about 5 to about 15 mol% of a helper lipid. In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 8 to about 12 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 10 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of DSPC. In some embodiments, a lipid nanoparticle comprises about 8 to about 12 mol% of DSPC. In some embodiments, a lipid nanoparticle comprises about 10 mol% of DSPC.
  • a PEG group that is part of a PEG-lipid has, on average in a composition comprising one or more PEG-lipid molecules, a number average molecular weight (M n ) of about 2000 g/mol.
  • M n number average molecular weight
  • a PEG-lipid is DMG-PEG.
  • a PEG-lipid is PEG2000-DMG:
  • a PEG-lipid is provided in WO 2021/026358, WO 2017/075531, or WO 2018/081480, each of which is incorporated by reference in its entirety.
  • a PEG-lipid is a compound of Formula PCL-I: or a pharmaceutically acceptable salt thereof, wherein, as applied to formula PGL-I, R 8 and R 9 are each independently C10-C30 aliphatic, optionally interrupted by one or more ester bonds, and w is an integer from 30 to 60.
  • a compound of Formula PCL-I is 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).
  • a compound of Formula PCL-I is: or a pharmaceutically acceptable salt thereof, where n’ is an integer from about 45 to about 50.
  • the PEG-lipid is represented by: wherein n has a mean value ranging from 30 to 60. In some embodiments, n is 50.
  • the PEG-conjugated lipid is PEG 2000 -C-DMA which preferably refers to 3-N-[( ⁇ -methoxy poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy- propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy) propylcarbamate (2000).
  • a PEG-lipid is selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG2000-DMG, ALC-159, PEG2000-C-DMA PEG-S-DMG, PEG-cer, and combinations thereof.
  • a PEG-lipid is ALC-0159 or PEG2000-DMG.
  • a PEG-lipid is ALC-0159.
  • a PEG-lipid is PEG2000- DMG.
  • a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid.
  • sarcosinylated lipid refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N-methylglycine)) portion.
  • a polymer-conjugated lipid is one described in WO 2024/028325, which is incorporated herein by reference in its entirety.
  • a polymer-conjugated lipid comprises monomers of 2-(2-(2- aminoethoxy)ethoxy)acetic acid.
  • the polymer of the polymer- conjugated lipid is or comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly- 2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof.
  • a polymer-conjugated lipid comprises monomers of unit PCL-II-1: II-1
  • a polymer-conjugated lipid comprises, 5 to 50, 5 to 25 or 10 to 25 monomers of PCL-II-1.
  • a polymer-conjugated lipid comprises 14 to 17 monomers of PCL-II-1. In some embodiments, a polymer-conjugated lipid comprises 8 to 14 monomers of PCL-II-1. In some embodiments, a polymer-conjugated lipid is selected from the table below: In some embodiments, an LNP comprises an polysarcosine-conjugated or a pAEEA/pMAEEA-conjugated lipid, as described herein.
  • nucleic acid particles e.g., DNA or RNA particles
  • nucleic acid particles described herein comprise a polysarcosine-conjugated or a pAEEA/pMAEEA-conjugated lipid and are substantially free of a pegylated lipid (or do not contain a pegylated lipid).
  • a lipid nanoparticle comprises about 0.5 to about 5.0 mol% of a polymer-conjugated lipid.
  • a lipid nanoparticle comprises about 1.0 to about 2.5 mol% of a polymer-conjugated lipid.
  • a lipid nanoparticle comprises about 1.5 to about 2.0 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.5 to about 1.8 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.5 mol% to about 1.8 mol% (relative to the total amount of lipids in a lipid nanoparticle) of a polymer-conjugated lipid selected from the group consisting of: DSPE-AEEA14-AC; VE-AEEA14-AC; ALC-0159 and PEG2000-DMG.
  • a lipid nanoparticle comprises about 1.5 mol% to about 1.8 mol% (relative to the total amount of lipids in a lipid nanoparticle) of a polymer-conjugated lipid selected from the group consisting of: DSPE-AEEA14-AC, VE-AEEA14-AC, and PEG2000- DMG.
  • a molar ratio of a cationic or cationically ionizable lipid to a polymer-conjugated lipid is from about 2:1 to about 8:1.
  • Steroids As described generally herein, lipid nanoparticles further comprise a steroid. In some embodiments, a steroid is a sterol.
  • a lipid nanoparticle comprises: about 40 to about 50 mol% of a cationically ionizable lipid; about 30 to about 45 mol% of a steroid (e.g., cholesterol); about 5 to about 15 mol% of a helper lipid (e.g., DSPC); and about 1 to about 2.5 mol% of a polymer-conjugated lipid.
  • a steroid e.g., cholesterol
  • helper lipid e.g., DSPC
  • a lipid nanoparticle comprises: about 47.5 mol% of ALC-366; about 10 mol% of DSPC; about 40.7 mol% of cholesterol; and about 1.8 mol% of ALC-159. In some embodiments, a lipid nanoparticle comprises about 50 mol% of SM-102; about 1.5 mol% of PEG2000-DMG; about 10 mol% of DSPC; and about 38.5 mol% of cholesterol.
  • cationically ionizable lipids, helper lipids, and steroids are solubilized in an organic solvent such as ethanol, at a predetermined weight or molar ratios/percentages (e.g., ones described herein).
  • a colloidal lipid dispersion comprising nucleic acids can be formed as follows: an ethanol solution comprising lipids, such as cationic lipids, helper lipids, steroids, and polymer-conjugated lipids, is combined with, e.g., injected into or continuously mixed with, an aqueous solution comprising nucleic acids.
  • lipids such as cationic lipids, helper lipids, steroids, and polymer-conjugated lipids
  • lipid and nucleic acid (e.g., RNA) solutions can be mixed at room temperature by pumping each solution (e.g., a lipid solution comprising a cationic lipid, a helper lipid, cholesterol, a conjugated lipid, and any other additives) at controlled flow rates into a mixing unit, for example, using piston pumps.
  • the flow rates of a lipid solution and a nucleic acid (e.g., RNA) solution into a mixing unit are maintained at a ratio of 1:3.
  • nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous nucleic acids (e.g., RNAs).
  • lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged nucleic acid (e.g., RNA).
  • a solution comprising nucleic acid (e.g., RNA)-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and/or filtration.
  • Pharmaceutical composition in one aspect, the invention provides a pharmaceutical composition comprising the polynucleotide, vector, or nucleic acid particle of the invention. In a further aspect, the invention provides a pharmaceutical composition comprising the polynucleotide according to the invention.
  • the pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • Such a formulation may, for example, be in a form suitable for intravenous infusion or intramuscular administration.
  • the pharmaceutical composition may be in a form not suitable for intratumoral administration.
  • Therapeutic use in a further aspect, provides the polynucleotide according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention for use as an immune inhibitory medicament.
  • the invention provides the use of provides the polynucleotide according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention for the manufacture of an immune inhibitory medicament.
  • the term “immune inhibitory medicament” refers to a medicament that is capable of reducing and/or inhibiting immune responses.
  • the immune inhibitory medicament is an anti-inflammatory medicament.
  • the invention provides the polynucleotide according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention for use as an anti- inflammatory medicament.
  • the invention provides the polynucleotide according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention for use in the prevention and/or treatment of an inflammatory disease.
  • the invention provides a method of preventing and/or treating an inflammatory disease in a subject, comprising administering the polynucleotide according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention to the subject. Treating an inflammatory disease relates to the therapeutic use of the polynucleotide, vector, nucleic acid particle, or pharmaceutical composition according to the invention.
  • the polynucleotide, vector, nucleic acid particle, or pharmaceutical composition according to the invention may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
  • Preventing an inflammatory disease relates to the prophylactic use of the polynucleotide, vector, nucleic acid particle, or pharmaceutical composition according to the invention.
  • the polynucleotide, vector, nucleic acid particle, or pharmaceutical composition according to the invention may be administered to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent development of at least one symptom associated with the disease.
  • the subject may have a predisposition for, or be thought to be at risk of developing, the disease.
  • the vector(s) or nucleic acids may be introduced by transduction.
  • the vector(s) or nucleic acids may be introduced by transfection.
  • the therapeutic and/or prophylactic uses described herein do not comprise intratumoral administration of the polynucleotide, vector, nucleic acid particle, or pharmaceutical composition according to the invention.
  • the polynucleotide, vector, nucleic acid particle, or pharmaceutical composition according to the invention is not administrated intratumorally.
  • the disease or condition to be treated and/or prevented may be cancer, a cardiovascular disease, atherosclerosis, Alzheimer's disease, an autoimmune inflammatory disease, a liver disease, or an inflammatory skin condition.
  • the disease is atherosclerosis or a cardiovascular disease.
  • the cardiovascular disease is heart failure or pericarditis.
  • the autoimmune inflammatory disease is multiple sclerosis or rheumatoid arthritis.
  • the inflammatory skin condition is psoriasis.
  • the liver disease is non-alcoholic fatty liver disease.
  • the subject is a mammalian subject. In one embodiment, the subject is a human.
  • any nucleic acid sequences are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino (N) to carboxy (C) orientation, respectively.
  • a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed.
  • Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure.
  • polypeptide is used in the conventional sense to mean a series of amino acids, typically L-amino acids, connected one to the other, typically by peptide bonds between the ⁇ - amino and carboxyl groups of adjacent amino acids.
  • polypeptide is used interchangeably with the terms “amino acid sequence”, “peptide” and/or “protein”.
  • nucleic acid sequence refers to amino acids in an amino acid sequence.
  • variant in relation to a polypeptide refers to a polypeptide that has an equivalent function to the amino acid sequences described herein, but which includes one or more amino acid substitutions, insertions or deletions.
  • polynucleotide refers to a polypeptide that has an equivalent function to the amino acid sequences described herein, but which includes one or more amino acid substitutions, insertions or deletions.
  • polynucleotide refers to a polypeptide that has an equivalent function to the amino acid sequences described herein, but which includes one or more amino acid substitutions, insertions or deletions.
  • polynucleotide refers to a polypeptide that has an equivalent function to the amino acid sequences described herein, but which includes one or more amino acid substitutions, insertions or deletions.
  • polynucleotide refers to a polypeptide that has an equivalent function to the amino acid sequences described herein, but which includes one
  • a fragment or variant of an amino acid sequence is preferably a "functional fragment” or “functional variant".
  • the term "functional fragment” or “functional variant” of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent.
  • one particular function is the anti-inflammatory antagonistic function displayed by the amino acid sequence from which the fragment or variant is derived and/or binding to the receptor(s) the amino acid sequence from which the fragment or variant is derived binds to.
  • a functional variant of an immune inhibitory e.g.
  • IL-1 interleukin-1
  • IL-1 ⁇ IL-1 ⁇
  • IL-18 IL-18
  • IL-33 IL-36 ⁇
  • IL 36 ⁇ and IL-36 ⁇ 7 pro-inflammatory cytokines of the IL-1 family
  • the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the binding characteristics of the molecule or sequence.
  • Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences.
  • the optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the global homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol.48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci.
  • NCBI National Center for Biotechnology Information
  • the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used.
  • the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment. Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.
  • the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence.
  • the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides.
  • the degree of similarity or identity is given for the entire length of the reference sequence.
  • isolated means removed (e.g., purified) from the natural state or from an artificial composition, such as a composition from a production process.
  • a nucleic acid, peptide or polypeptide naturally present in a living animal is not “isolated”, but the same nucleic acid, peptide or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated”.
  • An isolated nucleic acid, peptide or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • expression as used herein is defined as the transcription and/or translation of a particular nucleotide sequence.
  • a nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule.
  • a nucleic acid can be isolated.
  • nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose)
  • a nucleotide is composed of a nucleoside and one or more phosphate groups.
  • nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.
  • the five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine.
  • the five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively.
  • thymidine is more commonly written as “dT” (“d” represents “deoxy”) as it contains a 2’-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine.
  • d represents “deoxy”
  • uridine is found in RNA and not DNA.
  • the remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG.
  • DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA.
  • a molecule contains “a majority of deoxyribonucleotide residues” if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule.
  • the total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).
  • DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA.
  • the cDNA may be obtained by reverse transcription of RNA.
  • RNA relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues.
  • ribonucleotide refers to a nucleotide with a hydroxyl group at the 2’-position of a ⁇ -D- ribofuranosyl group.
  • RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides.
  • altered/modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered/modified nucleotides (i.e., altered/modified RNAs) can be referred to as analogs of naturally occurring RNAs.
  • a molecule contains “a majority of ribonucleotide residues” if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule.
  • the total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).
  • RNA includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self- amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA).
  • RNA refers to mRNA. It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
  • Example 1 Characterization of HEK-Blue TM reporter cell lines To study the IL-1 family the functionality of three HEK-Blue TM IL-1R family reporter cell lines was validated. These cell lines were then used to study cross-reactivities within the IL-1 subfamilies and the functionality of mRNA variants encoding the anti-inflammatory IL-1 family members.
  • IL-1 receptor family members IL-1R1, IL-1RAP, IL-18R1, IL-18R2 and IL-36R
  • IL-1R1, IL-1RAP, IL-18R1, IL-18R2 and IL-36R The surface expression of the IL-1 receptor family members was quantified via multiparametric flow cytometry in the reporter cell lines HEK-BlueTM IL-1R ( Figure 1), HEK-BlueTM IL-18 ( Figure 2), and HEK-BlueTM IL-36 ( Figure 3).
  • the receptors described to be activated in each reporter cell line (such as IL-1R1 and IL-1RAP in HEK-BlueTM IL-1R cells) were overexpressed to varying degrees.
  • HEK-BlueTM IL-1R cells exhibited high surface expression of IL-1R1 and IL-1RAP.
  • IL-18R1 High expression of IL-18R1 was observed in HEK-BlueTM IL-18 cells ( Figure 2).
  • high expression of IL-1RAP and IL-36R was detected in HEK-Blue TM IL-36R cells ( Figure 3).
  • Bioactivity assay The functionality of the reporter cell lines in detecting bioactive cytokines of the IL-1 family was investigated by monitoring the activation of the NF ⁇ B and AP-1 pathways and resulting in the production of SEAP.
  • the HEK-Blue TM reporter cell lines were stimulated with a titration series of recombinant human IL-1 ⁇ , IL-1 ⁇ , IL-18, IL-36 ⁇ , IL-36 ⁇ and IL-36 ⁇ .
  • the cytokine IFN ⁇ was used as a negative control. After 24 h, the NF ⁇ B and AP-1 response was quantified using QUANTI- BlueTM solution, a SEAP detection reagent ( Figure 4 and Table 1). Table 1: EC50 values of HEK-Blue TM IL-1R, IL-18 and IL-36 cells for the detection of bioactive IL1 cytokines in the SEAP assay.
  • the HEK-Blue TM reporter cell lines responded to their respective cytokines with a sigmoidal dose-response curve (Figure 4A-C) and EC50 values ranging from 1.5 to 466.7 pg/ml (Table 1).
  • the HEK-BlueTM IL-1R cells responded to IL-1 ⁇ with an EC50 of 1.60 ⁇ 0.69 pg/ml and to IL-1 ⁇ with an EC 50 of 19.91 ⁇ 2.29 pg/ml.
  • the HEK-Blue TM IL-18 cells reacted to IL-18 with an EC 50 of 10.91 ⁇ 3.67 pg/ml.
  • the following cytokine concentrations were used for further experiments analyzing the antagonistic activity of anti-inflammatory mRNA family members: IL-1 ⁇ 8 pg/ml, IL-18 20 pg/ml, and IL-36 ⁇ 500 pg/ml.
  • the cytokine concentrations were chosen to be above the detected EC 50 values of the respective stimulated HEK-Blue TM reporter cell lines. Summary The present data indicate that the pathways within the IL-1 family are highly specific and do not cross-react between subfamilies. The data also demonstrate the suitability of mRNA- encoded anti-inflammatory IL-1 family members.
  • Example 2 Inhibitory activity of anti-inflammatory IL-1 family members Recombinant proteins The inhibitory effect of IL-1RA, IL-18BP, IL-36RA, IL-37, and IL-38 on IL-1 induced signaling in HEK-Blue TM reporter cell lines was quantified using recombinant versions of these anti- inflammatory members of the IL-1 family ( Figure 5 and Table 2).
  • Table 2 IC50 values of HEK-Blue TM IL-1R, IL-18 and IL-36 cells for the inhibitory effect of anti-inflammatory IL-1 cytokines in the SEAP assay.
  • Cytokine-mediated stimulation of HEK-Blue TM reporter cell lines was inhibited in a dose- dependent manner by the respective anti-inflammatory members ( Figure 5A-C) with IC50 values ranging from 232.2 to 584.3 ng/ml (Table 2).
  • agonists are nearly 1000 to 10000 times more active than the antagonists (Table 1 and Table 2).
  • IL-1RA inhibited IL-1 ⁇ - mediated stimulation of HEK-Blue TM IL-1R cells (Table 3).
  • IL-18BP inhibited IL-18-mediated stimulation of HEK-Blue TM IL-18 cells.
  • Human THP-1 monocytes were differentiated into macrophages by incubation with 100 nM phorbol-12-myristate-13-acetate (PMA) for 72 h followed by 24 h wash in RPMI medium. Differentiated macrophages were treated with 100 ⁇ g/ml oxLDL for 24 h to form foam cells.
  • PMA phorbol-12-myristate-13-acetate
  • the surface and gene expression of the IL-1 receptor family members IL-1R1, IL-1RAP, IL-18R1, IL-18R2 and IL-36R was quantified in foam cells compared to THP-1 macrophages (treated with 100 nM PMA) and THP-1 monocytes (untreated).
  • Gene expression was quantified by RT-qPCR ( Figure 7) and receptor surface expression by multiparametric flow cytometry ( Figure 8).
  • RT-qPCR the expression of IL-1 receptor family genes during foam cell formation was compared (see Figure 7). After 4 h, a significant dose-dependent increase in IL36R (Figure 7E) expression was detected.
  • Macrophage-derived foam cells are essential for atherosclerosis development and progression from early fatty streaks to advanced plaques. Assessment of foam cell formation is crucial to evaluate atherosclerosis and serves as an important endpoint. Therefore, a macrophage-mediated foam cell formation model was established.
  • THP-1 monocytes THP-1 macrophages and foam cells
  • IL-18R2 and IL-36R expression were detected.
  • a significant decrease in the expression of both the gene and the protein of IL-1RAP was also observed.
  • a significant increase in the expression of IL36R mRNA after 4 h of exposure to oxLDL was observed.
  • Example 4 Surface expression of IL-1 family receptors To characterize cross-reactivity and bioactivity of IL-1 family members, the reporter cell lines HEK-Blue TM IL-1R, HEK-Blue TM IL-18 and HEK-Blue TM IL-36 were used. To investigate the effect of cytokine stimulation on the receptor surface expression of the reporter cells, unstimulated and IL-1 ⁇ stimulated HEK-BlueTM IL-1R cells were compared ( Figure 9).
  • the main receptor of HEK-Blue TM IL-1R, IL-1R1, and its coreceptor IL-1RAcP were expressed to the same level, but less than IL-18R ⁇ (Figure 9C).
  • the coreceptor of IL-18R ⁇ , IL-18R ⁇ was more highly expressed on the surface of unstimulated and stimulated cells (Figure 9B, C) compared to the other receptors.
  • the receptor IL-18R ⁇ was expressed at lower level on the surface of both unstimulated and IL-1 ⁇ stimulated cells ( Figure 9C). In general, the receptor expression after the IL-1 ⁇ stimulation was slightly reduced compared to the receptor expression on unstimulated cells (Figure 9C).
  • the bioactivity of the anti-inflammatory members of the IL-1 family was then demonstrated by using the HEK-Blue TM IL-1R and IL-18 reporter cell lines by monitoring the activation of the NF ⁇ B and AP-1 pathways.
  • IL-1RA and IL-18BP anti-inflammatory members of the IL-1 family
  • the standard calibrators were diluted in 300 ⁇ l of assay diluent in a 5-folds serial dilution (7-steps and blank). Then, 50 ⁇ l of the samples and calibrators were transferred into the MSD plate in technical duplicates and incubated (2 hours, RT, shaking at 750 rpm). After washing the plates, 50 ⁇ l of the detection antibody mix diluted in assay diluent (Buffer 3) was added in each well and incubated (1 hour, RT, shaking at 750 rpm). The plates were then washed, and 150 ⁇ l of MSD Gold Read buffer B was added to the wells. The measurements were immediately performed in the MSD reader.

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Abstract

The present invention provides an isolated RNA encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof.

