Lipid nanoparticles containing nucleic acid-binding proteins

Abstract

The present disclosure relates to lipid nanoparticles and uses thereof for delivering RNA, where the lipid nanoparticles include therein a nucleic acid binding protein or peptide (eg, an RNA binding protein or peptide) that is bound to the RNA.

Description

[Technical field]

[0001] Related Application Data This application claims priority to Australian Patent Application No. 2021903192, entitled "Lipid nanoparticle comprising a RNA-binding protein", filed on October 6, 2021, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is filed with a Sequence Listing in electronic format, the entire contents of which are incorporated herein by reference.

[0003] The present disclosure relates to lipid nanoparticles and uses thereof for delivering RNA, where the lipid nanoparticles include therein a nucleic acid binding protein or peptide (eg, an RNA binding protein) that is bound to the RNA. [Background technology]

[0004] Nucleic acid vaccines have recently emerged as a promising approach for the treatment and prevention of various diseases, including nucleic acid vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the ongoing severe acute respiratory syndrome coronavirus 2019 (COVID-19) global pandemic.

[0005] mRNA vaccines rely on the delivery of mRNA into the cytoplasm of host cells, where it is transcribed into antigenic proteins to induce the production of neutralizing antibodies. However, the large size and negative charge of mRNA hinder cellular uptake. Therefore, lipid delivery vehicles, such as liposomes or lipid nanoparticles, are used to encapsulate mRNA, inhibiting RNA degradation in plasma while promoting cellular uptake to efficiently deliver mRNA in vivo.

[0006] Lipid delivery vehicles are usually formed from cationic lipids and other ionizable lipid components, such as ionizable lipids, neutral lipids, cholesterol, and PEGylated lipids.