Description

RNA ENCODING AN IMMUNE INHIBITORY IL-1 FAMILY MEMBER FIELD OF THE INVENTION The present invention provides an isolated RNA encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof. The invention also relates to a nucleic acid particle or a pharmaceutical composition comprising the RNA of the invention. Therapeutic uses of the RNA, nucleic acid particle and pharmaceutical composition of the invention are also provided. BACKGROUND TO THE INVENTION A large number of diseases are significantly exacerbated by ongoing inflammatory processes. These include atherosclerosis. The interleukin (IL) 1 (IL-1) family of cytokines plays a central role in the regulation of both innate and adaptive immunity, and particularly in inflammation. Tight regulation of the pro-inflammatory effects of IL-1 family members by receptor antagonists and decoy receptors provides a balance between enhancement of immunity and uncontrolled inflammation, which is the cause of a variety of diseases and contributes to their development and maintenance. The different pro-inflammatory IL-1 family members have been associated with a variety of diseases. For example, the pro-inflammatory influence of the IL-1 family increases the risk and severity of cardiovascular disease (CVD) and atherosclerosis. One of the most important inflammatory mediators within this family is IL-1β, which is a secreted cytokine that triggers the production of further inflammatory mediators and also activates other immune cells. Despite the major influence of IL-1β on the inflammatory process, inhibition of this cytokine at the receptor is clinically less successful than expected. For example, the IL-1R1 receptor antagonist IL-1RA (also known as anakinra) has been associated with a number of problems, which limit the effectiveness of the antagonist. There is therefore a need in the art for improved (immune inhibitory (e.g. anti-inflammatory) IL-1 family drug candidates. SUMMARY OF THE INVENTION Accordingly, in a first aspect the invention provides an isolated RNA encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof. In another aspect, the invention provides a polynucleotide encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof. In some embodiments, the polynucleotide is RNA or DNA. In some embodiments, the polynucleotide is RNA. Where further aspects of the invention described herein refer to polynucleotide, the polynucleotide is preferably an RNA. In a further aspect, the invention provides an RNA encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof. In some embodiments, the polynucleotide may be an isolated polynucleotide. In some embodiments, the immune inhibitory protein of the IL-1 family is selected from the group consisting of: IL-1 receptor antagonist (IL-1RA), interleukin-18 binding protein (IL- 18BP), interleukin 36 receptor antagonist (IL-36RA), interleukin 37 (IL-37), and interleukin 38 (IL-38). In some embodiments, the immune inhibitory protein of the IL-1 family is IL-1RA, IL-18BP or IL-36RA. In some embodiments, the immune inhibitory protein of the interleukin-1 (IL-1) family further comprises the cognate of a binding moiety. In some embodiments, the cognate of a binding moiety is an antigen, an epitope or a polypeptide tag. In a preferred embodiment, the polypeptide tag is an ALFA tag. In some embodiments, the polynucleotide is mRNA. In some embodiments, the RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside is pseudouridine (ψ) or N1-methyl- pseudouridine (m1ψ). In some embodiments, the RNA comprises more than one type of modified nucleoside, wherein the modified nucleosides are independently selected from pseudouridine (ψ), N1- methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside is pseudouridine (ψ) or N1-methyl- pseudouridine (m1ψ). In some embodiments, the RNA comprises a 5’ cap analog. In some embodiments, the RNA comprises the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG or 3´- O-Me-m7G(5')ppp(5')G. In some embodiments, the RNA comprises the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG. In some embodiments, the RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12 and 31, or a nucleotide sequence having at least 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12 and 31. In some embodiments, the RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 32, or a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 32. In some embodiments, the RNA comprises a poly-A tail. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises the poly-A tail shown in SEQ ID NO: 14. In some embodiments, the polynucleotide comprises a sequence as set forth in any one of SEQ ID NOs: 16 to 25 or a sequence having at least 80% identity thereto. In a further aspect, the invention provides a vector comprising the polynucleotide according to the invention. In a further aspect, the invention provides a nucleic acid particle comprising the polynucleotide, preferably the RNA, according to the invention or the vector according to the invention. In a further aspect, the invention provides a pharmaceutical composition comprising the polynucleotide, preferably the RNA, according to the invention, the vector according to the invention or the nucleic acid particle according to the invention. In one aspect, the invention provides a pharmaceutical composition comprising the polynucleotide, preferably the RNA, according to the invention. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients. In a further aspect, the invention provides the polynucleotide, preferably the RNA, according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention for use as an immune inhibitory medicament. In a further aspect, the invention provides the polynucleotide, preferably the RNA, according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention for use as an anti- inflammatory medicament. In a further aspect, the invention provides the polynucleotide, preferably the RNA, according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention for use in the prevention and/or treatment of a disease or condition selected from the group consisting of: cancer, a cardiovascular disease, atherosclerosis, Alzheimer's disease, an autoimmune inflammatory disease, a liver disease, or an inflammatory skin condition. Preferably, the disease is atherosclerosis or a cardiovascular disease. In some embodiments, the cardiovascular disease is heart failure or pericarditis. In some embodiments, the autoimmune inflammatory disease is multiple sclerosis or rheumatoid arthritis. In some embodiments, the inflammatory skin condition is psoriasis. In some embodiments, the liver disease is non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE FIGURES Figure 1: Receptor expression on HEK-BlueTM IL-1R cells. Surface expression was quantified by staining with the corresponding antibodies against the receptors IL-1R1, IL-18R1 and IL-36R and co-receptors IL-1RAP and IL-18R2 by flow cytometric analysis. Shown are representative histograms (A) and the normalized MFI relative to the respective Isotype (B). Isotype staining and unstained cells served as control (n = 3 ± SD). Figure 2: Receptor expression on HEK-BlueTM IL-18 cells. Surface expression was quantified by staining with the corresponding antibodies against the receptors IL-1R1, IL-18R1 and IL-36R and co-receptors IL1-RAP and IL-18R2 by flow cytometric analysis. Shown are representative histograms (A) and the normalized MFI relative to the respective Isotype (B). Isotype staining and unstained cells served as control (n = 3 ± SD). Figure 3: Receptor expression on HEK-BlueTM IL-36 cells. Surface expression was quantified by staining with the corresponding antibodies against the receptors IL-1R1, IL-18R1 and IL-36R and co-receptors IL-1RAP and IL-18R2 by flow cytometric analysis. Shown are representative histograms (A) and the normalized MFI relative to the respective Isotype (B). Isotype staining and unstained cells served as control (n = 3 ± SD). Figure 4: Dose-response of HEK-BlueTM IL-1R, IL-18 and IL-36 cells to recombinant IL-1 cytokine family members. HEK-BlueTM IL-1R (A), IL-18 (B) and IL-36 (C) cells were stimulated with increasing concentrations of recombinant human IL-1α, IL-1β, IL-18, IL-36α, IL-36β and IL-36γ. After 24 h of incubation, the NFκB and AP-1 response was determined by adding QUANTI-Blue™ Solution, a SEAP detection reagent. After 1.5 h of incubation, the absorbance was measured at 620 nm. IFNγ was used as a negative control. The activity in % of the different reporter cell lines was calculated. The highest response of either IL-1α, IL-18 or IL-36α was set at 100% activity. (n = 3 ± SD). D HEK-BlueTM IL-36 cells were treated with 1 µg/ml IL-1RA or IL-36RA for 30 min before being stimulated with 100 pg/ml IL-1α or 500 pg/ml IL-36γ. After 24 h of incubation, the NFκB and AP-1 response was determined by adding QUANTI-Blue™ Solution. After 1.5 h of incubation, the absorbance was measured at 620 nm. IFNγ was used as a negative control. The activity in % was calculated. The highest response of either IL-1α or IL-36γ was set at 100% activity. (n = 4 ± SD). Statistical significance was determined by one-way ANOVA with Dunnett’s post hoc multiple comparisons test. **** = p ≤ 0.0001. Figure 5: Inhibitory effect of anti-inflammatory members of the IL-1 family on stimulated HEK-BlueTM reporter cell lines. HEK-BlueTM IL-1R (A), IL-18 (B) and IL-36 (C) cells were stimulated with recombinant human IL-1α (8 pg/ml), IL-18 (20 pg/ml) or IL-36γ (500 pg/ml) in the presence of serially diluted IL-1RA, IL-18BP, IL-36RA, IL-37 and IL-38. After 24 h of incubation, the NFκB and AP-1 response was determined by adding QUANTI-Blue™ Solution, a SEAP detection reagent. After 1 h of incubation, the absorbance was measured at 620 nm. IFNγ was used as a negative control. The activity in % of the different reporter cell lines was calculated. The highest response of either IL-1α, IL-18 or IL-36γ was set at 100% activity (n = 3 ± SD). Figure 6: Inhibitory effect of mRNA-encoded anti-inflammatory IL-1 cytokines on stimulated HEK-BlueTM reporter cell lines. IL-1RA- or IL-18BP-encoding mRNA was lipofected into HEK 293T/17 cells and the IL-1RA or IL-18BP concentration in the supernatant was quantified by ELISA. HEK-BlueTM IL-1R (A) and IL-18 (B) cells were stimulated with IL- 1α (8 pg/ml) or IL-18 (20 pg/ml) in the presence of serially diluted mRNA-encoded IL-1RA or IL-18BP supernatant or recombinant IL-1RA (purchased, Anakinra) or IL-18BP (purchased) as a reference. After 24 h of incubation, the NFκB and AP-1 response was determined by adding QUANTI-Blue™ Solution, a SEAP detection reagent. After 1 h of incubation, the absorbance was measured at 620 nm. IFNγ was used as a negative control. Shown are representative graphs for in total four individual experiments. The activity in % of the different reporter cell lines was calculated. The lowest concentration of either IL-1RA or IL-18BP was set to 100% activity (n = 2 ± SD of technical replicates). Figure 7: IL-1 receptor family gene expression. THP-1 cells were incubated for 72 h in the presence of 100 nM PMA and then in RPMI medium for further 24 h. Macrophages were stimulated in presence of 100 µg/ml oxLDL for 24 h and RNA expression of IL-1 receptor family genes IL1R1 (A), IL1RAP (B), IL18R1 (C), IL18R2 (D) and IL36R (E) was quantified by RT-qPCR and normalized to GAPDH expression. Data presented as ΔΔCt values relative to vehicle control of the respective time point (n = 4 ± SD). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc multiple comparisons test. * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001. Figure 8: IL-1 receptor family expression of THP-1 cells. THP-1 cells were incubated for 72 h in the presence of 100 nM PMA and then in RPMI medium for further 24 h. Macrophages were stimulated in presence of 100 µg/ml oxLDL for 24 h, then detached with a cell scraper, collected, and washed. Expression was quantified by staining with the corresponding antibodies against the receptors IL-1R1, IL-18R1 and IL-36R and co-receptors IL 1RAP and IL-18R2 by flow cytometric analysis. Shown are representative histograms of IL-1R1 (A), IL- 1RAP (C), IL-18R1 (E), IL-18R2 (G) and IL-36R (I) and the normalized MFI relative to the respective Isotype of IL-1R1 (B), IL-1RaP (D), IL-18R1 (F), IL-18R2 (H) and IL-36R (J). Isotype staining and unstained cells served as control (n = 3 ± SD). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc multiple comparisons test. * = p ≤0.05, *** = p ≤ 0.001, **** = p ≤ 0.0001. Figure 9: Comparison of receptor expression on the surface of unstimulated and stimulated HEK-BlueTM IL-1R cells. The receptor expression was quantified by flow cytometry staining with the corresponding antibodies against the receptors IL-1R1, IL-18Rα and IL-36R and the co-receptors IL-1RAcP and IL-18Rβ. Shown is the quantified receptor expression on HEK-BlueTM IL-1R cells. As a control, the cells were also stained with the corresponding isotype of IL-1R1, IL-18Rα, IL-1RAcP and IL-18Rβ (1:100). For IL-36R a mixture of Aqua Zombie (1:1,000) and streptavidin FITC (1:100) was used as a control. A Histogram of the unstimulated HEK-BlueTM IL-1R cells receptor expression compared to the unstained cells and isotypes. B Histogram of the IL-1α stimulated HEK-BlueTM IL-1R cells receptor expression compared to the unstained cells and isotypes. C Comparison of the normalized MFI of the receptor surface expression of unstimulated and IL-1α stimulated HEK- BlueTM IL-1R (n = 3 ± SD). Figure 10: Comparison of receptor expression on the surface of unstimulated and stimulated HEK-BlueTM IL-18 cells. The receptor expression was quantified by staining with the corresponding antibodies against the receptors IL-1R1, IL-18Rα and IL-36R and the co- receptors IL-1RAcP and IL-18Rβ using flow cytometry. Shown is the quantified receptor expression on HEK-BlueTM IL-18 cells. As a control, the cells were also stained with the corresponding isotype of IL-1R1, IL-18Rα, IL-1RAcP and IL-18Rβ (1:100). For IL-36R a mixture of Aqua Zombie (1:1,000) and streptavidin FITC (1:100) was used as a control. A Histograms of the receptor expression of the unstimulated HEK-BlueTM IL-18 cells receptor expression compared to the unstained cells and isotypes. B Histograms of the receptor expression of the IL-18 stimulated HEK-BlueTM IL-18 cells compared to the unstained cells and isotypes. C Comparison of the normalized MFI of the surface expression of the receptors of unstimulated and IL-18 stimulated HEK-BlueTM IL-18 (n = 3 ± SD). Figure 11: Comparison of receptor expression on the surface of unstimulated and stimulated HEK-BlueTM IL-36 cells. The receptor expression was quantified by staining with the corresponding antibodies against the receptors IL-1R1, IL-18Rα and IL-36R and the co- receptors IL-1RAcP and IL-18Rβ using flow cytometry. Shown is the quantified receptor expression on HEK-BlueTM IL-36 cells. Isotype staining was used as a control for IL-1R1 (1:8), IL-18Rα, IL-1RAcP and IL-18Rβ (1:100). For IL-36R a mixture of Aqua Zombie (1:1,000) and streptavidin FITC (1:100) was used as a control. A Histograms of the receptor expression of the unstimulated HEK-BlueTM IL-36 cells receptor expression compared to the unstained cells and isotypes. B Histograms of the receptor expression of the IL-36γ stimulated HEK-BlueTM IL-36 cells compared to the unstained cells and isotypes. C Comparison of the normalized MFI of the surface expression of the receptors of unstimulated and IL-36γ stimulated HEK- BlueTM IL-36 (n = 3 ± SD). Figure 12: Responses of the HEK-BlueTM reporter cell lines after cytokine stimulation. The bioactivity of the cytokines of the IL-1 family was quantified using SEAP assay. Therefore, the HEK-BlueTM cells were first stimulated for 24 h at 37°C and 5% CO2 with a x/(√10) dilution series of IL-1α IL-1β, IL-18, IL-36α, IL-36γ (1 ng/ml) or IL-36β (100 ng/ml) or IFN-γ (1 ng/ml) as a control. Afterwards the supernatant was diluted 1:3 in Quanti-BlueTM solution and incubated for 1 h at 37°C and 5% CO2 and the absorption was measured at 620 nm. A Response of HEK-BlueTM IL-1R cells normalized to IL-1α. B Response of HEK-BlueTM IL-18 cells normalized to IL-18. C Response of HEK-BlueTM IL-36 cells normalized to IL-36γ. The strongest response of either IL-1α, IL-18 or IL 36α was set at 100% activity (n = 3 ± SD). Figure 13: Inhibitory ability of anti-inflammatory IL-1 family cytokines to the bioactivity on the HEK-BlueTM reporter cell lines. The inhibitory activity of anti-inflammatory cytokines of the IL-1 family was determined using HEK-BlueTM reporter cells. Therefore, the cells were first stimulated for 30 min at 37°C and 5% CO2 with a dilution series of IL-1RA, IL-18BP, IL- 36RA, IL-37, IL-38 or IL-37 with IL-18BP (10 ng/ml) and afterward with a constant concentration of IL-1α (8 pg/ml), IL-18 (20 pg/ml) or IL-36γ ( 500 pg/ml). After 24 h at 37°C and 5% CO2 the supernatant was diluted 1:3 in Quanti-BlueTM solution and incubated for 1 h at 37°C and 5% CO2 and the absorption was measured at 620 nm. The strongest response of either IL-1α, IL-18 or IL-36γ was set at 100% activity (n = 3 ± SD). A Bioactivity of HEK-BlueTM IL-1R after stimulation with IL-1α and different anti-inflammatory members. B Bioactivity of HEK-BlueTM IL-18 after stimulation with IL-18 and different anti-inflammatory members. C Bioactivity of HEK-BlueTM IL-36 after stimulation with IL-36γ and different anti-inflammatory members. Figure 14: Inhibitory ability of IL-1RA and IL-36RA to IL-1α or IL-36γ stimulation on HEK- BlueTM IL-36 cells. The inhibitory activity of anti-inflammatory cytokines of the IL-1 family was determined using HEK-BlueTM IL-36 reporter cells. Therefore, the cells were first stimulated for 30 min at 37°C and 5% CO2 with 1 μg/ml of IL-1RA or IL-36RA. Afterwards, the cells were stimulated with either 100 pg/ml IL-1α or 500 pg/ml IL-36γ. After 24 h at 37°C and 5% CO2 the supernatant was diluted 1:3 in Quanti-BlueTM solution, incubated for 1 h at 37°C and 5% CO2 and the absorption was measured at 620 nm. The strongest response of either IL-1α or IL- 36γ was set at 100% activity (n = 4 ± SD). Statistical significance was determined by one-way ANOVA with Dunnett’s post hoc multiple comparisons test. **** = p ≤ 0.0001. Figure 15: Impact of IL-1 stimulation to the HUVEC cytokine release. For measurement of the IL-1 family surface expression on HUVEC cells, the cells were stained with the corresponding antibodies against the receptors IL-1R1, IL-18Rα and IL-36R and the co- receptors IL-1RAcP and IL-18Rβ using flow cytometry. Shown is the quantified receptor expression on HUVEC cells. Isotype staining was used as a control for IL-1R1 (1:8), IL-18Rα, IL-1RAcP and IL-18Rβ (1:100). For IL-36R the combination of Aqua Zombie with streptavidin FITC (1:100) were used as a control. For the gene expression analysis, the HUVEC cells were stimulated for 4 or 24 h with either IL-1α (10 ng/ml) or IL-1β (10 ng/ml). Gene expression of IL-6 (IL6), ICAM-1 (ICAM1), MCP-1 (MCP-1) and E-Selectin (SELE) was analyzed in HUVEC cells. The expression was compared to gene expression of GAPDH, used as a control. A Quantification of the normalized MFI of the surface expression of the receptors of HUVEC cells. B Quantification of the gene expression after IL-1 stimulation after 4 h and 24 h normalized to GAPDH (n = 3 ± SD). Figure 16: Binding and bioactivity studies using mRNA-encoded cytokine-ALFA-tag variants. For the transfection mRNA, coding for IL-1RA, IL-18BP and IL-36RA fused to an ALFA-tag, of HEK 293/T17 cells, the LipofectamineTM MessengerMaxTM Reagent was used and the supernatant was harvested after 24 h. A Binding of IL-1RA_ALFA to IL-1R1 (1 μg/ml) or IL-1RAcP (1 μg/ml) and IL-18BP_ALFA binding to IL-18Rα (1 μg/ml) or IL-18Rβ (1 μg/ml) was determined by ELISA using the sdAB α ALFA-HRP (1:10,000 dilution). The highest concentration of IL-1RA_ALFA or IL-18BP_ALFA was set at 100% binding (n = 2). B Binding of ALFA-tagged proteins (IL-1RA 8 μg/ml, IL-18BP 8μg/ml and IL-36RA 0,7 μg/ml) was quantified by flow cytometry using HEK-BlueTM IL-1R, IL-18 or IL-36 cells. As the antibody the universal α-ALFA AF647 (1:500 dilution) was used to measure the ALFA-tagged proteins. The highest concentration of IL-1RA_ALFA and IL-18BP_ALFA was set at 100% binding (n = 2). C Comparison of the functionality of the ALFA-protein with the translated protein (IL-1RA 8 μg/ml, IL-18BP 1 μg/ml and IL-36RA 0,7 μg/ml) and commercially available control proteins IL-1RA, IL-18BP and IL-36RA (10 μg/ml) using a SEAP assay and HEK-BlueTM IL-1R, IL-18 or IL-36 cells (n = 3). The lowest concentration of IL-1RA_ALFA, IL-18BP_ALFA or IL-36RA_ALFA was set at 100% cytokine activity. For all experiments (A to C) representative results from independent experiments are shown. Figure 17: Inhibitory effect of mRNA-encoded anti-inflammatory IL-1 family members formulated in LPX using HEK BlueTM reporter cell lines. (A) IL-1RA- and (B) IL-18BP encoding mRNA formulated in F12-Lipoplex particles displayed bioactivity by inhibiting IL-1α and IL-18 effects on HEK-BlueTM cells. (n = 2-3 ± SD). Figure 18: Inhibitory effect of IL-1RA on HUVEC cells. IL-1RA-encoding mRNA formulated in LipofectamineTM MessengerMaxTM (MM) exhibits a dose-dependent ability to neutralize the effects of IL-1α in HUVEC cells, by reducing (A) IL-6 (B) TNF-α and (C) IL-1β secretion. These inhibitory effects were comparable to those observed with the recombinant IL-1RA (Anakinra) (n = 1). Figure 19: Inhibitory effect of IL-1RA on monocytes. Pro-inflammatory cytokines release was analyzed in CD14+ monocytes with MSD. Both mRNA-encoded IL-1RA and recombinant IL-1RA (Anakinra) neutralized, in a dose-dependent, IL-1α-increased release of (A) IL-6 and (B) TNF-α. The mRNA-encoded IL-1RA demonstrated superior inhibitory dose-dependent effects in comparison to the recombinant protein (Anakinra), by decreasing the secretion of (C) IL-1β and (D) IFN-γ. (n = 1-2). Interleukin-1α (IL-1α), Interleukin-6 (IL-6), Tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β), Interferon-γ (IFN-γ), Lipoplex (LPX), LipofectamineTM MessengerMaxTM (MM). DETAILED DESCRIPTION OF THE INVENTION Polynucleotide Accordingly, in one aspect the invention provides a polynucleotide encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof. In one aspect, the invention provides a polynucleotide encoding an anti-inflammatory protein of the interleukin-1 (IL-1) family or functional variant thereof. According to the present disclosure, terms such as “capable of expressing”, “polynucleotide (e.g. RNA) expressing” and “polynucleotide (e.g. RNA) encoding” or similar terms are used interchangeably herein and with respect to a particular peptide or polypeptide mean that the polynucleotide, if present in the appropriate environment, e.g. within a cell, can be expressed to produce said peptide or polypeptide. It will be understood by the skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Suitably, the polynucleotides of the present invention are codon optimised to enable expression in a mammalian cell, in particular a cell as described herein. Polynucleotides according to the invention may comprise DNA and/or RNA. The polynucleotides may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. Herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest. In some embodiments, the polynucleotide is DNA and/or RNA. In one embodiment, the polynucleotide is DNA. In one embodiment, the polynucleotide is RNA. In a further aspect, the invention provides an RNA encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof. In some embodiments, the polynucleotide may be an isolated polynucleotide. In a further aspect, the invention provides an isolated RNA encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof. In one embodiment, the polynucleotide encodes one or more immune inhibitory protein(s) of the interleukin-1 (IL-1) family or functional variant(s) thereof. Suitably, the polynucleotide encodes IL-18BP and IL-37. Suitably, the polynucleotide may be a bicistronic or multicistronic cassette. IL-1 family The IL-1 family of cytokines plays a central role in the regulation of both innate and adaptive immunity, and in particular in inflammation. The IL-1 family includes 10 receptors and 11 cytokines. The cytokines in this family include 7 ligands with pro-inflammatory agonistic activity (namely, IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL 36β and IL-36γ) and 4 proteins with anti-inflammatory antagonistic functions (either receptor antagonists (RA) or antagonistic or anti-inflammatory cytokines) (namely, IL-1RA, IL-36RA, IL-37 and IL-38). In addition, there are other negative regulatory receptors in the family with anti-inflammatory properties. These include IL-1R2, IL-1R8, IL-1R9 and IL-18 binding protein (IL-18BP). IL-1R2 acts as a decoy receptor for IL-1 cytokines, while IL-1R8 and IL-1R9 are co receptors for IL-37 and IL-38. IL-18BP is neither a classical receptor nor a classified cytokine. IL-18BP functions in a similar way to the receptor antagonists in the family by binding to the corresponding receptor, i.e. it acts as an antagonist for the pro-inflammatory IL-18. A fundamental process of signal transduction within the IL-1 family is the formation of heterotrimeric complexes of ligand, receptor and its co-receptor. Thus, the IL-1 family members mediate their effects via different heterodimeric IL-1 receptor (IL-1R) complexes. Many well-defined interactions within the IL-1 family are known in the art, such as the binding of IL-1α and IL-1β to IL-1R, IL-18 to IL-18R, and IL-36α, IL-36β, and IL-36γ to IL-36R. The different pro-inflammatory IL-1 family members have been associated with a variety of diseases. For example, the pro-inflammatory influence of the IL-1 family increases the risk and severity of cardiovascular disease (CVD). Modulating IL-1 signaling by administering immune inhibitory or anti-inflammatory IL-1 family members or neutralizing antibodies has been shown to be therapeutically effective in various autoimmune inflammatory diseases and inflammatory conditions such as cardiovascular disease. For example, blocking IL-1β is the current standard of care for some autoimmune inflammatory diseases and IL-1 antagonists such as canakinumab or anakinra are used clinically for the treatment of CVD. Furthermore, an anti-atherosclerotic effect was observed when anakinra was administrated in ApoE KO mice. In addition to its use in cardiovascular diseases, neutralizing anti-IL-1β antibodies have been used to demonstrate the central role of IL-1β in the development and pathogenesis of atherosclerosis, a chronic inflammatory vascular disease. The pathological process of atherosclerosis increases the risk of developing CVD such as AMI. Immune inhibitory proteins of the IL-1 family Suitably, the term “immune inhibitory protein of the IL-1 family” refers to an immunomodulatory protein of the IL-1 family capable of reducing and/or inhibiting immune responses. Suitably, the immunomodulatory protein of the IL-1 family is an anti-inflammatory protein. Suitably, an anti-inflammatory protein of the IL-1 family has anti-inflammatory and/or antagonistic functions. Suitably, an anti-inflammatory protein of the IL-1 family has anti-inflammatory, antagonistic functions. Suitably, the immune inhibitory (e.g. anti-inflammatory protein) of the IL-1 family acts as an antagonist for at least one of the 7 pro-inflammatory cytokines of the IL- 1 family (namely, IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL 36β and IL-36γ). Suitably, the immune inhibitory (e.g. anti-inflammatory) protein of the IL-1 family may be an inflammatory cytokine binding protein (e.g. IL-18BP), a decoy receptor (e.g. IL-1R2), a receptor antagonist (e.g. IL- 1RA or IL-36RA) or an anti-inflammatory cytokine (e.g. IL-37 or IL-38) as described herein. In some embodiments, the immune inhibitory protein of the IL-1 family is selected from the group consisting of: IL-1RA, IL-18BP, IL-36RA, IL-37, and IL-38. In some preferred embodiments, the immune inhibitory protein of the IL-1 family is IL-1RA, IL- 18BP or IL-36RA. In one preferred embodiment, the immune inhibitory protein of the IL-1 family is IL-1RA. In one preferred embodiment, the immune inhibitory protein of the IL-1 family is IL-18BP. In one preferred embodiment, the immune inhibitory protein of the IL-1 family is IL-36-RA. In one embodiment, the immune inhibitory protein of the IL-1 family is IL-37. In one embodiment, the immune inhibitory protein of the IL-1 family is IL-38. In some embodiments, the immune inhibitory protein of the interleukin-1 (IL-1) family further comprises the cognate of a binding moiety. As used herein, the term “cognate of a binding moiety” refers to any motif that is specifically recognised by a binding moiety. Numerous cognates of binding moieties are provided in the art. In some embodiments, the cognate is an antigen, an epitope or a polypeptide tag that is specifically recognised by an antibody or an antigen-binding fragment of an antibody. In a preferred embodiment, the cognate is a polypeptide tag. In a preferred embodiment, the cognate is a polypeptide tag that is specifically recognised by an antigen-binding fragment of an antibody. Suitably, the cognate is an ALFA tag (as described in Götzke et al., Nat Commun, 2019, 10:4403). In an embodiment, the cognate is a polypeptide comprising or consisting of the sequence SRLEEELRRRLTE (SEQ ID NO: 1). As used herein, the term “specifically recognised” refers to binding with an affinity equivalent to that of a functional antibody fragment. IL-1RA IL-1α and IL-1β mediate their effects through formation of a heterotrimeric complex with IL-1R and the co-receptor. IL-1α and IL-1β are known to play a role in several diseases such as CVD. IL-1α is present in its precursor variant in mesenchymal tissues such as the myocardium or endothelium, and triggers the secretion of IL-6, which further drives the inflammatory process during CVD. Although IL-1β is not widely present as a precursor variant in the myocardium, viral infection triggers IL-1β activation which in turn causes necrosis of cardiomyocytes, resulting in acute myocardial infarction (AMI). The inflammation-induced production of IL-1β by monocytes and macrophages represents a link between the innate and adaptive immune systems. As a typical inflammatory cytokine, it induces the formation of the inflammatory T cell subunit Th17 cells, which can lead to chronic tissue inflammation and subsequent organ failure. At the same time, IL-1β (and IL-1α) in turn activates IL-6 which inhibits Treg differentiation, reducing the protection provided by Tregs against autoimmunity and tissue injury. Thus, IL-6 functions as a regulator of the Treg and Th17 balance in the adaptive immune system. In addition to IL-6, NF-kB activation by IL-1β results in the expression of other inflammatory cytokines, such as IL-1β itself and IL-18. The pro-inflammatory activity of both IL-1 cytokines is regulated by the expression of IL-1RA, a naturally occurring inhibitor of IL-1 cytokines, or by the formation of an alternative non- signaling complex with IL-1R2 instead of IL-1R1. Since IL-1RA binds to IL-1R1 with a higher affinity than IL-1α or IL-1β, activation of the receptor is prevented in its presence and the subsequent pro-inflammatory signal is suppressed. Hence, disturbances in the balance between IL-1 (α and β) and IL-1RA influence the course, susceptibility, and severity of many diseases. While IL-1RA prevents both the activation of IL-1R1 by IL-1α and IL-1β, IL-1R2, a decoy receptor without a cytoplasmic domain, specifically blocks the activation triggered by IL-1β. IL-18BP and IL-37 IL-18 mediates its effects through formation of a heterotrimeric complex with IL-18R and a co- receptor. IL-18 acts as an inflammatory mediator in many diseases, including neurodegenerative diseases such as Alzheimer's neuroinflammation and multiple sclerosis. In the secreted form, active IL-18 is tightly regulated by IL-18BP. Similar to IL-1RA for IL-1R, the decoy receptor IL-18 binding protein (IL-18BP) negatively regulates IL-18. As a secretagogue antagonist, it binds with high affinity to IL-18 and blocks the activation of IL-18R. Disruption of this regulation results in enhanced IL-18-driven inflammatory responses, which may play an important role in disease. Accordingly, the use of IL-18BP may be appropriate in diseases where further inflammation mediated by IL-18 is to be prevented. Compared to the specific anti-inflammatory effects related to their respective receptors of IL-1RA and IL-36RA, the anti-inflammatory effect of IL-37 and IL-38 is nonspecific and has an influence on the innate and acquired immune systems. IL-37 belongs to the IL-18 subfamily as it binds to IL-18Rα. As an anti-inflammatory cytokine, IL-37 is produced by immune cells, such as monocytes, B cells or natural killer cells, as well as non-immune cells, such as keratinocytes. By inhibiting the production of pro-inflammatory cytokines such as IL-6, IL-1α, IL-1β or TNF-α, IL-37 provides a protective effect on both the innate and acquired immune system, inflammatory or autoimmune diseases and cancer. Triggered by a pro-inflammatory stimulus (such as IL-1β or TLR agonists), IL-37 is expressed and acts as a self-protective mechanism against uncontrolled inflammation and extreme tissue damage. An anti- inflammatory signal occurs when the cytokine IL-37 binds to its receptor IL-18Rα and forms a complex with the co receptor IL-1R8. This includes, for example, inhibiting the assembly and activation of the inflammasome, which blocks the proteolytic maturation of IL-1β and IL-18. IL-36RA and IL-38 IL-36 mediates its effects through formation of a heterotrimeric complex with IL-36R and a co- receptor. IL-36 cytokine release is strongly induced by psoriasis-associated cytokines such as IL-17 or IL-22 in keratinocytes and thus plays a specific role in skin inflammation. The binding of IL-36RA to IL-36R prevents recruitment of the co-receptor IL-1RAcP and inhibits the subsequent signaling cascade. IL-36RA has a higher affinity for IL-36R than IL-36α or IL-36γ. Therefore, simultaneous stimulation of IL-36RA and IL-36α/IL-36γ inhibits receptor activity. IL-38 is assigned to the IL-36 subfamily, as it binds to IL-36R and thus blocks NF-κB and MAPK activation. Besides IL-36R, it can also inhibit the binding of agonistic ligands of IL-1R1 and IL-1RAcP, which results in the inhibition of pro-inflammatory signaling pathways. IL-38 is thus the only member of the IL-1 family that is able to inhibit several receptors by binding, indicating possible cross-reactivity within the family. This anti-inflammatory interleukin is secreted by peripheral blood mononuclear cells (PBMCs), keratinocytes or B cells, but is also strongly expressed in various tissues such as skin, lungs or heart. Illustrative protein sequences Illustrative sequences of immune inhibitory proteins of the IL-1 family for use according to the invention are provided below. Thus, the polynucleotide (e.g. the isolated RNA) of the invention may encode any of the following illustrative protein sequences or a functional variant thereof. Illustrative IL-1RA sequence (NCBI database NP_776214.1, human IL-1RA precursor): MEICRGLRSHLITLLLFLFHSETICRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLE EKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSF ESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO: 2) Illustrative IL-18BP sequence (NCBI database NP_001034748.1, human IL-18BP precursor): MTMRHNWTPDLSPLWVLLLCAHVVTLLVRATPVSQTTTAATASVRSTKDPCPSQPPVFPAAKQCPALE VTWPEVEVPLNGTLSLSCVACSRFPNFSILYWLGNGSFIEHLPGRLWEGSTSRERGSTGTQLCKALVL EQLTPALHSTNFSCVLVDPEQVVQRHVVLAQLWAGLRATLPPTQEALPSSHSSPQQQG (SEQ ID NO: 3) Illustrative IL-36RA sequence: VLSGALCFRMKDSALKVLYLHNNQLLAGGLHAGKVIKGEEISVVPNRWLDASLSPVILGVQGGSQCLS CGVGQEPTLTLEPVNIMELYLGAKESKSFTFYRRDMGLTSSFESAAYPGWFLCTVPEADQPVRLTQLP ENGGWNAPITDFYFQQCD (SEQ ID NO: 4) Further illustrative IL36-RA sequence (NCBI database NP_036407.1, human IL-36RA): MVLSGALCFRMKDSALKVLYLHNNQLLAGGLHAGKVIKGEEISVVPNRWLDASLSPVILGVQGGSQCL SCGVGQEPTLTLEPVNIMELYLGAKESKSFTFYRRDMGLTSSFESAAYPGWFLCTVPEADQPVRLTQL PENGGWNAPITDFYFQQCD (SEQ ID NO: 5) Illustrative IL-37 sequence: VHTSPKVKNLNPKKFSIHDQDHKVLVLDSGNLIAVPDKNYIRPEIFFALASSLSSASAEKGSPILLGV SKGEFCLYCDKDKGQSHPSLQLKKEKLMKLAAQKESARRPFIFYRAQVGSWNMLESAAHPGWFICTSC NCNEPVGVTDKFENRKHIEFSFQPVCKAEMSPSEVSD (SEQ ID NO: 6) Further illustrative IL-37 sequence (NCBI database NP_055254.2, human IL-37): MSFVGENSGVKMGSEDWEKDEPQCCLEDPAGSPLEPGPSLPTMNFVHTSPKVKNLNPKKFSIHDQDHK VLVLDSGNLIAVPDKNYIRPEIFFALASSLSSASAEKGSPILLGVSKGEFCLYCDKDKGQSHPSLQLK KEKLMKLAAQKESARRPFIFYRAQVGSWNMLESAAHPGWFICTSCNCNEPVGVTDKFENRKHIEFSFQ PVCKAEMSPSEVSD (SEQ ID NO: 7) Illustrative IL-38 sequence: CSLPMARYYIIKYADQKALYTRDGQLLVGDPVADNCCAEKICILPNRGLARTKVPIFLGIQGGSRCLA CVETEEGPSLQLEDVNIEELYKGGEEATRFTFFQSSSGSAFRLEAAAWPGWFLCGPAEPQQPVQLTKE SEPSARTKFYFEQSW (SEQ ID NO: 8) Further illustrative IL-38 sequence (NCBI database NP_115945.4, human IL-38): MCSLPMARYYIIKYADQKALYTRDGQLLVGDPVADNCCAEKICILPNRGLARTKVPIFLGIQGGSRCL ACVETEEGPSLQLEDVNIEELYKGGEEATRFTFFQSSSGSAFRLEAAAWPGWFLCGPAEPQQPVQLTK ESEPSARTKFYFEQSW (SEQ ID NO: 9) In some embodiments, the immune inhibitory protein of the IL-1 family comprises a sequence as set forth in any one of SEQ ID NOs: 2 to 9 or a functional variant thereof having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the immune inhibitory protein of the IL-1 family consists of a sequence as set forth in any one of SEQ ID NOs: 2 to 9 or a functional variant thereof having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto In some preferred embodiments, the immune inhibitory protein of the IL-1 family comprises a sequence as set forth in any one of SEQ ID NOs: 2 to 5 or a functional variant thereof having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the immune inhibitory protein of the IL-1 family consists of a sequence as set forth in any one of SEQ ID NOs: 2 to 5 or a functional variant thereof having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the immune inhibitory protein of the IL-1 family comprises a sequence as set forth in SEQ ID NO: 2 or a functional variant thereof having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the immune inhibitory protein of the IL-1 family comprises a sequence as set forth in SEQ ID NO: 3 or a functional variant thereof having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the immune inhibitory protein of the IL-1 family comprises a sequence as set forth in SEQ ID NO: 4 or a functional variant thereof having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the immune inhibitory protein of the IL-1 family comprises a sequence as set forth in SEQ ID NO: 5 or a functional variant thereof having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the immune inhibitory protein of the IL-1 family comprises a sequence as set forth in SEQ ID NO: 6 or a functional variant thereof having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the immune inhibitory protein of the IL-1 family comprises a sequence as set forth in SEQ ID NO: 7 or a functional variant thereof having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the immune inhibitory protein of the IL-1 family comprises a sequence as set forth in SEQ ID NO: 8 or a functional variant thereof having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the immune inhibitory protein of the IL-1 family comprises a sequence as set forth in SEQ ID NO: 9 or a functional variant thereof having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. The immune inhibitory protein of the IL-1 family as described herein may comprise a signal peptide to aid in its production. The signal peptide may cause the protein to be secreted by a host cell, such that the protein can be harvested from the host cell supernatant. The core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases. The signal peptide may be at the amino terminus of the immune inhibitory protein of the IL-1 family. The signal peptide may comprise or consist of: MRVMAPRTLILLLSGALALTETWA (SEQ ID NO: 33) Accordingly, in some embodiments, the inhibitory protein of the IL-1 family comprises a sequence as set forth in SEQ ID NO: 33. mRNA In some embodiments, the polynucleotide is mRNA. Suitably, the RNA (e.g. the mRNA) is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA. Methods for making IVT-RNA are known in the art. Any suitable method for producing IVT-RNA may be used in accordance with the present invention (see, for example, Ziegenhals et al., 2023, Front. Mol. Biosci., 10:1291045). In one embodiment, the RNA described herein may have modified nucleosides. In some embodiments, the RNA comprises a modified nucleoside in place of at least one uridine. Suitably, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is independently selected from pseudouridine (φ), N1-methyl-pseudouridine (m1φ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside is pseudouridine (ψ) or N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises pseudouridine (φ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1φ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). Suitably, the RNA comprises a pseudouridine (φ) or N1-methyl-pseudouridine (m1φ) in place of each uridine. In some embodiments, RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (φ), N1-methyl- pseudouridine (m1φ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside is pseudouridine (ψ) or N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleosides comprise pseudouridine (φ) and N1-methyl-pseudouridine (m1φ). In some embodiments, the modified nucleosides comprise pseudouridine (φ) and 5-methyl- uridine (m5U). In some embodiments, the modified nucleosides comprise N1-methyl- pseudouridine (m1φ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (φ), N1-methyl-pseudouridine (m1φ), and 5-methyl- uridine (m5U). In some embodiments, the modified nucleoside replacing one or more uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6- aza-uridine, 2-thio-5-azauridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo- uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5- oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyluridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5- methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio- uridine (mnm5s2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-carbamoylmethyl- uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5- carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1- propynylpseudouridine, 5-taurinomethyl-uridine (rm5U), 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thiouridine (rm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio- uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4φ), 4-thio-1-methyl-pseudouridine, 3- methyl-pseudouridine (m3φ), 2-thio-1-methylpseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, N 1-methyl-pseudouridine, 3-(3-amino-3- carboxypropyl) uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 φ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O- methyl-pseudouridine (φm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'- O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5- carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'- F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E- propenylamino) uridine, or any other modified uridine known in the art. In some embodiments, the RNA according to the present disclosure comprises a 5' cap. In one embodiment, the RNA of the present disclosure does not have uncapped 5'-triphosphates. In one embodiment, the RNA may be modified by a 5' cap analog. The term "5' cap" refers to a structure found on the 5'-end of an mRNA molecule (including a 5’ cap analog) and generally consists of a guanosine nucleotide connected to the mRNA via a 5'- to 5'-triphosphate linkage. In one embodiment, this guanosine is methylated at the 7- position. Providing an RNA with a 5' cap or 5' cap analog may be achieved by in vitro transcription, in which the 5' cap or 5’ cap analog is co-transcriptionally expressed into the RNA strand, or may be attached to RNA posttranscriptionally using capping enzymes. The term “5’ cap analog” refers to a synthetic 5’ cap, e.g. to a non-naturally occurring 5' cap. In some embodiments, the RNA according to the present disclosure comprises a 5’ cap analog. In some embodiments, the 5’ cap analog for RNA is m27,3'-0Gppp(m12'-0)ApG (also sometimes referred to as m27,3'0G(5')ppp(5')m2'-0ApG), which has the following structure: In some embodiments, the 5’ cap analog for RNA is 3´-O-Me-m7G(5')ppp(5')G. In some embodiments, the RNA comprises the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG or 3´- O-Me-m7G(5')ppp(5')G. In some embodiments, the RNA comprises the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG. In some embodiments, RNA according to the present disclosure comprises a 5'-UTR and/or a 3'-UTR. The term "untranslated region" or "UTR" may refer to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and/or 3' (downstream) of an open reading frame (3'-UTR). A 5'-UTR, if present, is located at the 5' end, upstream of the start codon of a protein-encoding region. A 5'-UTR is downstream of the 5'cap (if present), e.g. directly adjacent to the 5' cap. A 3'-UTR, if present, is located at the 3' end, downstream of the termination codon of a protein-encoding region. Suitably, the term "3'-UTR" may encompass a poly(A) sequence. Alternatively, the term "3'-UTR" may not include a poly(A) sequence. Suitably, the 3'-UTR may be upstream of the poly(A) sequence (if present), e.g. directly adjacent to the poly(A) sequence. An illustrative 5’UTR sequence is provided below: GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 10) A further illustrative 5’UTR sequence is provided below: GGAAUAAACUAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUC UAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGA ACGAUAGCC (SEQ ID NO: 11) A further illustrative 5’UTR sequence is provided below: AGACGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC(SEQ ID NO: 12) A further illustrative 5’UTR sequence is provided below: AUUCUUCUGGUCCCCACAGACUCAGAGAGAACCC (SEQ ID NO: 31) In some embodiments, the RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12 and 31, or a nucleotide sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12 and 31. In some preferred embodiments, the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. An illustrative 3’UTR sequence is provided below: CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCC CCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGAC ACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAG UGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGU GCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 13) A further illustrative 3’UTR sequence is provided below: CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACC UCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUA ACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGC CACACC (SEQ ID NO: 32) In some embodiments, the RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 32, or a nucleotide sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 31. In some preferred embodiments, the RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the RNA according to the present disclosure comprises a 3'-poly(A) sequence. As used herein, the term "poly(A) sequence" or "poly-A tail" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3' end of an RNA molecule. Poly(A) sequences are known to those of skill in the art and may follow the 3' UTR in the RNAs described herein. Suitably, the poly(A) sequence may also be defined as part of the 3' UTR. The poly(A) sequence may be of any length. In some embodiments, a poly(A) sequence comprises or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides, and, in particular, about 110 nucleotides. In some embodiments, a poly(A) sequence comprises or consists of from about 80 to about 150 nucleotides, optionally from about 110 to about 120 nucleotides. In some embodiments, the poly(A) sequence only consists of A nucleotides. In some embodiments, the poly(A) sequence comprises or consists of about 120 A nucleotides. In some embodiments, the poly(A) sequence essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, and U), as disclosed in WO 2016/005324 A1, hereby incorporated by reference. Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. A poly(A) cassette present in the coding strand of DNA that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g. 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency. In some embodiments, no nucleotides other than A nucleotides flank a poly(A) sequence at its 3' end, i.e., the poly(A) sequence is not masked or followed at its 3' end by a nucleotide other than A. In some embodiments, the RNA comprises a poly-A tail. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail is an interrupted poly-A tail. An illustrative poly-A tail sequence is provided below: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 14) In some embodiments, the poly-A tail comprises the poly-A tail shown in SEQ ID NO: 14. In some embodiments, the RNA of the invention may comprise a poly-A tail shown in SEQ ID NO: 14 and the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG. In some embodiments, the RNA of the invention may comprise a poly-A tail shown in SEQ ID NO: 14, and the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG, and a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the RNA of the invention may comprise a poly-A tail shown in SEQ ID NO: 14, and the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG, and a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the nucleotide sequence of SEQ ID NO: 31. In some embodiments, the RNA of the invention may comprise a poly-A tail shown in SEQ ID NO: 14, and the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG, and a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto, and a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the nucleotide sequence of SEQ ID NO: 31. Illustrative polynucleotide sequences Illustrative RNA backbone sequence (5’UTR shown in bold, Kozak sequence shown in italics, 3’UTR shown underlined, and poly-A tail shown in italics and underlined): AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC – [coding sequence] - CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCC CCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGAC ACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAG UGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGU GCCAGCCACACCCUGGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAA Illustrative sequences encoding an immune inhibitory protein of the IL-1 family for use according to the invention are provided below. For example, these coding sequences may be inserted into an RNA backbone sequence, such as SEQ ID NO: 15. Illustrative RNA sequence encoding IL-1RA: AUGGAAAUCUGCAGAGGCCUGCGGAGCCACCUGAUUACCCUGCUGCUGUUCCUGUUCCACAGCGAGAC AAUCUGCAGGCCCAGCGGCAGAAAGUCCAGCAAGAUGCAGGCCUUCCGGAUCUGGGACGUGAACCAGA AAACCUUCUACCUGCGGAACAAUCAGCUGGUGGCCGGCUAUCUGCAGGGCCCCAAUGUGAACCUGGAA GAAAAGAUCGACGUGGUGCCCAUCGAGCCCCACGCUCUGUUUCUGGGAAUUCACGGCGGCAAGAUGUG CCUGAGCUGUGUGAAGUCUGGCGACGAGACACGGCUGCAGCUGGAAGCCGUGAACAUCACCGACCUGA GCGAGAACCGGAAGCAGGACAAGAGAUUCGCCUUCAUCAGAAGCGACAGCGGCCCCACCACAAGCUUU GAGUCUGCUGCUUGCCCUGGCUGGUUCCUGUGUACAGCCAUGGAAGCCGACCAGCCUGUGUCUCUGAC CAACAUGCCUGACGAGGGCGUGAUGGUCACCAAGUUCUACUUCCAAGAGGACGAGUGAUGA (SEQ ID NO: 16) Illustrative RNA sequence encoding IL-18BP: AUGACCAUGCGGCACAACUGGACCCCUGAUCUGUCUCCUCUGUGGGUGCUGCUGCUGUGUGCCCACGU UGUGACACUGCUUGUCAGAGCCACACCUGUGUCUCAGACCACCACAGCCGCUACAGCCUCUGUGCGGA GCACCAAGGAUCCCUGUCCUUCUCAGCCUCCUGUGUUCCCUGCCGCCAAACAGUGUCCUGCUCUGGAA GUGACAUGGCCCGAGGUGGAAGUGCCUCUGAAUGGCACACUGAGCCUGAGCUGCGUGGCCUGCAGCAG AUUCCCCAACUUCAGCAUCCUGUACUGGCUCGGCAACGGCAGCUUCAUCGAGCAUCUGCCUGGCAGAC UGUGGGAGGGCAGCACAUCUAGAGAGAGAGGCAGCACCGGAACUCAGCUGUGCAAAGCCCUGGUGCUG GAACAGCUGACACCAGCUCUGCACAGCACCAAUUUCAGCUGCGUGCUGGUGGACCCCGAACAGGUGGU GCAGAGACAUGUGGUUCUGGCCCAACUGUGGGCCGGACUGAGAGCUACACUGCCUCCUACACAAGAGG CCCUGCCUAGCUCUCACUCUAGCCCUCAGCAACAGGGAUGAUGA (SEQ ID NO: 17) Illustrative RNA sequence encoding IL-36RA: AUGAGAGUGAUGGCCCCUCGGACACUGAUCCUGCUGCUUUCUGGUGCCCUGGCUCUGACAGAAACAUG GGCUGUUCUGUCUGGCGCCCUGUGCUUCAGAAUGAAGGACAGCGCCCUGAAGGUGCUGUACCUGCACA ACAACCAGCUGCUGGCUGGCGGACUGCAUGCCGGCAAAGUUAUCAAGGGCGAAGAAAUCAGCGUGGUG CCCAACCGGUGGCUGGAUGCUUCUCUGUCUCCUGUGAUCCUGGGCGUGCAAGGCGGAAGCCAGUGUCU GUCUUGUGGCGUGGGACAAGAGCCCACACUGACCCUGGAACCUGUGAACAUCAUGGAACUGUACCUGG GCGCCAAAGAGAGCAAGAGCUUCACCUUCUAUCGGAGAGACAUGGGCCUGACCAGCAGCUUCGAGUCU GCCGCUUAUCCUGGCUGGUUCCUGUGUACAGUGCCCGAGGCUGACCAGCCUGUCAGACUGACACAGCU GCCUGAGAACGGCGGAUGGAAUGCCCCUAUCACCGACUUCUACUUCCAACAGUGCGACUGAUGA (SEQ ID NO: 18) Illustrative RNA sequence encoding IL-37: AUGAGAGUGAUGGCCCCUCGGACACUGAUCCUGCUGCUUUCUGGUGCCCUGGCUCUGACAGAAACAUG GGCCGUGCACACAAGCCCCAAAGUGAAGAAUCUGAACCCCAAGAAGUUCAGCAUCCACGACCAGGACC ACAAGGUGCUGGUGCUGGAUAGCGGCAACCUGAUCGCCGUGCCUGACAAGAACUAUAUCAGACCCGAG AUCUUCUUCGCCCUGGCCAGCUCUCUGUCUAGCGCCUCUGCCGAGAAGGGCUCUCCUAUCCUGCUGGG AGUGUCCAAGGGCGAGUUCUGCCUGUACUGCGACAAGGACAAGGGCCAGUCUCACCCUAGCCUGCAGC UGAAGAAAGAAAAGCUGAUGAAGCUGGCCGCUCAGAAAGAGAGCGCCAGACGGCCCUUCAUCUUCUAC AGAGCCCAAGUCGGCAGCUGGAACAUGCUGGAAUCUGCCGCUCAUCCCGGCUGGUUCAUCUGCACCAG CUGCAACUGCAAUGAGCCCGUGGGCGUGACCGACAAGUUCGAGAACAGAAAGCACAUCGAGUUCAGCU UCCAGCCUGUGUGCAAGGCCGAGAUGAGCCCAUCUGAGGUGUCCGAUUGAUGA (SEQ ID NO: 19) Illustrative RNA sequence encoding IL-38: AUGAGAGUGAUGGCCCCUCGGACACUGAUCCUGCUGCUUUCUGGUGCCCUGGCUCUGACAGAGACUUG GGCCUGUUCUCUGCCCAUGGCCAGAUAUUACAUCAUUAAGUACGCCGACCAGAAGGCCCUGUACACCA GAGAUGGACAGCUGCUCGUGGGAGAUCCCGUGGCCGAUAAUUGCUGCGCCGAGAAGAUCUGCAUCCUG CCUAACAGAGGCCUGGCCAGAACAAAGGUGCCCAUCUUCCUGGGCAUCCAAGGCGGCUCUAGAUGCCU GGCCUGUGUGGAAACAGAGGAAGGCCCUAGCCUGCAGCUGGAAGAUGUGAACAUCGAGGAACUGUACA AAGGCGGCGAGGAAGCCACCAGAUUCACAUUCUUUCAGAGCAGCAGCGGCAGCGCCUUCAGACUGGAA GCUGCUGCUUGGCCUGGCUGGUUUCUGUGUGGACCUGCUGAACCUCAGCAGCCCGUGCAGCUGACAAA AGAGAGCGAACCCAGCGCCAGGACCAAGUUCUACUUCGAGCAGAGCUGGUGAUGA (SEQ ID NO: 20) Illustrative mRNA sequence encoding IL-1RA: AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGAAAUCUGCAGA GGCCUGCGGAGCCACCUGAUUACCCUGCUGCUGUUCCUGUUCCACAGCGAGACAAUCUGCAGGCCCAG CGGCAGAAAGUCCAGCAAGAUGCAGGCCUUCCGGAUCUGGGACGUGAACCAGAAAACCUUCUACCUGC GGAACAAUCAGCUGGUGGCCGGCUAUCUGCAGGGCCCCAAUGUGAACCUGGAAGAAAAGAUCGACGUG GUGCCCAUCGAGCCCCACGCUCUGUUUCUGGGAAUUCACGGCGGCAAGAUGUGCCUGAGCUGUGUGAA GUCUGGCGACGAGACACGGCUGCAGCUGGAAGCCGUGAACAUCACCGACCUGAGCGAGAACCGGAAGC AGGACAAGAGAUUCGCCUUCAUCAGAAGCGACAGCGGCCCCACCACAAGCUUUGAGUCUGCUGCUUGC CCUGGCUGGUUCCUGUGUACAGCCAUGGAAGCCGACCAGCCUGUGUCUCUGACCAACAUGCCUGACGA GGGCGUGAUGGUCACCAAGUUCUACUUCCAAGAGGACGAGUGAUGACUCGAGCUGGUACUGCAUGCAC GCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUG CUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUG CAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAAC GAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAG AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 21) Illustrative mRNA sequence encoding IL-18BP: AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGACCAUGCGGCAC AACUGGACCCCUGAUCUGUCUCCUCUGUGGGUGCUGCUGCUGUGUGCCCACGUUGUGACACUGCUUGU CAGAGCCACACCUGUGUCUCAGACCACCACAGCCGCUACAGCCUCUGUGCGGAGCACCAAGGAUCCCU GUCCUUCUCAGCCUCCUGUGUUCCCUGCCGCCAAACAGUGUCCUGCUCUGGAAGUGACAUGGCCCGAG GUGGAAGUGCCUCUGAAUGGCACACUGAGCCUGAGCUGCGUGGCCUGCAGCAGAUUCCCCAACUUCAG CAUCCUGUACUGGCUCGGCAACGGCAGCUUCAUCGAGCAUCUGCCUGGCAGACUGUGGGAGGGCAGCA CAUCUAGAGAGAGAGGCAGCACCGGAACUCAGCUGUGCAAAGCCCUGGUGCUGGAACAGCUGACACCA GCUCUGCACAGCACCAAUUUCAGCUGCGUGCUGGUGGACCCCGAACAGGUGGUGCAGAGACAUGUGGU UCUGGCCCAACUGUGGGCCGGACUGAGAGCUACACUGCCUCCUACACAAGAGGCCCUGCCUAGCUCUC ACUCUAGCCCUCAGCAACAGGGAUGAUGACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCC UUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGC CCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAG CCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCU AUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 22) Illustrative mRNA sequence encoding IL-36RA: AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCC CCUCGGACACUGAUCCUGCUGCUUUCUGGUGCCCUGGCUCUGACAGAAACAUGGGCUGUUCUGUCUGG CGCCCUGUGCUUCAGAAUGAAGGACAGCGCCCUGAAGGUGCUGUACCUGCACAACAACCAGCUGCUGG CUGGCGGACUGCAUGCCGGCAAAGUUAUCAAGGGCGAAGAAAUCAGCGUGGUGCCCAACCGGUGGCUG GAUGCUUCUCUGUCUCCUGUGAUCCUGGGCGUGCAAGGCGGAAGCCAGUGUCUGUCUUGUGGCGUGGG ACAAGAGCCCACACUGACCCUGGAACCUGUGAACAUCAUGGAACUGUACCUGGGCGCCAAAGAGAGCA AGAGCUUCACCUUCUAUCGGAGAGACAUGGGCCUGACCAGCAGCUUCGAGUCUGCCGCUUAUCCUGGC UGGUUCCUGUGUACAGUGCCCGAGGCUGACCAGCCUGUCAGACUGACACAGCUGCCUGAGAACGGCGG AUGGAAUGCCCCUAUCACCGACUUCUACUUCCAACAGUGCGACUGAUGACUCGAGCUGGUACUGCAUG CACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGU AUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCA AUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUA AACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGC AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 23) Illustrative mRNA sequence encoding IL-37: AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCC CCUCGGACACUGAUCCUGCUGCUUUCUGGUGCCCUGGCUCUGACAGAAACAUGGGCCGUGCACACAAG CCCCAAAGUGAAGAAUCUGAACCCCAAGAAGUUCAGCAUCCACGACCAGGACCACAAGGUGCUGGUGC UGGAUAGCGGCAACCUGAUCGCCGUGCCUGACAAGAACUAUAUCAGACCCGAGAUCUUCUUCGCCCUG GCCAGCUCUCUGUCUAGCGCCUCUGCCGAGAAGGGCUCUCCUAUCCUGCUGGGAGUGUCCAAGGGCGA GUUCUGCCUGUACUGCGACAAGGACAAGGGCCAGUCUCACCCUAGCCUGCAGCUGAAGAAAGAAAAGC UGAUGAAGCUGGCCGCUCAGAAAGAGAGCGCCAGACGGCCCUUCAUCUUCUACAGAGCCCAAGUCGGC AGCUGGAACAUGCUGGAAUCUGCCGCUCAUCCCGGCUGGUUCAUCUGCACCAGCUGCAACUGCAAUGA GCCCGUGGGCGUGACCGACAAGUUCGAGAACAGAAAGCACAUCGAGUUCAGCUUCCAGCCUGUGUGCA AGGCCGAGAUGAGCCCAUCUGAGGUGUCCGAUUGAUGACUCGAGCUGGUACUGCAUGCACGCAAUGCU AGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACC UCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAA AACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUU AACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 24) Illustrative mRNA sequence encoding IL-38: AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCC CCUCGGACACUGAUCCUGCUGCUUUCUGGUGCCCUGGCUCUGACAGAGACUUGGGCCUGUUCUCUGCC CAUGGCCAGAUAUUACAUCAUUAAGUACGCCGACCAGAAGGCCCUGUACACCAGAGAUGGACAGCUGC UCGUGGGAGAUCCCGUGGCCGAUAAUUGCUGCGCCGAGAAGAUCUGCAUCCUGCCUAACAGAGGCCUG GCCAGAACAAAGGUGCCCAUCUUCCUGGGCAUCCAAGGCGGCUCUAGAUGCCUGGCCUGUGUGGAAAC AGAGGAAGGCCCUAGCCUGCAGCUGGAAGAUGUGAACAUCGAGGAACUGUACAAAGGCGGCGAGGAAG CCACCAGAUUCACAUUCUUUCAGAGCAGCAGCGGCAGCGCCUUCAGACUGGAAGCUGCUGCUUGGCCU GGCUGGUUUCUGUGUGGACCUGCUGAACCUCAGCAGCCCGUGCAGCUGACAAAAGAGAGCGAACCCAG CGCCAGGACCAAGUUCUACUUCGAGCAGAGCUGGUGAUGACUCGAGCUGGUACUGCAUGCACGCAAUG CUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCA CCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUC AAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGU UUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 25) Illustrative DNA sequence encoding IL-1RA: AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGAAATCTGCAGA GGCCTGCGGAGCCACCTGATTACCCTGCTGCTGTTCCTGTTCCACAGCGAGACAATCTGCAGGCCCAG CGGCAGAAAGTCCAGCAAGATGCAGGCCTTCCGGATCTGGGACGTGAACCAGAAAACCTTCTACCTGC GGAACAATCAGCTGGTGGCCGGCTATCTGCAGGGCCCCAATGTGAACCTGGAAGAAAAGATCGACGTG GTGCCCATCGAGCCCCACGCTCTGTTTCTGGGAATTCACGGCGGCAAGATGTGCCTGAGCTGTGTGAA GTCTGGCGACGAGACACGGCTGCAGCTGGAAGCCGTGAACATCACCGACCTGAGCGAGAACCGGAAGC AGGACAAGAGATTCGCCTTCATCAGAAGCGACAGCGGCCCCACCACAAGCTTTGAGTCTGCTGCTTGC CCTGGCTGGTTCCTGTGTACAGCCATGGAAGCCGACCAGCCTGTGTCTCTGACCAACATGCCTGACGA GGGCGTGATGGTCACCAAGTTCTACTTCCAAGAGGACGAGTGATGACTCGAGCTGGTACTGCATGCAC GCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATG CTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATG CAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAAC GAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAG AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 26) Illustrative DNA sequence encoding IL-18BP: AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGACCATGCGGCAC AACTGGACCCCTGATCTGTCTCCTCTGTGGGTGCTGCTGCTGTGTGCCCACGTTGTGACACTGCTTGT CAGAGCCACACCTGTGTCTCAGACCACCACAGCCGCTACAGCCTCTGTGCGGAGCACCAAGGATCCCT GTCCTTCTCAGCCTCCTGTGTTCCCTGCCGCCAAACAGTGTCCTGCTCTGGAAGTGACATGGCCCGAG GTGGAAGTGCCTCTGAATGGCACACTGAGCCTGAGCTGCGTGGCCTGCAGCAGATTCCCCAACTTCAG CATCCTGTACTGGCTCGGCAACGGCAGCTTCATCGAGCATCTGCCTGGCAGACTGTGGGAGGGCAGCA CATCTAGAGAGAGAGGCAGCACCGGAACTCAGCTGTGCAAAGCCCTGGTGCTGGAACAGCTGACACCA GCTCTGCACAGCACCAATTTCAGCTGCGTGCTGGTGGACCCCGAACAGGTGGTGCAGAGACATGTGGT TCTGGCCCAACTGTGGGCCGGACTGAGAGCTACACTGCCTCCTACACAAGAGGCCCTGCCTAGCTCTC ACTCTAGCCCTCAGCAACAGGGATGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCC TTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGC CCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAG CCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCT ATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 27) Illustrative DNA sequence encoding IL-36RA: AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGATGGCC CCTCGGACACTGATCCTGCTGCTTTCTGGTGCCCTGGCTCTGACAGAAACATGGGCTGTTCTGTCTGG CGCCCTGTGCTTCAGAATGAAGGACAGCGCCCTGAAGGTGCTGTACCTGCACAACAACCAGCTGCTGG CTGGCGGACTGCATGCCGGCAAAGTTATCAAGGGCGAAGAAATCAGCGTGGTGCCCAACCGGTGGCTG GATGCTTCTCTGTCTCCTGTGATCCTGGGCGTGCAAGGCGGAAGCCAGTGTCTGTCTTGTGGCGTGGG ACAAGAGCCCACACTGACCCTGGAACCTGTGAACATCATGGAACTGTACCTGGGCGCCAAAGAGAGCA AGAGCTTCACCTTCTATCGGAGAGACATGGGCCTGACCAGCAGCTTCGAGTCTGCCGCTTATCCTGGC TGGTTCCTGTGTACAGTGCCCGAGGCTGACCAGCCTGTCAGACTGACACAGCTGCCTGAGAACGGCGG ATGGAATGCCCCTATCACCGACTTCTACTTCCAACAGTGCGACTGATGACTCGAGCTGGTACTGCATG CACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGT ATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCA ATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATA AACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGC AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 28) Illustrative DNA sequence encoding IL-37: AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGATGGCC CCTCGGACACTGATCCTGCTGCTTTCTGGTGCCCTGGCTCTGACAGAAACATGGGCCGTGCACACAAG CCCCAAAGTGAAGAATCTGAACCCCAAGAAGTTCAGCATCCACGACCAGGACCACAAGGTGCTGGTGC TGGATAGCGGCAACCTGATCGCCGTGCCTGACAAGAACTATATCAGACCCGAGATCTTCTTCGCCCTG GCCAGCTCTCTGTCTAGCGCCTCTGCCGAGAAGGGCTCTCCTATCCTGCTGGGAGTGTCCAAGGGCGA GTTCTGCCTGTACTGCGACAAGGACAAGGGCCAGTCTCACCCTAGCCTGCAGCTGAAGAAAGAAAAGC TGATGAAGCTGGCCGCTCAGAAAGAGAGCGCCAGACGGCCCTTCATCTTCTACAGAGCCCAAGTCGGC AGCTGGAACATGCTGGAATCTGCCGCTCATCCCGGCTGGTTCATCTGCACCAGCTGCAACTGCAATGA GCCCGTGGGCGTGACCGACAAGTTCGAGAACAGAAAGCACATCGAGTTCAGCTTCCAGCCTGTGTGCA AGGCCGAGATGAGCCCATCTGAGGTGTCCGATTGATGACTCGAGCTGGTACTGCATGCACGCAATGCT AGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACC TCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAA AACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTT AACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 29) Illustrative DNA sequence encoding IL-38: AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGATGGCC CCTCGGACACTGATCCTGCTGCTTTCTGGTGCCCTGGCTCTGACAGAGACTTGGGCCTGTTCTCTGCC CATGGCCAGATATTACATCATTAAGTACGCCGACCAGAAGGCCCTGTACACCAGAGATGGACAGCTGC TCGTGGGAGATCCCGTGGCCGATAATTGCTGCGCCGAGAAGATCTGCATCCTGCCTAACAGAGGCCTG GCCAGAACAAAGGTGCCCATCTTCCTGGGCATCCAAGGCGGCTCTAGATGCCTGGCCTGTGTGGAAAC AGAGGAAGGCCCTAGCCTGCAGCTGGAAGATGTGAACATCGAGGAACTGTACAAAGGCGGCGAGGAAG CCACCAGATTCACATTCTTTCAGAGCAGCAGCGGCAGCGCCTTCAGACTGGAAGCTGCTGCTTGGCCT GGCTGGTTTCTGTGTGGACCTGCTGAACCTCAGCAGCCCGTGCAGCTGACAAAAGAGAGCGAACCCAG CGCCAGGACCAAGTTCTACTTCGAGCAGAGCTGGTGATGACTCGAGCTGGTACTGCATGCACGCAATG CTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCA CCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTC AAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGT TTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 30) In some embodiments, the polynucleotide comprises a sequence as set forth in any one of SEQ ID NOs: 16 to 30 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the polynucleotide consists of a sequence as set forth in any one of SEQ ID NOs: 16 to 30 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the polynucleotide comprises a sequence as set forth in any one of SEQ ID NOs: 16 to 20 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the polynucleotide comprises a sequence as set forth in any one of SEQ ID NOs: 16 to 18 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 16 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 17 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 18 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 19 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 20 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the polynucleotide comprises a sequence as set forth in any one of SEQ ID NOs: 21 to 25 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the polynucleotide comprises a sequence as set forth in any one of SEQ ID NOs: 21 to 23 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 21 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 22 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 23 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 24 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 25 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the polynucleotide comprises a sequence as set forth in any one of SEQ ID NOs: 26 to 30 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the polynucleotide comprises a sequence as set forth in any one of SEQ ID NOs: 26 to 28 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 26 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 27 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some preferred embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 28 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 29 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 30 or a sequence having at least 80% (suitably, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. Vector In one aspect, the invention provides a vector comprising the polynucleotide of the invention. In an embodiment, the polynucleotide described herein is in the form of a vector. In an embodiment, the present invention provides a vector comprising a nucleic acid sequence described herein. Such a vector may be used to introduce the nucleic acid sequence into a host cell so that it expresses the immune inhibitory protein of the IL-1 family as described herein. In an embodiment, the vector comprises one or more polynucleotides of the invention which encode different immune inhibitory proteins of the IL-1 family. For example, in an embodiment the vector comprises a first polynucleotide sequence which encodes a first immune inhibitory protein of the IL-1 family as described herein and a second polynucleotide which encodes a second immune inhibitory protein of the IL-1 family as described herein, optionally as well as a third polynucleotide which encodes a third immune inhibitory protein of the IL-1 family as described herein. Suitably, the vector comprises polynucleotides encoding IL-18BP and IL-37. In embodiments, the vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA. In an embodiment, the vector is capable of transfecting or transducing a cell. Nucleic acid particle In one aspect, the invention provides a nucleic acid particle comprising the polynucleotide according to the invention or the vector according to the invention. In an embodiment, the nucleic acid particle comprises one or more polynucleotides of the invention which encode different immune inhibitory proteins of the IL-1 family. For example, in an embodiment the nucleic acid particle comprises a first polynucleotide sequence which encodes a first immune inhibitory protein of the IL-1 family as described herein and a second polynucleotide which encodes a second immune inhibitory protein of the IL-1 family as described herein, optionally as well as a third polynucleotide which encodes a third immune inhibitory protein of the IL-1 family as described herein. Suitably, the nucleic acid particle comprises polynucleotides encoding IL-18BP and IL-37. A nucleic acid particle may be a particle comprising at least one cationic or cationically ionizable compound such as a polymer or lipid. Suitably, the particle may comprise at least one cationic or cationically ionizable compound such as a polymer or lipid complexing a payload, for example a polynucleotide. Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acid are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles comprising the one or more nucleic acid sequences. Different types of nucleic acid containing particles have been described previously to be suitable for delivery of nucleic acid (e.g. RNA) in particulate form (cf., e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral delivery vehicles, nanoparticle encapsulation of nucleic acids physically protects nucleic acids from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape. In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance possesses both hydrophilic and lipophilic properties. The envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic. Thus, the particle may be a monolamellar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids) optionally in combination with additional substances (e.g., additional lipids) which do not have to be amphiphilic. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles. A "nucleic acid particle" can be used to deliver nucleic acid sequences to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may be formed from lipids comprising at least one cationic or cationically ionizable lipid or lipid-like material. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid or lipid-like material combines together with the nucleic acids to form aggregates, and this aggregation results in colloidally stable particles. Nucleic acid particles (such as RNA particles and/or DNA particles) include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations. In general, a lipoplex (LPX) is obtainable from mixing two aqueous phases, namely a phase comprising nucleic acid (such as RNA and/or DNA) and a phase comprising a dispersion of lipids. In some embodiments, the lipid phase comprises liposomes. In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase. A prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous environment. In some embodiments, liposomes comprise unilamellar or multilamellar phospholipid bilayers enclosing an aqueous core (also referred to herein as an aqueous lumen). They may be prepared from materials possessing polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids employed in formulating liposomes designed for the delivery of nucleic acids are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked to a hydrocarbon chain or cholesterol derivative via glycerol. In some embodiments, lipoplexes are multilamellar liposome-based formulations that form upon electrostatic interaction of cationic liposomes with nucleic acids (such as RNAs and/or DNAs). In some embodiments, formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact nucleic acid-lipoplexes (such as RNA– and/or DNA–lipoplexes). In some embodiments, an LPX particle comprises an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, and nucleic acid (such as RNA and/or DNA, especially mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, in particular cationic or cationically ionizable amphiphilic lipids) and negatively charged nucleic acid (especially mRNA) results in complexation and spontaneous formation of nucleic acid lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic or cationically ionizable amphiphilic lipid, such as DOTMA and/or DODMA, and additional lipids, such as DOPE. In some embodiments, a nucleic acid (such as RNA and/or DNA, especially mRNA) lipoplex particle is a nanoparticle. In general, a lipid nanoparticle (LNP) is obtainable from direct mixing of nucleic acid (such as RNA and/or DNA) in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol. In that case, lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water. In some embodiments, LNPs comprise or consist of a cationic/ionisable lipid and helper lipids such as phospholipids, cholesterol, and/or polymer-conjugated lipids (i.e., stealth lipids), such as polyethylene glycol (PEG) lipids. In some embodiments, in the nucleic acid LNPs (such as RNA/DNA LNPs) described herein the nucleic acid (such as mRNA) is bound by ionisable lipid that occupies the central core of the LNP. In some embodiments, PEG lipid forms the surface of the LNP, along with phospholipids. In some embodiments, the surface comprises a bilayer. In some embodiments, cholesterol and ionisable lipid in charged and uncharged forms can be distributed throughout the LNP. In some embodiments, nucleic acid (such as RNA and/or DNA, e.g., mRNA) may be noncovalently associated with a particle as described herein. In embodiments, the nucleic acid (such as RNA and/or DNA, especially mRNA) may be adhered to the outer surface of the particle (surface nucleic acid) and/or may be contained in the particle (encapsulated nucleic acid (such as encapsulated DNA or mRNA). In some embodiments, the particles (e.g., LNPs and LPXs) described herein have a size (such as a diameter) in the range of about 10 to about 2000 nm, such as at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and/or at most 1900 nm (e.g., at most about 1900 nm, at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1200 nm, at most about 1100 nm, at most about 1000 nm, at most about 950 nm, at most about 900 nm, at most about 850 nm, at most about 800 nm, at most about 750 nm, at most about 700 nm, at most about 650 nm, at most about 600 nm, at most about 550 nm, or at most about 500 nm), such as in the range of about 20 to about 1500 nm, such as about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about 900 nm, about 70 to 800 nm, about 80 to 700 nm, about 90 to 600 nm, or about 50 to 500 nm or about 100 to 500 nm, such as in the range of 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, 50 to 250 nm, 60 to 200 nm, or 70 to 150 nm. In some embodiments, the particles described herein are nanoparticles. The term “nanoparticle” relates to a nano-sized particle comprising nucleic acid (especially RNA and/or DNA) as described herein and at least one cationic or cationically ionisable lipid, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least about 1 nm and below about 1000 nm. Preferably, the size of a particle is its diameter. Lipoplexes A nucleic acid particle of the disclosure may be a particle comprising at least one lipid. In some embodiments, the particles formed from the polynucleotides (e.g., RNA) of the disclosure and at least one lipid are lipid nanoparticles (LNP), lipoplexes (LPX) or liposomes. Preferably, the particles are LPX particles. Preferably the polynucleotide encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof of the disclosure is an RNA or mRNA molecule. In some embodiments, an LPX particle comprises an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, and RNA (especially mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, in particular cationic or cationically ionizable amphiphilic lipids) and negatively charged nucleic acid (especially mRNA) results in complexation and spontaneous formation of nucleic acid lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic or cationically ionizable amphiphilic lipid, such as DOTMA and/or DODMA, and additional lipids, such as DOPE. In some embodiments, an RNA (especially mRNA) lipoplex particle is a nanoparticle. Preferably, the at least one lipid comprises at least one cationic or cationically ionizable lipid, preferably is a cationic lipid. As used herein, a "cationic lipid" refers to a lipid or lipid-like material having a net positive charge. Cationic lipids bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic or cationically ionizable lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge. In other embodiments, the particles according to the present disclosure comprises RNA in the format of a lipoplex. The term, “lipoplex” or “RNA lipoplex” refers to a complex of lipids and nucleic acids such as RNA. Lipoplexes can be formed of cationic (positively charged) liposomes and the anionic (negatively charged) nucleic acid. The cationic liposomes can also include a neutral “helper” lipid. In the simplest case, the lipoplexes form spontaneously by mixing the nucleic acid with the liposomes with a certain mixing protocol, however various other protocols may be applied. It is understood that electrostatic interactions between positively charged liposomes and negatively charged nucleic acid are the driving force for the lipoplex formation (WO 2013/143555 A1). In one embodiment of the present disclosure, the net charge of the RNA lipoplex particles is close to zero or negative. It is known that electro- neutral or negatively charged lipoplexes of RNA and liposomes lead to substantial RNA expression in spleen dendritic cells (DCs) after systemic administration and are not associated with the elevated toxicity that has been reported for positively charged liposomes and lipoplexes (cf. WO 2013/143555 A1). Therefore, in one embodiment of the present disclosure, the composition according to the present disclosure comprises RNA encoding an immune inhibitory protein of the IL-1 family or functional variant thereof, in the format of nanoparticles, preferably lipoplex nanoparticles, in which (i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles and/or (ii) the nanoparticles have a net negative charge and/or (iii) the charge ratio of positive charges to negative charges in the nanoparticles is 1.4:1 or less and/or (iv) the zeta potential of the nanoparticles is 0 or less. As described in WO 2013/143555 A1, zeta potential is a scientific term for electrokinetic potential in colloidal systems. In the present disclosure, (a) the zeta potential and (b) the charge ratio of the cationic or cationically ionizable lipid to the RNA in the nanoparticles can both be calculated as disclosed in WO 2013/143555 A1. In summary, particles which are nanoparticulate lipoplex formulations with a defined particle size, wherein the net charge of the particles is negative, as disclosed in WO 2013/143555 A1, are preferred particles in the context of the present disclosure. In other embodiments, the lipoplexes are obtained according to a method as disclosed in WO 2019/077053 A1. According to WO 2019/077053 A1, lipoplexes can be obtained by adding liposome colloid with a solution comprising RNA. The liposome colloid, according to WO 2019/077053 A1, can be obtained by a method comprising injecting a lipid solution in ethanol into an aqueous phase to produce the liposome colloid, wherein the concentration of at least one of the lipids in the lipid solution corresponds to or is higher than the equilibrium solubility of the at least one lipid in ethanol. A particularly preferred method of producing a liposome colloid comprises injecting a lipid solution comprising DOTMA and DOPE in a molar ratio of about 2:1 in ethanol into water stirred at a stirring velocity of about 150 rpm to produce the liposome colloid, wherein the concentration of DOTMA and DOPE in the lipid solution is about 330 mM. In some embodiments, the particles formed are lipoplex particles, preferably negatively charged lipoplex particles. Without being bound by theory the RNA particles (e.g., the RNA- lipoplex particles) specifically target an immune cell, in particular in the spleen, which immune cell is selected from the group consisting of neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, T lymphocytes, preferably cytotoxic T cells or regulatory T cells, and B lymphocytes. Preferably the immune cell is a monocyte or macrophage. In some embodiments, the RNA particles (e.g., RNA-LPX particles) target the spleen, in particular professional antigen presenting cells, preferably monocytes and/or macrophages or their precursors. In some embodiments, provided herein is a composition comprising nanoparticles which comprise at least one cationic lipid, at least one neutral helper lipid and RNA encoding an immune inhibitory protein of the IL-1 family, or functional variant thereof, of the present disclosure, where the charge ratio of positive charges to negative charges in the nanoparticles can be between 1:1 and 1:8 (such as between 1:1 and 1:4, optionally between 1:2 and 1:1.2). Typically, the positive charges are contributed by the at least one cationic lipid and the negative charges are contributed by the RNA. Typically, the charge ratio is the charge ratio at physiological pH. In some embodiments, the at least one cationic or cationically ionizable lipid can comprise 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleyloxy-3- dimethylaminopropane (DODMA), and/or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In some embodiments, the at least one helper lipid (e.g., neutral helper lipid) can comprise 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and/or 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC). In some embodiments, the molar ratio of the at least one cationic or cationically ionizable lipid to the at least one helper lipid can be from 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1:1. In some embodiments, the molar ratio of the at least one cationic lipid to the at least one neutral helper lipid is from about 9:1 to about 3:7. In some embodiments, the nanoparticles can be lipoplexes comprising DODMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DODMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2. In some embodiments, the nanoparticles can be lipoplexes comprising DODMA and Cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DODMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2. In some embodiments, the nanoparticles can be lipoplexes comprising DODMA and DSPC in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DODMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2. In some embodiments, the nanoparticles can be lipoplexes comprising DOTMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2. In some embodiments, the nanoparticles can be lipoplexes comprising DOTMA and Cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2. In some embodiments, the nanoparticles can be lipoplexes comprising DOTAP and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2. In some embodiments, RNA lipoplex particles target immune cells, preferably monocytes and/or macrophages. In some embodiments the composition comprises RNA lipoplex particles as described in WO2022069632A1 or WO2019077053A1. In some embodiments, the composition comprises: RNA lipoplex particles comprising: RNA encoding an immune inhibitory protein of the IL-1 family, or functional variant thereof, according to the present disclosure, and a cationic or cationically ionizable lipid (such as DOTMA) and neutral lipid (such as DOPE) in a molar ratio of from about 3:1 to about 1:1 (optionally about 2:1), optionally wherein the ratio of positive charges to negative charges in the composition is from about 1:2 to about 1.9:2 (optionally about 1.3:2.0). In some embodiments , the composition comprises: RNA lipoplex particles comprising: RNA encoding an immune inhibitory protein of the IL-1 family, or functional variant thereof, according to the present disclosure, and DOTMA and DOPE in a molar ratio of about 2:1, wherein the ratio of positive charges to negative charges in the composition is about 1.3:2.0; sodium chloride at a concentration of about 8.2 mM; sucrose at a concentration of about 13% (w/v); HEPES at a concentration of about 5 mM with a pH of about 6.7, and EDTA at a concentration of about 2.5 mM. In one embodiment, the RNA lipoplex particle according to the present disclosure comprises: a RNA molecule encoding an immune inhibitory protein of the IL-1 family, or functional variant thereof, according to the present disclosure; and at least one cationic or cationically ionizable lipid and at least one neutral lipid; wherein the charge ratio of positive charges to negative charges in the RNA lipoplex particles is between 1:1 and 1:8. In one embodiment, there is provided a composition (such as a pharmaceutical composition) comprising RNA lipoplex particles comprising: a RNA molecule encoding an immune inhibitory protein of the IL-1 family, or functional variant thereof, according to the present disclosure; and at least one cationic or cationically ionizable lipid and at least one neutral lipid; wherein the charge ratio of positive charges to negative charges in the RNA lipoplex particles is between 1:1 and 1:8. In one embodiment, the lipid particle according to the present disclosure or composition according to the present disclosure, such as pharmaceutical composition according to the present disclosure for use in therapy comprises: a RNA molecule encoding an immune inhibitory protein of the IL-1 family, or functional variant thereof, according to the present disclosure; and at least one cationic or cationically ionizable lipid and at least one neutral lipid; wherein the charge ratio of positive charges to negative charges in the RNA lipoplex particles is between 1:1 and 1:8. Suitably, the charge ratio of positive charges to negative charges in the RNA lipoplex particles may be between 1:1 and 1:4. Suitably, the charge ratio of positive charges to negative charges in the RNA lipoplex particles may be from about 1:2 to about 1.9:2, or about 1.3:2.0. Suitably, the lipoplex particles may comprise DOTMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2. Suitably, the lipoplex particles may comprise DOTMA and DOPE in a molar ratio of from about 10:0 to 1:9, from about 4:1 to 1:2, from about 3:1 to about 1:1, or about 2:1; optionally wherein the ratio of positive charges to negative charges in the composition is from about 1:2 to about 1.9:2, or about 1.3:2.0. Suitably, the RNA lipoplex particles may have an average diameter that ranges from about 200 to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In other embodiments, the lipoplexes are RNA lipoplex particles according to WO 2020/069632 A1 comprising RNA, and at least one cationic or cationically ionizable lipid and at least one additional lipid, sodium chloride at a concentration of about 10 mM or less, a stabilizer at a concentration of more than about 10% weight by volume percent (% w/v) and less than about 15% weight by volume percent (% w/v), and a buffer. Preferably the lipoplexes according to the present disclosure are RNA lipoplex particles comprising DOTMA and DOPE in a molar ratio of about 2:1, wherein the ratio of positive charges to negative charges in the composition is about 1.3:2.0, sodium chloride at a concentration of about 8.2 mM, sucrose at a concentration of about 13% (w/v), HEPES at a concentration of about 5 mM with a pH of about 6.7, and EDTA at a concentration of about 2.5 mM, as described in WO 2020/069632 A1. Lipid Nanoparticles (LNPs) In some embodiments, also provided is a composition comprising nanoparticles which comprise at least one cationic or cationically ionizable lipid, and a polynucleotide or RNA molecule encoding an immune inhibitory protein of the IL-1 family or functional variant thereof, of the disclosure, wherein the nanoparticles are lipid nanoparticles (LNPs). The LNP may comprise any lipid capable of forming a particle to which the nucleic acid molecules are attached, or in which the nucleic acid molecules are encapsulated. In one embodiment, the LNP comprises a cationic or cationically ionizable lipid, a neutral lipid, a steroid, a polymer conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle. In some embodiments, a particle described herein is a lipid nanoparticle (LNP). LNPs have emerged as particularly useful vehicles for delivery of nucleic acids, for example as described in Theranostics, 2022 Oct 24;12(17):7509-7531. It is understood that a LNP is structurally distinct from other nanoparticles previously used for nucleic acid delivery, such as a liposome, or a lipoplex. LNPs, as described herein, typically do not comprise a bilayer (uni-lamellar), or a concentric series of multiple bilayers (multi-lamellar) separated by aqueous compartments, enclosing a central aqueous compartment. Moreover, LNPs, as described herein, typically do not comprise a central aqueous core or compartment. LNPs as described herein typically comprise nucleic acids (e.g., DNA or RNA such as mRNA) and lipids forming a disordered, non-lamellar phase. LNPs as described herein may be considered as oil-in-water emulsions in which the LNP core materials are preferably in liquid state and hence have a melting point below body temperature. See, e.g., ACS Nano 2021, 15, 11, 16982–17015; Aldosari, et al., Pharmaceutics, 2021, 13, 206. In some embodiments, the LNPs further comprise a targeting moiety, such as an antibody or a fragment thereof. The targeting moiety may be a protein, an antibody, an scFv, a nanobody or a VHH. The targeting moiety may be conjugated or otherwise bound to a lipid comprised within the LNP. The targeting moiety may be displayed on the surface of the LNP. The targeting moiety may be capable of binding (i.e., specifically binding) to a cell surface antigen expressed by a cell type of interest (i.e., a protein displayed on the cell surface of a target cell type). The cell type of interest may be an immune cell. The cell type of interest may be a neutrophil, eosinophil, basophil, mast cell, monocyte, macrophage, dendritic cell, natural killer cell, T lymphocyte, cytotoxic T cell, regulatory T cell, or B lymphocyte. Preferably the cell type of interest is a T cell, a B cell, a monocyte or a macrophage. Without being bound by theory the LNPs comprising the targeting moiety may be preferentially delivered to the cell type of interest in the subject to which the LNPs are delivered. LNPs described herein generally comprise four categories of lipids in addition to a polynucleotide of the disclosure (e.g., DNA or RNA such as mRNA): a cationic or cationically ionizable lipid (typically a cationically ionizable lipid), a polymer-conjugated lipid, a helper lipid, and a steroid. A person of skill in the art will understand that various combinations of these four categories of lipids can be used to prepare lipid nanoparticles for use in delivering nucleic acid agents. (i) Cationic or cationically ionizable lipids As described generally herein, a nucleic acid particle comprises a nucleic acid and a cationic or a cationically ionizable lipid. In some embodiments, a cationic or cationically ionizable lipid useful for incorporation into a nucleic acid particle are those lipids having a polar head group and an aliphatic tail. In some embodiments, a cationic lipid is one where the polar head group has a permanently positive charge (for example, comprising a quaternary ammonium group). In some embodiments, a cationically ionizable lipid is a lipid wherein, at a given pH and in the context of an LNP, the lipid becomes positively charged, such as at below physiological pH (e.g., below pH about 7.4) or neutral pH (e.g., a pH around 7 to 7.5), or in some embodiments, at a pH of less than 7 (e.g., less than 6). In some embodiments, a cationically ionizable lipid is one comprising polar head group that comprises one or more a tertiary amine groups (or secondary or primary amine group) that can become positively charged. LNPs typically comprise cationically ionizable lipids. In some embodiments, a lipid nanoparticle comprises about 30 mol% to about 60 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 35 mol% to about 55 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 40 mol% to about 50 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 50 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises 47.5 mol% of a cationic or cationically ionizable lipid. Suitable cationic or cationically ionizable lipids are readily identified by those of skill in the art. In some embodiments, a cationic lipid or cationically ionizable lipid is one provided in WO 2010/144740 or WO 2012/016184, which are incorporated herein by reference in their entirety. For example, in some embodiments, a cationic lipid is selected from N-(2,3-dioleyloxypropyl)- N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(2,3-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride (DOTAP), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); 3-(N-(N′,N′dimethylaminoethane)- carbamoyl)cholesterol (DC-Chol), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide (DMRIE). In some embodiments, a cationically ionizable lipid is selected from 1,2-dioleoyl-3-dimethylammonium propane (DODAP); N,N-dimethyl- (2,3-dioleoyloxypropyl)amine (DODMA); and 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1- (9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (DLin-MC3-DMA). In some embodiments, a cationically ionizable lipid is a lipid described in WO 2017/075531 or WO 2018/081480, each of which is incorporated by reference herein in its entirety. In some embodiments, a cationically ionizable lipid is a lipid represented by formula CL-I: CL-I or a pharmaceutically acceptable salt thereof, wherein, as applied to formula CL-I: one of L1 or L2 is -O(C═O)-, -(C═O)O-, -C(═O)-, -O-, -S(O)x-, -S-S-, -C(═O)S-, -SC(═O)-, -NRaC(═O)-, -C(═O)NRa-, -NRaC(═O)NRa-, -OC(═O)NRa- or -NRaC(═O)O-, and the other of L1 or L2 is - O(C═O)-, -(C═O)O-, -C(═O)-, -O-, -S(O)x-, -S-S-, -C(═O)S-, -SC(═O)-, -NRaC(═O)-, - C(═O)NRa-, -NRaC(═O)NRa-, -OC(═O)NRa- or -NRaC(═O)O- or a direct bond; G1 and G2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; Ra is H or C1-C12 alkyl; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is H, OR5, CN, -C(═O)OR4, -OC(═O)R4 or -NR5C(═O)R4; R4 is C1-C12 alkyl; R5 is H or C1-C6 alkyl; and x is 0, 1 or 2. In some embodiments, a cationically ionizable lipid is ((4-hydroxybutyl)azanediyl)bis(hexane- 6,1-diyl) bis(2-hexyldecanoate) (ALC-0315) or ((3-hydroxypropyl)azanediyl)bis(nonane-9,1- diyl) bis(2-butyloctanoate) (ALC-366): ALC-0315 ALC-0366 In some embodiments, a lipid nanoparticle comprises about 40 mol% to about 50 mol% of a cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mol% of a cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises 47.5 mol% of a cationically ionizable lipid. In some embodiments, a cationic lipid is one described in WO 2017/049245, which is incorporated by reference in its entirety. In some embodiments, a cationic lipid is represented by formula CL-II or a pharmaceutically acceptable salt thereof, wherein, as applied to formula CL-II: R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR″, -YR″, and -R″M′R′; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, - R*YR″, -YR″, and -R*OR″, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQR2, and unsubstituted C1-6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH2)nNR2, -C(O)OR, OC(O)R, -CX3, -CX2H, -CXH2, - CN, -NR2, -C(O)NR2, -NRC(O)R, -NRS(O)2R, -NRC(O)NR2, -NRC(S)NR2, -NRR8, - O(CH2)nOR, -NRC(═NR9)NR2, -NRC(═CHR9)NR2, -OC(O)NR2, -NRC(O)OR, -N(OR)C(O)R, - N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)NR2, -N(OR)C(S)NR2, N(OR)C(═NR9)NR2, - N(OR)C(═CHR9)NR2, -C(═NR9)NR2, -C(═NR9)R, -C(O)NROR, and -CRNR2C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M′ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, - CH(OH)-, -P(O)(OR′)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycle and heterocycle; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2NR2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R′ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, - R*YR″, -YR″, and H; each R″ is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. In some embodiments, a cationically ionizable lipid is heptadecan-9-yl 8-{(2-hydroxyethyl)[6- oxo-6-(undecyloxy)hexyl]amino}octanoate) (SM-102): SM-102 In some embodiments, a cationically ionizable lipid is a lipid described in WO 2015/095340, which is incorporated by reference herein in its entirety. In some embodiments, a cationic lipid is represented by formula CL-III or a pharmaceutically acceptable salt thereof, wherein, as related to formula CL-III: n and p are each, independently, 0, 1 or 2; L1 is -OC(O)-, -C(O)O- or a bond; R1 is heterocyclyl, heterocyclyl-C1-8-alkyl or heterocyclyl-C1-8-alkoxyl, each of which may be optionally substituted with 1, 2, 3, 4 or 5 groups, independently selected from halogen, formidamidine, C1-8-alkyl, C3- 7-cycloalkyl, C3-7-cycloalkyl-C1-8-alkyl, heterocyclyl, -[(C1-C4)alkylene]v-N(R′)R″, -O-[(C1- C4)alkylene]v-N(R′)R″ or -N(H)-[(C1-C4)alkylene]v-N(R′)R″, where said (C1-C4)alkylene is optionally substituted with one or more R groups; v is 0, 1, 2, 3 or 4; R is hydrogen or -C1-8- alkyl or when v is 0 R is absent; R′ and R″, are each, independently, hydrogen, -C1-8-alkyl; or R′ and R″ combine with the nitrogen to which they are bound, and optionally including another heteroatom selected from N, O and S, to form a 5-8 membered heterocycle or heteroaryl, optionally substituted with an -C1-8-alkyl, hydroxy or cycloalkyl-C1-8-; R2 and R3 are each, independently, C7-22 alkyl, C12-22 alkenyl, C3-8 cycloalkyl optionally substituted with 1, 2, or 3 C1-8 alkyl groups, , R4 is selected from hydrogen, C1-14 alkyl, . In some embodiments, a cationically ionizable lipid is represented by In some embodiments, a cationic lipid is one described in WO 2018/087753, which is incorporated herein by reference in its entirety. In some embodiments, a cationic lipid is represented by formula CL-IV: or a pharmaceutically acceptable salt thereof, wherein, as applied for formula CL-IV: Y is O or NH; T is C or S; W is a bond, O, NH or S; R1 is selected from the group consisting of: (a) NR4R5, wherein R4 and R5 are each independently a C1-C4 alkyl; or R4 and R5 together with the nitrogen to which they are attached form a 5 or 6 membered heterocyclic or heteroaromatic ring, optionally containing one or more additional heteroatoms selected from the group consisting of O, N and S; or NR4R5 represent a guanidine group (-NHC(═NH)NH2); (b) the side chain of a natural or unnatural amino acid; and (c) a 5 or 6 membered heterocyclic or heteroaromatic ring containing one or more heteroatoms selected from the group consisting of O, N and S; R2 and R3 are selected from the group consisting of: (a) C10-C22 alkyl; (b) C10- C22 alkenyl; (c) C10-C22 alkynyl; (d) C4-C10 alkylene-Z-C4-C22 alkyl; and (e) C4-C10 alkylene-Z- C4-C22 alkenyl; Z is -O-C(═O)-, -C(═O)-O- or -O-; n is 0, 1, 2, 3, 4, 5 or 6; m is 0 or 1; p is 0 or 1; and z is 0 or 2. In some embodiments, a cationically ionizable lipid is selected from: In some embodiments, a cationically ionizable lipid is one described in WO 2022/081750, which is incorporated herein by reference in its entirety. In some embodiments, a cationically ionizable lipid is represented by formula CL-V-1: or a pharmaceutically acceptable salt thereof, wherein, as applied to formula CL-V-1: each R1 and each R2 is independently selected from the group consisting of H, an optionally substituted C1-C22 alkyl, optionally substituted C2-C22 alkenyl, optionally substituted C2- C22 alkynyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C4- C6 heterocycloalkyl, optionally substituted C4-C6 alkylcycloalkyl, optionally substituted C4-C6 aryl, optionally substituted C3-C6 heteroaryl, optionally substituted C4-C8 aryloxy, optionally substituted C7-C10 arylalkyl, optionally substituted C5-C10 heteroaryl alkyl group, optionally substituted amine; or R1 and R2 can together form a 3-7 membered heterocycloalkyl or heteroaryl ring; each R3, R4, R13 and R14 is independently selected from the group consisting of an optionally substituted C1-C22 alkyl, optionally substituted C2-C22 alkenyl, optionally substituted C2-C22 alkynyl; each R5, R6, R7, R8, R9, R10, R15, and R16 is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C1-C22 alkyl, optionally substituted C2-C22 alkenyl, optionally substituted C2-C22 alkynyl; each of w, x, y, and z is independently an integer from 0-10; each Q is independently an atom selected from O, NH, NR1, and S; each of m is an integer from 0 to 8, preferably 0, 1, or 2; and each of L1 and L2 is independently selected from the group consisting of -C(=O)-; -OC(=O)-; -OC(=O)O-; - C(=O)O-; -C(=O)O(CR5R6R7)-; -NH-C(=O)-; -C(=O)NH-; -SO-; - SO2-; -SO3-; -NSO2-; -SO2N- ; -NH((C1-C8)alkyl)-; -N((C1-C8)alkyl)2-; -NH((C6)aryl)-; -N((C6)aryl)2-; -NHC(=O)NH-; - NHC(=O)O-; -OC(=O)NH-; -NHC(=O)NR1-; -NHC(=O)O-; -OC(=O)NR1-; -C(=O)R1-; -CO((C1- C8)alkyl)-; -CO((C6)aryl)-; -CO2((C1-C8)alkyl)-; - CO2((C6)aryl)-; -SO2((C1-C8)alkyl)-; and - SO2((C6)aryl)-. In some embodiments, a cationically ionizable lipid is represented by formula CL-V-2: or a pharmaceutically acceptable salt thereof, wherein, as applied to CL-V-2: each R1’, R1, R2, R11, and R12 is independently selected from the group consisting of H, an optionally substituted C1-C22 alkyl, optionally substituted C2-C22 alkenyl, optionally substituted C2-C22 alkynyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C4- C6 heterocycloalkyl, optionally substituted C4-C6 alkylcycloalkyl, optionally substituted C4-C6 aryl, optionally substituted C3-C6 heteroaryl, optionally substituted C4-C8 aryloxy, optionally substituted C7-C10 arylalkyl, optionally substituted C5-C10 heteroarylalkyl group, optionally substituted amine; or R1 and R2 can together form cycloalkyl or heterocycloalkyl ring; if Q is S or O the R1 attached to the S or O is an electron pair; each R3 and R4 is independently selected from the group consisting of an optionally substituted C1-C22 alkyl, optionally substituted C2- C22 alkenyl, optionally substituted C2-C22 alkynyl; each R5, R6, R7, R8, R9, and R10 is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C1-C22 alkyl, optionally substituted C2-C22 alkenyl, optionally substituted C2- C22 alkynyl; each of x, y, and z is independently an integer from 0-10; G and Q are each independently an atom selected from CH, O, N, and S; each of m and n is an integer from 0- 8; and each of L1 and L2 is independently selected from the group consisting of -C(=O)-; - OC(=O)-; -OC(=O)O-; -C(=O)O-; -C(=O)O(CR1R2R3)-; -NH-C(=O)-; -C(=O)NH-; -SO-; -SO2-; - SO3-; -NSO2-; -SO2N-; -NH((C1-C8)alkyl)-; -N((C1-C8)alkyl)2-; -NH((C6)aryl)-; -N((C6)aryl)2-; - NHC(=O)NH-; -NHC(=O)O-; -OC(=O)NH-; -NHC(=O)NR1-; -NHC(=O)O-; -OC(=O)NR1-; - C(=O)R1-; -CO((C1-C8)alkyl)-; -CO((C6)aryl)-; -CO2((C1-C8)alkyl)-; -CO2((C6)aryl)-; -SO2((C1- C8)alkyl)-; and -SO2((C6)aryl)-. In some embodiments, a cationically ionizable lipid is selected from: di(heptadecan-9-yl) 3,3’- ((2-(4-methylpiperazin-1-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); bis(2- octyldodecyl) 3,3’-((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2- 1Me-Pyr); bis(2-octyldodecyl) 3,3’-((2-(pyrrolidin-1-yl)ethyl)azanediyl)dipropionate (BODD- C2C2-Pyr); bis(2-octyldodecyl) 3,3’-(((1-methylpiperidin-3-yl)methyl)azanediyl)dipropionate (BODD-C2C2-1Me-3PipD); bis(2-octyldodecyl) 3,3’-((2- (dimethylamino)ethyl)azanediyl)dipropionate (BODD-C2C2-DMA); bis(2-octyldodecyl) 3,3’- ((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BODD-C2C4-PipZ); bis(2- octyldodecyl) 3,3’-((4-(pyrrolidin-1-yl)butyl)azanediyl)dipropionate (BODD-C2C4-Pyr); and bis(2-hexyldecyl) 3,3’-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BHD-C2C4- PipZ). In some embodiments, a lipid nanoparticle (LNP) comprises a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ, BODD-C2C2-1Me-Pyr, ALC- 0315, ALC-366, SM-102, HY-501, EA-405, HY-405, DODMA, and Dlin-MC3-DMA. In some embodiments, a LNP comprises a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ, BODD-C2C2-1Me-Pyr, ALC-0315, SM-102, HY-501, and DODMA. In some embodiments, a LNP comprises about 40 mol% to about 50 mol% (e.g., about 47.5 mol%) (relative to the total amount of lipids in a LNP) of a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ; BODD-C2C2-1Me-Pyr; ALC-0315; ALC-0366; SM-102; HY-501; EA-405; HY-405; DODMA; and Dlin-MC3-DMA. In some embodiments, a LNP comprises about 40 mol% to about 50 mol% (e.g., about 47.5 mol%) (relative to the total amount of lipids in a LNP) of a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ; BODD-C2C2-1Me-Pyr; ALC-315, SM- 102; HY-501; and DODMA. (ii) Helper lipids As described herein, lipid nanoparticles of the present disclosure comprise a helper lipid. In some embodiments, a helper lipid is or comprises phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. In some embodiments, a helper lipid is a phospholipid. In some embodiments, a helper lipid is or comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), sphingomyelins, N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), 1,2-diacylglyceryl-3-O-4’-(N,N,N-trimethyl)-homoserine (DGTS), ceramides, and their derivatives. In some embodiments, a helper lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DSPE, and SM. In some embodiments, the helper lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the helper lipid is DSPC. Helper lipids may be synthetic or naturally derived. Other helper lipids suitable for use in a lipid nanoparticle are described in WO 2021/026358, WO 2017/075531, and WO 2018/081480, the entire contents of each of which are incorporated herein by reference. In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of a helper lipid. In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 8 to about 12 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 10 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of DSPC. In some embodiments, a lipid nanoparticle comprises about 8 to about 12 mol% of DSPC. In some embodiments, a lipid nanoparticle comprises about 10 mol% of DSPC. (iii) Polymer-conjugated lipids As described herein, LNPs of the present disclosure comprise a polymer-conjugated lipid. In some embodiments, a polymer-conjugated lipid is a lipid conjugated to polyethylene glycol (a “PEG-lipid”). In some embodiments, a PEG-lipid is selected from pegylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG- DMG) (e.g., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000- DMG)), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-1-O-(ω- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000 amine), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as ω- methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate, and 2,3- di(tetradecanoxy)propyl-Ν-(ω-methoxy(polyethoxy)ethyl)carbamate. In some embodiments, a PEG group that is part of a PEG-lipid has, on average in a composition comprising one or more PEG-lipid molecules, a number average molecular weight (Mn) of about 2000 g/mol. In some embodiments, a PEG-lipid is DMG-PEG. In some embodiments, a PEG-lipid is PEG2000-DMG: In some embodiments, a PEG-lipid is provided in WO 2021/026358, WO 2017/075531, or WO 2018/081480, each of which is incorporated by reference in its entirety. In some embodiments, a PEG-lipid is a compound of Formula PCL-I: or a pharmaceutically acceptable salt thereof, wherein, as applied to formula PGL-I, R8 and R9 are each independently C10-C30 aliphatic, optionally interrupted by one or more ester bonds, and w is an integer from 30 to 60. In some embodiments, a compound of Formula PCL-I is 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159). In some embodiments, a compound of Formula PCL-I is: or a pharmaceutically acceptable salt thereof, where n’ is an integer from about 45 to about 50. In some embodiments, the PEG-lipid is represented by: wherein n has a mean value ranging from 30 to 60. In some embodiments, n is 50. In one embodiment, the PEG-conjugated lipid (pegylated lipid) is PEG2000-C-DMA which preferably refers to 3-N-[(ω-methoxy poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy- propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy) propylcarbamate (2000). In some embodiments, a PEG-lipid is selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG2000-DMG, ALC-159, PEG2000-C-DMA PEG-S-DMG, PEG-cer, and combinations thereof. In some embodiments, a PEG-lipid is ALC-0159 or PEG2000-DMG. In some embodiments, a PEG-lipid is ALC-0159. In some embodiments, a PEG-lipid is PEG2000- DMG. In some embodiments, a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid. The term “sarcosinylated lipid” refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N-methylglycine)) portion. In some embodiments, a polymer-conjugated lipid is one described in WO 2024/028325, which is incorporated herein by reference in its entirety. In some embodiments, a polymer- conjugated lipid is represented by formula PCL-II: or a pharmaceutically acceptable salt thereof, wherein, as applied to formula PCL-II: X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester; Y is -CH2-, -(CH2)2-, or –(CH2)3-; z is 2 to 24; and n is 1 to 100. In some embodiments of formula PCL-II: (i) when X1 is -C(O)- then X2 is -NR1-; (ii) when X1 is -NR1- then X2 is -C(O)- ; (iii) when X1 is -C(S)- then X2 is -NR1-; (iv) when X1 is -NR1- then X2 is -C(S)-; (v) when X1 is -C(O)- then X2 is -O-; (vi) when X1 is -O- then X2 is -C(O)-; (vii) when X1 is -C(S)- then X2 is - O-; (viii) when X1 is -O- then X2 is -C(S)-; (ix) when X1 is -C(O)- then X2 is -S-; or (x) when X1 is -S- then X2 is -C(O)-; wherein R1 is hydrogen or C1-8 alkyl. In some embodiments of formula PCL-II: (i) when X1 is -C(O)- then X2 is -NR1-; (ii) when X1 is -NR1- then X2 is -C(O)-; (iii) when X1 is -C(S)- then X2 is -NR1-; (iv) when X1 is -NR1- then X2 is -C(S)-; (v) when X1 is -C(O)- then X2 is -O-; or (vi) when X1 is -O- then X2 is -C(O)-; wherein R1 is hydrogen or C1-8 alkyl. In some embodiments, a polymer-conjugated lipid comprises monomers of 2-(2-(2- aminoethoxy)ethoxy)acetic acid. In some embodiments, the polymer of the polymer- conjugated lipid is or comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly- 2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, a polymer-conjugated lipid comprises monomers of unit PCL-II-1: II-1 In some embodiments, a polymer-conjugated lipid comprises, 5 to 50, 5 to 25 or 10 to 25 monomers of PCL-II-1. In some embodiments, a polymer-conjugated lipid comprises 14 to 17 monomers of PCL-II-1. In some embodiments, a polymer-conjugated lipid comprises 8 to 14 monomers of PCL-II-1. In some embodiments, a polymer-conjugated lipid is selected from the table below: In some embodiments, an LNP comprises an polysarcosine-conjugated or a pAEEA/pMAEEA-conjugated lipid, as described herein. In some embodiments, nucleic acid particles (e.g., DNA or RNA particles) described herein comprise a polysarcosine-conjugated or a pAEEA/pMAEEA-conjugated lipid and are substantially free of a pegylated lipid (or do not contain a pegylated lipid). In some embodiments, a lipid nanoparticle comprises about 0.5 to about 5.0 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.0 to about 2.5 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.5 to about 2.0 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.5 to about 1.8 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.5 mol% to about 1.8 mol% (relative to the total amount of lipids in a lipid nanoparticle) of a polymer-conjugated lipid selected from the group consisting of: DSPE-AEEA14-AC; VE-AEEA14-AC; ALC-0159 and PEG2000-DMG. In some embodiments, a lipid nanoparticle comprises about 1.5 mol% to about 1.8 mol% (relative to the total amount of lipids in a lipid nanoparticle) of a polymer-conjugated lipid selected from the group consisting of: DSPE-AEEA14-AC, VE-AEEA14-AC, and PEG2000- DMG. In some embodiments, a molar ratio of a cationic or cationically ionizable lipid to a polymer-conjugated lipid is from about 2:1 to about 8:1. (iv) Steroids As described generally herein, lipid nanoparticles further comprise a steroid. In some embodiments, a steroid is a sterol. In some embodiments, a sterol is β-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or α-tocopherol. In some embodiments, a sterol is cholesterol. In some embodiments, a lipid nanoparticle comprises about 39 to about 49 mol% of a steroid. In some embodiments, a lipid nanoparticle comprises about 40 to about 46 mol% of a steroid. In some embodiments, a lipid nanoparticle comprises about 40 to about 44 mol% of a steroid. In some embodiments, a lipid nanoparticle comprises: about 30 to about 60 mol% of a cationically ionizable lipid; about 18.5 to about 48.5 mol% of a steroid (e.g., cholesterol); about 0 to about 30 mol% of a helper lipid (e.g., DSPC); and about 0 to about 10 mol% of a polymer- conjugated lipid. In some embodiments, a lipid nanoparticle comprises: about 35 to about 55 mol% of a cationically ionizable lipid; about 30 to about 40 mol% of a steroid (e.g., cholesterol); about 5 to about 25 mol% of a helper lipid (e.g., DSPC); and about 0 to about 10 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises: about 40 to about 50 mol% of a cationically ionizable lipid; about 30 to about 45 mol% of a steroid (e.g., cholesterol); about 5 to about 15 mol% of a helper lipid (e.g., DSPC); and about 1 to about 2.5 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises: 47.5 mol% di(heptadecan-9-yl) 3,3’-((2- (4-methylpiperazin-1-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% VE-AEEA14-AC. In some embodiments, a lipid nanoparticle comprises: 47.5 mol% di(heptadecan-9-yl) 3,3’-((2-(4-methylpiperazin-1- yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% PEG2000-DMG. In some embodiments, a lipid nanoparticle comprises: about 47.5 mol% of ALC-0315; about 10 mol% of DSPC; about 40.7 mol% of cholesterol; and about 1.8 mol% of ALC-159. In some embodiments, a lipid nanoparticle comprises: about 47.5 mol% of ALC-366; about 10 mol% of DSPC; about 40.7 mol% of cholesterol; and about 1.8 mol% of ALC-159. In some embodiments, a lipid nanoparticle comprises about 50 mol% of SM-102; about 1.5 mol% of PEG2000-DMG; about 10 mol% of DSPC; and about 38.5 mol% of cholesterol. In some embodiments, a lipid nanoparticle comprises: 47.5 mol% bis(2- octyldodecyl) 3,3’-((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2- 1Me-Pyr); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% VE-AEEA14-AC. In some embodiments, a lipid nanoparticle comprises: 47.5 mol% bis(2-octyldodecyl) 3,3’-((2-(1- methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1Me-Pyr); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% PEG2000-DMG. (v) Manufacturing Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U.S. Patent Publication Nos.2016/0009637, 2015/0273068, 2014/0200257, 2013/0338210, 2013/0245107, 2013/0123338, 2013/0017223, 2012/0183581, 2012/0027803, 2011/0311583, 2011/0216622, 2011/0117125, 2007/0042031, 2006/0083780, 2005/017054, 2004/0142025, 2007/0042031, 1999/009076 and PCT Pub. Nos. WO 99/39741, WO 2018/081480, WO 2017/004143, WO 2017/075531, WO 2015/199952, WO 2013/086322, WO 2013/016058, WO 2013/086373, WO 2011/141705, WO 2022/016089, WO 2022/081752, the full disclosures of which are herein incorporated by reference in their entirety for the purposes described herein. For example, in some embodiments, cationically ionizable lipids, helper lipids, and steroids are solubilized in an organic solvent such as ethanol, at a predetermined weight or molar ratios/percentages (e.g., ones described herein). In some embodiments, lipid nanoparticles are prepared at a total lipid to nucleic acid (e.g., RNA) weight ratio of approximately 10:1 to 50:1. In some embodiments, such nucleic acid (e.g., RNA) can be diluted to 0.1 to 1.0 mg/mL (e.g., 0.4 mg/mL) in an acidic buffer, such as citrate or acetate having a pH of between about 4 to about 6. In some embodiments, using an ethanol injection technique, a colloidal lipid dispersion comprising nucleic acids (e.g., RNAs) can be formed as follows: an ethanol solution comprising lipids, such as cationic lipids, helper lipids, steroids, and polymer-conjugated lipids, is combined with, e.g., injected into or continuously mixed with, an aqueous solution comprising nucleic acids. In some embodiments, lipid and nucleic acid (e.g., RNA) solutions can be mixed at room temperature by pumping each solution (e.g., a lipid solution comprising a cationic lipid, a helper lipid, cholesterol, a conjugated lipid, and any other additives) at controlled flow rates into a mixing unit, for example, using piston pumps. In some embodiments, the flow rates of a lipid solution and a nucleic acid (e.g., RNA) solution into a mixing unit are maintained at a ratio of 1:3. Upon mixing, nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous nucleic acids (e.g., RNAs). The lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged nucleic acid (e.g., RNA). In some embodiments, a solution comprising nucleic acid (e.g., RNA)-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and/or filtration. Pharmaceutical composition In one aspect, the invention provides a pharmaceutical composition comprising the polynucleotide, vector, or nucleic acid particle of the invention. In a further aspect, the invention provides a pharmaceutical composition comprising the polynucleotide according to the invention. In an embodiment, the pharmaceutical composition comprises one or more polynucleotides of the invention which encode different immune inhibitory proteins of the IL-1 family. For example, in an embodiment the pharmaceutical composition comprises a first polynucleotide sequence which encodes a first immune inhibitory protein of the IL-1 family as described herein and a second polynucleotide which encodes a second immune inhibitory protein of the IL-1 family as described herein, optionally as well as a third polynucleotide which encodes a third immune inhibitory protein of the IL-1 family as described herein. Suitably, the pharmaceutical composition comprises polynucleotides encoding IL-18BP and IL-37. The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion or intramuscular administration. The pharmaceutical composition may be in a form not suitable for intratumoral administration. Therapeutic use In a further aspect, the invention provides the polynucleotide according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention for use as an immune inhibitory medicament. In a further aspect, the invention provides the use of provides the polynucleotide according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention for the manufacture of an immune inhibitory medicament. Suitably, the term “immune inhibitory medicament” refers to a medicament that is capable of reducing and/or inhibiting immune responses. Suitably, the immune inhibitory medicament is an anti-inflammatory medicament. Accordingly, in a further aspect, the invention provides the polynucleotide according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention for use as an anti- inflammatory medicament. In a further aspect, the invention provides the use of provides the polynucleotide according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention for the manufacture of an anti-inflammatory medicament. Suitably, an anti-inflammatory medicament has anti-inflammatory antagonistic functions. Suitably, in the context of the present invention, the immune inhibitory (e.g. anti-inflammatory) medicament acts as an antagonist for at least one of the 7 pro-inflammatory cytokines of the IL-1 family (namely, IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL 36β and IL-36γ). In a further aspect, the invention provides the polynucleotide according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention for use in the prevention and/or treatment of an inflammatory disease. In a further aspect, the invention provides a method of preventing and/or treating an inflammatory disease in a subject, comprising administering the polynucleotide according to the invention, the vector according to the invention, the nucleic acid particle according to the invention, or the pharmaceutical composition according to the invention to the subject. Treating an inflammatory disease relates to the therapeutic use of the polynucleotide, vector, nucleic acid particle, or pharmaceutical composition according to the invention. In this respect, the polynucleotide, vector, nucleic acid particle, or pharmaceutical composition according to the invention may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease. Preventing an inflammatory disease relates to the prophylactic use of the polynucleotide, vector, nucleic acid particle, or pharmaceutical composition according to the invention. In this respect, the polynucleotide, vector, nucleic acid particle, or pharmaceutical composition according to the invention may be administered to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent development of at least one symptom associated with the disease. The subject may have a predisposition for, or be thought to be at risk of developing, the disease. Suitably, the vector(s) or nucleic acids may be introduced by transduction. Suitably, the vector(s) or nucleic acids may be introduced by transfection. Suitably, the therapeutic and/or prophylactic uses described herein do not comprise intratumoral administration of the polynucleotide, vector, nucleic acid particle, or pharmaceutical composition according to the invention. Thus, in some embodiments, the polynucleotide, vector, nucleic acid particle, or pharmaceutical composition according to the invention is not administrated intratumorally. The disease or condition to be treated and/or prevented may be cancer, a cardiovascular disease, atherosclerosis, Alzheimer's disease, an autoimmune inflammatory disease, a liver disease, or an inflammatory skin condition. Preferably, the disease is atherosclerosis or a cardiovascular disease. In some embodiments, the cardiovascular disease is heart failure or pericarditis. In some embodiments, the autoimmune inflammatory disease is multiple sclerosis or rheumatoid arthritis. In some embodiments, the inflammatory skin condition is psoriasis. In some embodiments, the liver disease is non-alcoholic fatty liver disease. In some embodiments, the subject is a mammalian subject. In one embodiment, the subject is a human. General definitions This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino (N) to carboxy (C) orientation, respectively. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure. The term “polypeptide” is used in the conventional sense to mean a series of amino acids, typically L-amino acids, connected one to the other, typically by peptide bonds between the α- amino and carboxyl groups of adjacent amino acids. The term “polypeptide” is used interchangeably with the terms “amino acid sequence”, “peptide” and/or “protein”. The term “residues” is used to refer to amino acids in an amino acid sequence. The term “variant” in relation to a polypeptide refers to a polypeptide that has an equivalent function to the amino acid sequences described herein, but which includes one or more amino acid substitutions, insertions or deletions. As used herein, the terms “polynucleotide”, “nucleotide”, “nucleic acid sequence” and “nucleic acid” are intended to be synonymous with each other. The terms “variant”, “homologue” or “derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence. In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to the immune inhibitory proteins described herein, one particular function is the anti-inflammatory antagonistic function displayed by the amino acid sequence from which the fragment or variant is derived and/or binding to the receptor(s) the amino acid sequence from which the fragment or variant is derived binds to. Suitably, a functional variant of an immune inhibitory (e.g. anti-inflammatory) protein of the IL-1 family acts as an antagonist for at least one of the 7 pro-inflammatory cytokines of the IL-1 family (namely, IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL 36β and IL-36γ). The term "functional fragment" or "functional variant", as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence as described herein. In one embodiment, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the binding characteristics of the molecule or sequence. In different embodiments, binding of the functional fragment or functional variant may be reduced but still significantly present, e.g ., binding of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, binding of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence. “Sequence identity” between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. The terms “% identical” and “% identity” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the global homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol.48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK _LOC=align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment. Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100. In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence. In some embodiments, “isolated” means removed (e.g., purified) from the natural state or from an artificial composition, such as a composition from a production process. For example, a nucleic acid, peptide or polypeptide naturally present in a living animal is not “isolated”, but the same nucleic acid, peptide or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated”. An isolated nucleic acid, peptide or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence. In the context of the present disclosure, the term “transcription” relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA may be translated into peptide or polypeptide. With respect to RNA, the term “expression” or “translation” relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide. The term “polynucleotide” comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid can be isolated. The term “isolated polynucleotide” means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis. The term “nucleoside” (abbreviated herein as “N”) relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine. The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively. However, thymidine is more commonly written as “dT” (“d” represents “deoxy”) as it contains a 2’-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG. A modified purine (A or G) or pyrimidine (C, T, or U) base moiety is preferably modified by one or more alkyl groups, more preferably one or more C1-4 alkyl groups, even more preferably one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N7-alkyl-guanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(1)- alkyl-uracil, such as N7-C1-4 alkyl-guanine, N6-C1-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5- C1-4 alkyl-uracil, and N(1)-C1-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl- adenine, 5-methyl-cytosine, 5-methyl-uracil, N1-methyl-pseudouridine, and N(1)-methyl- uracil. Herein, the term “DNA” relates to a nucleic acid molecule which includes deoxyribonucleotide residues. In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, “deoxyribonucleotide” refers to a nucleotide which lacks a hydroxyl group at the 2’-position of a β-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains “a majority of deoxyribonucleotide residues” if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof). DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA. The term “RNA” relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, “ribonucleotide” refers to a nucleotide with a hydroxyl group at the 2’-position of a β-D- ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered/modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered/modified nucleotides (i.e., altered/modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains “a majority of ribonucleotide residues” if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof). “RNA” includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self- amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, “RNA” refers to mRNA. It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms “comprising”, “comprises” and “comprised of” also include the term “consisting of’. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. EXAMPLES Example 1: Characterization of HEK-BlueTM reporter cell lines To study the IL-1 family the functionality of three HEK-BlueTM IL-1R family reporter cell lines was validated. These cell lines were then used to study cross-reactivities within the IL-1 subfamilies and the functionality of mRNA variants encoding the anti-inflammatory IL-1 family members. Receptor expression The surface expression of the IL-1 receptor family members (IL-1R1, IL-1RAP, IL-18R1, IL-18R2 and IL-36R) was quantified via multiparametric flow cytometry in the reporter cell lines HEK-Blue™ IL-1R (Figure 1), HEK-Blue™ IL-18 (Figure 2), and HEK-Blue™ IL-36 (Figure 3). The receptors described to be activated in each reporter cell line (such as IL-1R1 and IL-1RAP in HEK-BlueTM IL-1R cells) were overexpressed to varying degrees. HEK-BlueTM IL-1R cells exhibited high surface expression of IL-1R1 and IL-1RAP. High expression of IL-18R1 was observed in HEK-BlueTM IL-18 cells (Figure 2). In addition, high expression of IL-1RAP and IL-36R was detected in HEK-BlueTM IL-36R cells (Figure 3). Bioactivity assay The functionality of the reporter cell lines in detecting bioactive cytokines of the IL-1 family was investigated by monitoring the activation of the NFκB and AP-1 pathways and resulting in the production of SEAP. The HEK-BlueTM reporter cell lines were stimulated with a titration series of recombinant human IL-1α, IL-1β, IL-18, IL-36α, IL-36β and IL-36γ. The cytokine IFNγ was used as a negative control. After 24 h, the NFκB and AP-1 response was quantified using QUANTI- Blue™ solution, a SEAP detection reagent (Figure 4 and Table 1). Table 1: EC50 values of HEK-BlueTM IL-1R, IL-18 and IL-36 cells for the detection of bioactive IL1 cytokines in the SEAP assay. The HEK-BlueTM reporter cell lines responded to their respective cytokines with a sigmoidal dose-response curve (Figure 4A-C) and EC50 values ranging from 1.5 to 466.7 pg/ml (Table 1). The HEK-BlueTM IL-1R cells responded to IL-1α with an EC50 of 1.60 ± 0.69 pg/ml and to IL-1β with an EC50 of 19.91 ± 2.29 pg/ml. The HEK-BlueTM IL-18 cells reacted to IL-18 with an EC50 of 10.91 ± 3.67 pg/ml. Based on these data, the following cytokine concentrations were used for further experiments analyzing the antagonistic activity of anti-inflammatory mRNA family members: IL-1α 8 pg/ml, IL-18 20 pg/ml, and IL-36γ 500 pg/ml. The cytokine concentrations were chosen to be above the detected EC50 values of the respective stimulated HEK-BlueTM reporter cell lines. Summary The present data indicate that the pathways within the IL-1 family are highly specific and do not cross-react between subfamilies. The data also demonstrate the suitability of mRNA- encoded anti-inflammatory IL-1 family members. Example 2: Inhibitory activity of anti-inflammatory IL-1 family members Recombinant proteins The inhibitory effect of IL-1RA, IL-18BP, IL-36RA, IL-37, and IL-38 on IL-1 induced signaling in HEK-BlueTM reporter cell lines was quantified using recombinant versions of these anti- inflammatory members of the IL-1 family (Figure 5 and Table 2). Table 2: IC50 values of HEK-BlueTM IL-1R, IL-18 and IL-36 cells for the inhibitory effect of anti-inflammatory IL-1 cytokines in the SEAP assay. Cytokine-mediated stimulation of HEK-BlueTM reporter cell lines was inhibited in a dose- dependent manner by the respective anti-inflammatory members (Figure Figure 5A-C) with IC50 values ranging from 232.2 to 584.3 ng/ml (Table 2). Thus, agonists are nearly 1000 to 10000 times more active than the antagonists (Table 1 and Table 2). The IL-1α-mediated stimulation of HEK-BlueTM IL-1R cells was only inhibited by IL-1RA with an IC50 of 232.2 ± 72.95 ng/ml, whereas IL-18-mediated stimulation of HEK-BlueTM IL-18 cells was inhibited by IL-18BP (IC50 of 261.8 ± 149.24 ng/ml) or the combination of IL-18BP and IL-37 (IC50 of 386.5 ± 217.34 ng/ml), but not with IL-37 alone, showing no inhibitory activity. Similarly, IL-36γ stimulation of HEK-BlueTM IL-36 cells was only inhibited by IL-36RA with an IC50 of 584.3 ± 156.60 ng/ml. The inhibitory activity of the anti-inflammatory members of the IL-1 family was demonstrated by the bioactivity assay. No cross-reactivity between anti-inflammatory members of the IL-1 family and other IL-1R family members was observed. In this study, no inhibitory activity on either HEK-BlueTM IL-1R or HEK-BlueTM IL-36 cells was detected with two different recombinant IL-38 proteins. Suitability of anti-inflammatory mRNAs To test the bioactivity of mRNA-encoded anti-inflammatory members of the IL-1 family, their inhibitory activity was investigated in comparison to recombinant cytokines. IL-1RA and IL-18BP were selected to demonstrate the suitability of mRNA-encoded variants (Figure 6 and Table 3). Table 3: IC TM 50 values of HEK-Blue IL-1R and IL-18 cells for the inhibitory effect of mRNA-encoded versus purchased anti-inflammatory IL-1 cytokines in the SEAP assay. A dose-dependent inhibitory effect, comparable to the recombinant cytokines, by the mRNA-encoded cytokines was demonstrated, suggesting the bioactivity of mRNA-based anti-inflammatory members of the IL-1 family (Figures 6A and 6B). IL-1RA inhibited IL-1α- mediated stimulation of HEK-BlueTM IL-1R cells (Table 3). IL-18BP inhibited IL-18-mediated stimulation of HEK-BlueTM IL-18 cells.
Summary The bioactivity assay was used to demonstrate the inhibitory effect of the anti-inflammatory members of the IL-1 family. The data showed no cross-reactivity between the anti- inflammatory cytokines and other members of the IL-1R family. Cytokine-mediated stimulation of HEK-BlueTM cell lines was selectively inhibited only by the respective subfamily anti- inflammatory cytokine. Based on the data, no inhibitory effect of IL-38 on either HEK-BlueTM IL-36 or HEK-BlueTM IL-1R cells could be demonstrated. Both reporter cell lines were only inhibited by IL-36RA or IL-1RA. In addition, an inhibitory effect of IL-37 on IL-18 stimulated HEK-BlueTM IL-18 cells was not found. IL-37 forms a heterotrimeric complex with IL-18R1 and the co-receptor IL-1R8 instead of IL-18R2 and is therefore not a direct antagonist of IL-18. In vivo experiments indicate that the anti-inflammatory properties of IL-37 are mainly mediated by the co-receptor IL-1R8. Therefore, it is hypothesized that the co-receptor is not expressed by HEK-BlueTM IL-18 cells. Furthermore, the IL-18-mediated stimulation of HEK-BlueTM IL-18 cells was not inhibited to a greater extent when IL-37 was combined with IL-18 as compared to IL-18 alone. The suitability of mRNA-encoded variants of the anti-inflammatory IL-1 family members was demonstrated using IL-1RA and IL-18BP. The dose-dependent inhibitory effect of mRNA-encoded IL-1RA and IL-18BP was comparable to that of recombinant cytokines, indicating the bioactivity of the in house developed mRNA-based anti-inflammatory members of the IL-1 family. These results suggest that anti-inflammatory cytokines based on mRNA technology have potential to be therapeutically effective. In summary, the data indicate that the pathways within the IL-1 family are highly specific and do not cross-react between subfamilies. The suitability of mRNA-encoded anti-inflammatory IL-1 family members is also demonstrated. Example 3: Foam cell generation and analysis Atherosclerosis is a chronic, inflammatory disease of the arteries. One of the crucial steps is the differentiation of recruited monocytes into macrophages in the subendothelium. Macrophages are the most important immune cells in atherosclerotic plaques, when they appear as cholesterol-laden foam cells. To study mRNA-encoded anti-inflammatory IL-1 family members in the context of atherosclerosis and foam cell formation, a model of macrophage-mediated foam cell formation was established with human monocytes (THP-1 cells). Foam cell formation was detected by treating the cells with oxLDL or Dil-oxLDL using confocal microscopy, ORO staining and flow cytometry. Human THP-1 monocytes were differentiated into macrophages by incubation with 100 nM phorbol-12-myristate-13-acetate (PMA) for 72 h followed by 24 h wash in RPMI medium. Differentiated macrophages were treated with 100 µg/ml oxLDL for 24 h to form foam cells. To verify the formation of foam cells from THP-1 macrophages, the intracellular lipid accumulation after oxLDL treatment using the Oil Red O (ORO) staining method and the accumulation of lipids after 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate labeled oxLDL (Dil-oxLDL) treatment using confocal microscopy was measured. A significant dose-dependent lipid accumulation was observed using the ORO staining method and it was observed that Dil-oxLDL was taken up by THP-1 macrophages, indicating that the macrophages adopted a foam cell phenotype, and that the macrophage-mediated foam cell formation model was successful. To further characterize the foam cells and to investigate the role of the IL-1 family for atherosclerosis, the surface and gene expression of the IL-1 receptor family members IL-1R1, IL-1RAP, IL-18R1, IL-18R2 and IL-36R was quantified in foam cells compared to THP-1 macrophages (treated with 100 nM PMA) and THP-1 monocytes (untreated). Gene expression was quantified by RT-qPCR (Figure 7) and receptor surface expression by multiparametric flow cytometry (Figure 8). Using RT-qPCR, the expression of IL-1 receptor family genes during foam cell formation was compared (see Figure 7). After 4 h, a significant dose-dependent increase in IL36R (Figure 7E) expression was detected. After 24 h and 48 h, a significant dose-dependent decrease in IL1RAP (Figure 7B) expression was observed. Interestingly, the expression of IL18R1 (Figure 7C) and IL18R2 (Figure 7D) was significantly increased after treatment with 100 µg/ml for 48 h. Using multiparametric flow cytometry, the surface expression of IL-1 receptor family members was further quantified. An increase in IL-1R1, IL-18R2 and IL-36R was detected while a decrease in IL-1RAP expression was observed (see Figure 8). Summary Atherosclerosis is a chronic inflammatory disease that begins with the infiltration of monocytes into the subendothelium and their subsequent differentiation into macrophages. Macrophage-derived foam cells are essential for atherosclerosis development and progression from early fatty streaks to advanced plaques. Assessment of foam cell formation is crucial to evaluate atherosclerosis and serves as an important endpoint. Therefore, a macrophage-mediated foam cell formation model was established. When analyzing the protein levels of receptors on the cell surface of THP-1 monocytes, THP-1 macrophages and foam cells, a significant increase in IL-18R2 and IL-36R expression was detected. A significant decrease in the expression of both the gene and the protein of IL-1RAP was also observed. In this experimental setup, a significant increase in the expression of IL36R mRNA after 4 h of exposure to oxLDL was observed. After 24 h of oxLDL exposure, this was also reflected in protein expression. The up-regulation of IL-18R2 and IL-36R in foam cell formation suggests that the IL-18R and IL-36R signaling pathways may play a role in atherosclerosis. Potential anti-inflammatory IL-1 therapeutics and Outlook CVD is a serious health problem in the Western world and increasingly in developing countries. Current treatments are not sufficient to effectively reduce the risk of developing the disease. Therefore, there is a need for effective therapeutic treatments to prevent the clinical manifestation of atherosclerosis. The mRNA technology offers a promising method for anti-inflammatory IL-1 cytokine therapeutics. It can overcome many of the problems associated with existing protein therapeutics, such as short half-life and systemic effects of IL-1 neutralization. The data from this study suggest that mRNA-encoded IL-1RA and IL-18BP are similarly as active as recombinant IL-1RA (Anakinra) or IL-18BP. Therefore, it is reasonable to utilize the advantages of mRNA-based therapeutics for therapeutic development. Example 4: Surface expression of IL-1 family receptors To characterize cross-reactivity and bioactivity of IL-1 family members, the reporter cell lines HEK-BlueTM IL-1R, HEK-BlueTM IL-18 and HEK-BlueTM IL-36 were used. To investigate the effect of cytokine stimulation on the receptor surface expression of the reporter cells, unstimulated and IL-1^ stimulated HEK-BlueTM IL-1R cells were compared (Figure 9). The main receptor of HEK-BlueTM IL-1R, IL-1R1, and its coreceptor IL-1RAcP were expressed to the same level, but less than IL-18Rβ (Figure 9C). The coreceptor of IL-18Rα, IL-18Rβ, was more highly expressed on the surface of unstimulated and stimulated cells (Figure 9B, C) compared to the other receptors. The receptor IL-18Rα was expressed at lower level on the surface of both unstimulated and IL-1α stimulated cells (Figure 9C). In general, the receptor expression after the IL-1α stimulation was slightly reduced compared to the receptor expression on unstimulated cells (Figure 9C). The receptor surface expression of the HEK-BlueTM IL-18 cells were compared before and after the stimulation with IL-18 using flow cytometry (Figure 10). Both unstimulated and IL-18 stimulated HEK-BlueTM IL-18 cells expressed IL-18Rα at the highest levels (Figure 10C), which was also confirmed with the histograms (Figure 10A, B). Interestingly, the surface expression levels of I IL-18Rα was decreased after stimulation with IL-18 (Figure 10C). All other receptors were expressed on HEK-BlueTM IL-18 cells, but not regulated after IL-18 stimulation. The surface expression of IL-1 receptors on the HEK-BlueTM IL-36 cells before and after stimulation with IL-36γ was also compared by flow cytometry (Figure 11). IL-36R was highly expressed on HEK-BlueTM IL-36 cells (Figure 11). All other receptors were expressed to lower degrees. Interestingly, the expression of all receptors slightly decreased after IL-36γ stimulation. Summary HEK-BlueTM reporter cell lines expressing the three main receptors IL-1R1, IL-18 and IL-36 from InvivoGen were used to investigate the surface receptor expression the ligand/receptor interactions within the IL-1 family and the bioactivity of mRNA variants of anti-inflammatory family members. The cell lines expressing the reporter gene, overexpress their specific receptor (e.g. HEK-BlueTM IL-1R the IL-1R1, Figures 9-11) but the remaining receptors of the family were also expressed. Stimulation of the HEK-BlueTM reporter cell lines with IL-1α, IL-18 or IL-36γ did not lead to a relevant increased expression of the respective receptor on the surface but tended to decrease after stimulation. Example 5: Bioactivity of recombinant IL-1 family cytokines and bioactivity of the anti- inflammatory IL-1 family members After quantification of receptor surface expression on the HEK-BlueTM reporter cell lines, their functionality to respond to cytokine stimulation was evaluated. The three HEK-BlueTM reporter cell lines were used to investigate cross-reactions within the IL-1 family. To demonstrate the bioactivity of each reporter cell line in response to IL-1 family cytokines, the secreted embryonic alkaline phosphatase (SEAP) assay was used. Cytokine- induced activation of NF-κB and AP-1 initiates the production of SEAP, which can be quantified using QUANTI-BlueTM solution (Figure 12). EC50 values of IL-1 cytokines used for each reporter cell line are listed below (Table 4). Table 4: EC50 values of HEK-BlueTM IL-1R, IL-18 and IL-36 cells for the detection of bioactive IL-1 cytokines using SEAP assay. The HEK-BlueTM IL-1R cells responded in a sigmoidal dose-response curve to both IL-1α and IL-1β stimulation. As listed in Table 4, the EC50 value of IL-1α (1.73 ± 0.69 pg/ml) was more than tenfold lower than IL-1β (20.26 ± 2.29 pg/ml), which indicates a higher potency of IL-1α (Figure 12A). However, no signal was induced by all other IL-1 family cytokines (Figure 12A). Similarly, for the HEK-BlueTM IL-18 cells, an activation was only observed after IL-18 stimulation (Figure 12B) with an EC50 value of 11.03 ± 3.68 pg/ml (Table 4). Unexpectedly, the HEK-BlueTM IL-36 cells not only responded to the stimulation with IL-36 cytokines (Figure 12C). The SEAP release was also triggered due to IL-1α and IL-1β stimulation with even a higher potency of IL-1α (76.38 ± 26.50 pg/ml) compared to IL-36γ (274.6 ± 73.3 pg/ml). The highest potency of bioactivity was recorded using the IL-36α cytokine with a more than sixfold lower EC50value of 11.22 ± 0.73 pg/ml, compared to the IL-1α (76.38 ± 26.50 pg/ml) (Table 4). With a more than fivefold higher EC50 value of IL-1β (471.1 ± 169.10 pg/mL) compared to IL-1α, the HEK-BlueTM IL-36 cells were able to respond sufficiently for an EC50value, unlike IL- 36β (Table 4). The bioactivity of the anti-inflammatory members of the IL-1 family was investigated using the HEK-BlueTM reporter cells. In addition to the well-known inhibitory cytokine IL-1RA, which is used under the brand name of Anakinra, IL-18BP, IL-37, IL-38 and IL-36RA are other anti- inflammatory IL-1 family cytokines. As mentioned, cross-reactions within the IL-1 family are already known, like for IL-38. Until now, IL-38 is the only member of the IL-1 family that is known to be able to inhibit several receptors by binding, indicating possible cross-reactivity within the family. Activation of HEK-BlueTM reporter cells was induced by IL-1α for IL-1R cells, IL-18 for IL-18 cells and IL-36γ for IL-36 cells. To obtain information on possible cross- reactivity within the IL-1R family, all anti-inflammatory IL-1 family cytokines were applied to all three HEK-BlueTM cell lines in combination with the corresponding pro-inflammatory cytokines (Figure 13). IC50 values of IL-1 cytokines used for each reporter cell line are listed below (Table 5). Table 5: IC val TM 50 ues of HEK-Blue IL-1R, IL-18 and IL-36 cells for the detection of the inhibitory ability of anti-inflammatory IL-1 family members using SEAP assay. Using HEK-BlueTM IL-1R cells, only IL-1RA inhibited IL-1α induced activity with an IC50 value of 256.7 ± 64.6 ng/ml (Figure 13A). The IL-18 induced activity in HEK-BlueTM IL-18 cells was inhibited by IL-18BP with an IC50 of 113.8 ± 93.3 ng/ml and the combination of IL-18BP and IL-37 with an IC50 of 480.53 ± 218.9 ng/ml for HEK-BlueTM IL-18 (Figure 13B). For the HEK-BlueTM IL-36 cells just IL-36RA was able to inhibit the IL-36-induced response with an IC50 of 428.1 ± 63.1 ng/ml (Figure 13C). Various combinations of inhibitory cytokines were tested. After receptor inhibition using the IL- 1R1 antagonist IL-1RA or the IL-36R antagonist IL-36RA, the cells were stimulated with either IL-1α or IL-36γ and the inhibitory effect was examined (Figure 14Figure ). The IL-1α-induced signals in HEK-BlueTM IL-36 cells were completely inhibited using IL-1RA within contrast, IL-36RA reduced the IL-1α induced signal by about 30% (Figure 14). Furthermore, the presence of IL-1RA led to a 15% reduced IL-36γ signal, while IL-36RA almost completely inhibited the IL-36 activity (Figure 14). Summary The inhibitory IL-1 family members only suppressed signaling via their cognate receptor, i.e. IL-1RA (IL-1R1 receptor antagonist) for IL-1R1, IL-18BP for IL-18 and IL-36RA (IL-36 receptor antagonist) for IL-36R. However, administration of IL-37 did not result in inhibition of IL-18 activity. In contrast to IL-18BP, IL-37 forms a heterotrimeric complex with IL-18Rα and the co- receptor IL-1R8 instead of IL-18Rβ. Since the anti-inflammatory property of IL-37 is mainly mediated by the co-receptor IL1R8, the lack of co-receptor IL-1R8 on the surface of HEK- BlueTM IL-18 may prevent the inhibitory effect of IL-37. The combination of IL-18BP and IL-37 did not show any increased inhibitory activity compared to IL-18BP alone (Figure 13). The experiments with the HEK-BlueTM reporter cell lines provided an insight into the lack of potential cross-reactivity within the IL-1 family and confirmed the selectivity of the IL-1 subfamily cytokines for their cognate receptors. Example 6: Endothelial cell expression of IL-1 family receptors Atherosclerosis is caused by IL-1α and sterile vascular inflammation in an inflammasome- independent manner. The expression of adhesion molecules such as MCP-1 and ICAM1 promotes the migration and infiltration of monocytes to the site of inflammation. The extent to which the gene expression of these adhesion molecules changes after IL-1 stimulation was investigated in human umbilical vein endothelial cells (HUVEC). In addition, the gene expression of IL-6 as an immune cell trigger factor was examined after stimulation with IL-1α or IL-1β. To determine the extent to which IL-1 family receptors are expressed on the surface of HUVECs, they were measured by flow cytometry (Figure 15A). The determination of the gene expression level was performed using RT-qPCR (Figure 15B). Compared to the other receptors, IL-1R1 and its co-receptor IL-1RAcP were expressed the most on the surface of HUVEC cells (Figure 15A). Since IL-1R1 had the highest expression on the surface of HUVEC cells, they were stimulated with IL-1α and IL-1β to determine important adhesions mediators and IL-6, known to be upregulated on endothelial cells. In general, the stimulation with IL-1 cytokines increased the gene expression level of all cytokines, except for ICAM-1 after 4 h stimulation with IL-1β, for both IL-1α and IL-1β stimulation for 4 h (Figure 15B). The gene expression of MCP-1 was increased the most after 4 h stimulation with IL-1α. Besides the ICAM1 level after 24 h stimulation, the stimulation with IL-1α led to a higher gene expression of all cytokines, after both 4 or 24 h stimulation. Especially the stimulation with IL-1β had a higher impact of the gene expression increase after 24 h compared to 4 h stimulation. Summary The major pro-inflammatory impact of the IL-1 family in the course of many diseases naturally raises the question of the extent to which specific and influential cell types express the IL-1 family members and respond to them. HUVEC cells were used as an endothelial cell model to investigate the IL-1 family receptor expression. Since IL-1R1 was highly expressed on the surface of the HUVEC cells (Figure 15), the IL-1α and IL-1β-mediated responses were analyzed and a strong upregulation of the gene expression of IL-6 (IL6), ICAM-1 (ICAM1), MCP-1 (MCP-1) and E-selectin (SELE) was observed. In summary, the HUVEC cell line, as endothelial cells, not only express IL-1 receptor members on the surface, but is also a good model to study leukocyte adhesion and migration as shown by the increased expression of adhesion molecules such as ICAM1, SELE and MCP-1 after IL-1α or IL-1β stimulation. Stimulation with IL-1α or IL-1β provided insight into the behavior of endothelial cells with a dysfunction and could thus illustrate its effect on leukocyte migration as well as IL6 production. Especially the importance of IL-1α released by endothelial cells was emphasized, since insufficient data sets are available. In addition, the release of IL-6 as a prominent inflammatory mediator intensifies and further drives an already existing inflammation. Example 7: Binding studies using mRNA-encoded IL-1-ALFA-tag cytokines To study the binding of IL-1 family members to the different IL-1R family members, novel mRNA-encoded cytokines were genetically engineered, where the cytokine is fused to an ALFA-tag. In particular, novel mRNA variants of IL-1RA, IL-18BP and IL-36RA were used that contain the ALFA-tag sequence at the C-terminal end. The corresponding proteins were generated after transfection of HEK 293/T17 cells. For testing the binding ability, an ELISA and flow cytometry was performed. The bioactivity of the protein with ALFA-tag was tested using the SEAP assay (Figure 16). The corresponding EC50 and IC50 values of ALFA-tagged IL-1 cytokines of each experimental trial from Figure 16 each are listed in the following Table 6. Table 6: EC50 or IC50 values of HEK-BlueTM IL-1R and IL-18 cells for the detection of inhibitory ability of the ALFA-tagged proteins for A ELISA, B flow cytometry and C SEAP assay. For getting information about the binding ability using the ALFA-Tag variants an ELISA and flow cytometry binding assay was performed. The results of the ELISA measurement showed a binding of IL-1RA_ALFA to IL-1R1 receptor with an average EC50 value of 47.08 ng/ml (SD = 31.26) (Table 6). No interaction of the IL-1RA_ALFA with the co-receptor IL-1RAcP was observed (Figure 16A). IL-18BP_ALFA showed binding to both IL-18Rα and IL-18Rβ, but no saturated binding could be achieved (Figure 16A). For the flow cytometry binding assay, the ALFA-tagged variants of IL-1RA, IL-18BP and IL-36RA interacted with the corresponding receptor of the HEK-BlueTM IL-1R, IL-18 and IL-36 reporter cell line. For IL-1RA_ALFA there was an average EC50 value of 102.67 ng/ml (SD = 49.74) (Table 6), while for the binding studies of IL-18BP and IL-36RA with HEK-BlueTM IL-18 and IL-36 there was no saturation observed and therefore no EC50 was determined (Figure 16 B). The mRNA variants of IL-1RA_ALFA inhibited IL-1 signaling in HEK-BlueTM IL-1R cells with an IC50 value of 690.3 ng/ml (Table 6).The self-produced variants of IL-18BP, with and without ALFA-tag, showed an approximately six times lower IC50 value (12.5 ng/ml vs 84.17 ng/ml) for the HEK-BlueTM IL-18 cells than the purchased variant of IL-18BP (Table 6, Figure 16C). Summary The successful use of ALFA-tagged mRNAs of IL-1RA and IL-18BP in binding studies such as ELISA and flow cytometry was demonstrated in this study. The specific binding of IL-1RA to IL-1R1, but not to the co-receptor IL-1RAcP, could be confirmed. Unexpectedly, binding of IL-18BP to both IL-18Rα and IL-18Rβ was detected by both ELISA and flow cytometry. The inhibitory effect of IL-18BP on IL-18 by binding of IL-18BP to IL-18 and not to the primary or co-receptor has been described in the literature. Binding to free IL-18 prevents the interaction of IL-18 with the receptors. The data of this study suggest that IL-1RA produced by mRNA is able to inhibit IL-1α activity at comparable concentrations to the recombinantly produced protein (Figure 16). IL-18BP led to a blockage of IL-18 signaling even at a lower dose (1 μg/ml) compared to the other inhibitory IL-1 members (8 μg/ml) (Figure 16). In this work, it was confirmed that an ALFA-coupled mRNA variant for IL-1RA, IL-18BP and IL-36RA did not change bioactivity of the translated protein. Thus, the addition of the ALFA- tag did not lead to a deterioration in the bioactivity of the inhibitory IL-1 family members. Detection in binding assays using flow cytometry and ELISA also proved successful for exemplary IL-1RA_ALFA (Figure 16). The ALFA-tag proved to be a very easy to use, efficient and successful tool to quantify the receptor binding of the inhibitory members of the IL-1 family without the presence of the ALFA-tag affecting the functional stability of the interleukins (Figure 16). In addition, the ALFA-tag proved to be very useful as an analysis tool for various binding assays due to the variability of detection systems. Example 8: Inhibitory activity of mRNAs-encoded anti-inflammatory IL-1 family members in a pharmaceutical formulation To test the bioactivity of IL-1RA- and IL-18BP-encoding mRNA formulated in F12-Lipoplex particles (LPX) (Fig. 17A, B, respectively), their inhibitory activity was investigated in comparison to recombinant anti-inflammatory IL-1 family cytokines (Table 7). To achieve this, HEK293T/17 cells were transfected with IL-1RA- or IL-18BP-encoding mRNA formulated in F12-Lipoplex particles. HEK 293T/17 were transfected with 0.24 ml of IL-1RA- encoding mRNA formulated in F12-Lipoplex. After 42 hours, the concentration of anti- inflammatory IL-1 cytokines produced by HEK293T/17 cells was determined by ELISA (enzyme-linked immunosorbent assay) (R&D Systems), following the manufacturer’s instructions. The bioactivity of the anti-inflammatory members of the IL-1 family (IL-1RA and IL-18BP) was then demonstrated by using the HEK-BlueTM IL-1R and IL-18 reporter cell lines by monitoring the activation of the NFκB and AP-1 pathways. Initially, 0.05 x 106 cells were plated in a 96-well plate. The cells were incubated with 100 µl of ^ a √ ^^ titration series of mRNA-encoded or recombinant IL-1RA or IL-18BP. After 30 minutes, cells were stimulated with either 8 pg/ml IL-1α (HEK-BlueTM IL-1R) or 20 pg/ml IL-18 (HEK- BlueTM IL-18) and incubated for 24 hours. QUANTI-BlueTM Solution, a colorimetric enzyme assay for the detection of secreted embryonic alkaline phosphatase (SEAP), was used to quantify SEAP activity. The SEAP secreted by the HEK-BlueTM reporter cell lines into the cell culture supernatant, turns from pink to blue/purple and can be quantified using a spectrophotometer. The QUANTI-BlueTM Solution was prepared according to the manufacturer’s instructions. Briefly, 150 µl of the solution and 50 µl of supernatant from the HEK-BlueTM reporter cell lines were added per well. Following 1.5 hour incubation at 37°C and 5% CO2, absorbance was measured at 620 nm using the CLARIOStar® microplate reader. The activity was calculated in % by setting the highest response of either IL-1 α for HEK-BlueTM IL-1R or IL-18 for HEK-BlueTM IL-18 as 100% activity. Data was processed and IC50 values were calculated using GraphPad Prism 9. Table 7: IC50 values of HEK-BlueTM cells for the inhibitory effect of mRNA-encoded IL-1 family cytokines versus recombinant IL-1RA and IL-18BP in the SEAP assay. A dose-dependent inhibitory effect by the mRNA-encoded cytokines was demonstrated, comparable or superior to the recombinant cytokines, confirming the bioactivity of the mRNA- based anti-inflammatory IL-1RA and IL-18BP (Fig.17A, B, Table 7). mRNA-based IL-1RA inhibited IL-1α-mediated stimulation of HEK-BlueTM IL-1R cells slightly more potently than the recombinant protein (Anakinra) (Fig.17A), whereas IL-18BP demonstrated superior inhibition in comparison to recombinant IL-18BP (Peprotech) (Fig.17B). These findings demonstrate that anti-inflammatory IL-1 family members can be encoded using suitable mRNA expression vectors leading to secretion via the ER/Golgi-apparatus via selected signaling peptides of functional proteins. The translated proteins from mRNAs that are complexed in a suitable pharmaceutical formulation show superior inhibitory activity compared to recombinant controls, suggesting enhanced therapeutic potential of this format, as compared to protein-based therapeutics. Example 9: Inhibitory activity of mRNA-encoded anti-inflammatory IL-1RA using pro- inflammatory responses in endothelial HUVEC cells. To demonstrate the anti-inflammatory effect of IL-1RA in a relevant in vitro model, we investigated the inhibition of the IL-1α-induced secretion of pro-inflammatory cytokines, in HUVEC (human umbilical vein endothelial) cells (Fig.18). IL-1RA-encoding mRNA formulated in LipofectamineTM MessengerMaxTM Reagent was transfected into HEK293T/17 cells. To prepare the transfection mixture, per well, 1.5 µl LipofectamineTM MessengerMaxTM Reagent was diluted in 25 µl Opti-MEMTM medium, and incubated for 10 minutes, RT. The mRNA master mix was prepared by diluting 1 µg mRNA in 25 µl Opti-MEMTM medium. The mRNA master mix was thoroughly mixed, centrifuged, and incubated at RT for 5 min. The LipofectamineTM mix was added gently to the diluted mRNA master mix. This final transfection mixture was added to the HEK 293T/17 cells. After 42 hours, the concentration of anti-inflammatory IL-1 cytokines produced by HEK293T/17 cells was determined by performing an ELISA (R&D Systems) following the manufacturer’s instructions, as described below. Then, HUVEC cells were stimulated with IL-1α (1 or 10 ng/ml) in the presence of serially diluted (1:10 dilution) recombinant IL-1RA (Anakinra) or supernatant containing the translated mRNA-encoded IL-1RA formulated in LipofectamineTM MessengerMaxTM. For stimulating, 0.05 x 106 cells were plated in 50 µl of endothelial cell growth medium in a 96-well plate. After 24 hours of incubation, the pro-inflammatory cytokines concentration in the supernatant of the HUVEC cells was determined with an IL-6 ELISA kit (BioLegend) or MSD® U-PLEX biomarker multiplex assay, following the manufacturer’s instructions. A similar ELISA protocol was used to that described above, but with the following changes: capture antibody was diluted in 100 µl coating buffer.200 µl assay diluent was added to block nonspecific binding.100 µl of a 2- fold dilution of the harvested HUVEC supernatant or a 2-fold serial dilution of the standard was added. Both recombinant IL-1RA (Anakinra) and mRNA-encoded IL-1RA (LipofectamineTM MessengerMaxTM) successfully neutralized the IL-1α stimulated release of IL-6, TNF-α and IL- 1β in HUVEC cells (Fig. 18A-C). The mRNA-encoded IL-1RA showed a dose-dependent inhibitory effect that was comparable to the recombinant IL-1RA (Anakinra), indicating the bioactivity of the anti-inflammatory mRNA-encoded IL-1RA. Thus, the effectiveness of mRNA- encoded anti-inflammatory IL-1 family members in a relevant in vitro model was demonstrated. These findings suggest that anti-inflammatory cytokines based on mRNA technology have the potential to be therapeutically effective. Example 10: Inhibitory activity of mRNA-encoded anti-inflammatory IL-1RA on human CD14+ monocytes. To demonstrate the anti-inflammatory effects of mRNA-encoded IL-1RA in another relevant cell type, we investigated the levels of pro-inflammatory cytokines secreted by CD14+ monocytes isolated from human PBMCs stimulated with IL-1α (10 ng/ml) in presence or absence of IL-1RA. This experiment was performed with the IL-1RA-encoding mRNA formulated in either F12- Lipoplex particles or Messenger Max lipofectamineTM, transfected into the HEK293T/17 cells, in comparison to the recombinant IL-1RA (Anakinra). The transfection of the HEK 293T/17 was performed as described above. After supernatant harvesting, the concentration of anti- inflammatory IL-1 cytokines produced by HEK293T/17 cells was determined by ELISA (R&D Systems) following the manufacturer’s instructions, as described below. The CD14+ cells isolation was performed using positive MACS selection. First, human PBMCs from healthy donors were obtained by density gradient centrifugation. Cells were centrifuged at 300g for 8 min at room temperature with complete removal of supernatant. Cells were resuspended in 40µl of MACS buffer and 10µl of CD14 Microbeads (human) per 10e7 total cells incubating for 15 min at 4°C. Subsequently the cells were washed with MACS buffer and centrifuged for 8 minutes at room temperature with complete removal of supernatant. The cell pellet was resuspended in 500 µl of MACS buffer and sorted in with magnetic columns in a magnetic MACS separator. Afterwards the magnetic labeled cells were collected by firmly pushing the plunger into the column. The cells were seeded at a density of 0.05x10e6 cells per well into a 96 well plate, in 50 µl Advanced DMEM + 1% Glutamax. Subsequently, the CD14+ monocytes were treated with 100 µl of a 1:10 titration series of recombinant IL-1RA (purchased, Anakinra) or mRNA-encoded IL-1RA (formulated in LPX or with Messenger Max lipofectamineTM). After 30 minutes of incubation, the cells were challenged with 10 ng/ml IL-1α and incubated for approximately 24 hours at 37°C at 5% CO2. After incubation, the pro-inflammatory cytokines concentration in the supernatant of monocytes were determined using an MSD® U-PLEX biomarker multiplex assay following the manufacturer’s instructions, as described below. Interestingly, mRNA-encoded IL-1RA (formulated in LPX or LipofectamineTM MessengerMaxTM) efficiently inhibited the effects of IL-1α, decreasing IL-6 and TNF-α secretion by the CD14+ cells (Fig. 19A, B). These promising dose-dependent effects promoted by mRNA-encoded IL-1RA were comparable to the recombinant IL-1RA (Anakinra), suggesting suitability of mRNA-based anti-inflammatory cytokines. Surprisingly, mRNA- encoded IL-1RA in both formulations successfully demonstrated superior dose-dependent effects to those of recombinant IL-1RA (Anakinra - which IC50 was not possible to be determined), inhibiting the release of IL-1β and IFN-γ by IL-1α-challenged CD14+ monocytes (Fig. 19C, D). Importantly, monocytes are key cells involved in the immune responses of several inflammatory diseases. Monocytes are key players in inflammatory conditions, they release pro-inflammatory factors that can exacerbate the condition. However, the effectiveness of IL-1RA in inhibiting the secretion of several pro-inflammatory cytokines by IL- 1α-challenged monocytes suggests their potential to be therapeutically successful. These results indicate that mRNA-encoded anti-inflammatory IL-1RA has the potential to be developed as a therapeutic approach for halting inflammatory diseases, especially by targeting and regulating CD14+ monocytes. Additional methods used in Examples 8-10 ELISA protocol Briefly, the appropriate capture antibody was immobilized overnight at room temperature (RT) on 96-well flat bottom Maxisorp Nunc-Immuno™ plates. Addition of assay diluent (300 µl, 1 hour, RT), was used to block nonspecific binding. A 1:10 dilution series of the HEK 293T/17 supernatant or a 2-fold dilution series of the standard, each in duplicate, was added and incubated for 2 hours, RT. To detect bound proteins, the corresponding detection antibody was added and incubated for 2 hours, RT, , followed by 20 minutes incubation of streptavidin coupled with horseradish peroxidase (HRP). After every step, the plate was washed (3 x 400 µl, 0.05% TWEEN® 20, DPBS). Subsequently, 3,3’,5,5’-Tetramethylbenzidin (TMB) substrate solution was added to each well. After 20 minutes of incubation in darkness, the reaction was stopped by adding 1 M H2SO4. Absorbance was measured at 450 nm using the CLARIOStar® microplate reader. GraphPad Prism 9 was used to calculate the protein concentration from the known concentrations of the protein recombinant standards. By plotting the mean absorbance values (y-axis) against the protein concentration (x-axis), a standard curve was generated. The standard curve was used to determine the protein concentration in each sample. Using the regression equation from the standard curve, the concentration of the absorbance in the area of the standard curve was calculated using GraphPad Prism 9. MSD® U-PLEX biomarker multiplex assay protocol To perform MSD® U-PLEX biomarker multiplex assay, the plates were coated with respective linkers bound to specific biotinylated antibodies (1 hour, RT, shaking). Subsequently the plates were washed 3 times with 1x MSD wash buffer. The cell supernatants were diluted 2-fold in a final volume of 100 µl with assay diluent (Buffer 57). The standard calibrators were diluted in 300 µl of assay diluent in a 5-folds serial dilution (7-steps and blank). Then, 50 µl of the samples and calibrators were transferred into the MSD plate in technical duplicates and incubated (2 hours, RT, shaking at 750 rpm). After washing the plates, 50 µl of the detection antibody mix diluted in assay diluent (Buffer 3) was added in each well and incubated (1 hour, RT, shaking at 750 rpm). The plates were then washed, and 150 µl of MSD Gold Read buffer B was added to the wells. The measurements were immediately performed in the MSD reader. Finally, MSD Discovery workbench® was used to calculate the analytes concentration in pg/ml from the calibrator standard curve. GraphPad Prism 9 was used to analyze and plot the graphs from the extracted data. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS 1. An isolated RNA encoding an immune inhibitory protein of the interleukin-1 (IL-1) family or functional variant thereof. 2. The isolated RNA according to claim 1, wherein the immune inhibitory protein of the IL-1 family is selected from the group consisting of: IL-1 receptor antagonist (IL-1RA), interleukin-18 binding protein (IL-18BP), interleukin 36 receptor antagonist (IL-36RA), interleukin 37 (IL-37), and interleukin 38 (IL-38). 3. The isolated RNA according to claim 1 or claim 2, wherein the immune inhibitory protein of the IL-1 family is IL-1RA, IL-18BP or IL-36RA. 4. The isolated RNA according to any one of the preceding claims, wherein the immune inhibitory protein of the IL-1 family is IL-1RA or IL-18BP. 5. The isolated RNA according to any one of the preceding claims, wherein the immune inhibitory protein of the IL-1 family is IL-1RA. 6. The isolated RNA according to any one of the preceding claims, wherein the RNA is mRNA. 7. The isolated RNA according to any one of the preceding claims, wherein the RNA comprises a modified nucleoside in place of at least one uridine, preferably in place of each uridine. 8. The isolated RNA according to claim 7, wherein the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5- methyl-uridine (m5U), preferably wherein the modified nucleoside is pseudouridine (ψ) or N1- methyl-pseudouridine (m1ψ). 9. The isolated RNA according to any one of the preceding claims, wherein the RNA comprises a 5’ cap analog, optionally the 5’ cap analog m27,3'0G(5')ppp(5')m2'-0ApG or 3´-O- Me-m7G(5')ppp(5')G, preferably m27,3'0G(5')ppp(5')m2'-0ApG. 10. The isolated RNA according to any one of the preceding claims, wherein the RNA comprises a 5’ UTR comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12 and 31, or a nucleotide sequence having at least 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12 and 31. 11. The isolated RNA according to any one of the preceding claims, wherein the RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 32, or a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 32. 12. The isolated RNA according to any one of the preceding claims, wherein the RNA comprises a poly-A tail, preferably wherein the poly-A tail comprises the poly-A tail shown in SEQ ID NO: 14. 13. The isolated RNA according to any one of the preceding claims, wherein the RNA comprises a sequence as set forth in any one of SEQ ID NOs: 16 to 25 or a sequence having at least 80% identity thereto. 14. A vector comprising the RNA according to any one of the preceding claims. 15. A lipid particle comprising the RNA according to any one of claims 1-13 or the vector according to claim 14. 16. The lipid particle according to claim 15, wherein the particle is a lipoplex (LPX), a lipid nanoparticle (LNP), or a liposome. 17. The lipid particle according to claim 15 or 16, wherein the particle is an RNA lipoplex particle comprising: the RNA, at least one cationic or cationically ionizable lipid, and at least one neutral lipid, wherein the charge ratio of positive charges to negative charges in the RNA lipoplex particles is between 1:1 and 1:8. 18. The lipid particle according to claim 17, wherein the charge ratio of positive charges to negative charges in the RNA lipoplex particle is between 1:1 and 1:4, between 1:2 to 1.9:2, or about 1.3:2.0. 19. The lipid particle according to claim 17 or 18, wherein the at least one cationic or cationically ionizable lipid, and the at least one neutral lipid are in the RNA lipoplex particle in a molar ratio of from about 10:0 to 1:9, from about 4:1 to 1:2, from about 3:1 to 1:1, or about 2:1. 20. The lipid particle according to any one of claims 17 to 19, wherein the at least one cationic or cationically ionizable lipid is DOTMA. 21. The lipid particle according to any one of claims 17 to 20, wherein the at least one neutral lipid is DOPE. 22. A pharmaceutical composition comprising the RNA according to any one of claims 1 to 13, or the vector according to claim 14, or the lipid particle according to claim 15 to 21, preferably wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients. 23. The RNA according to any one of claims 1 to 13, the vector according to claim 14, the lipid particle according to claim 15 to 21, or the pharmaceutical composition according to claim 22 for use as an immune inhibitory or anti-inflammatory medicament. 24. The polynucleotide according to any one of claims 1 to 13, the vector according to claim 14, the lipid particle according to claim 15 to 21, or the pharmaceutical composition according to claim 22 for use in the prevention and/or treatment of atherosclerosis or a cardiovascular disease, preferably wherein the cardiovascular disease is heart failure or pericarditis.
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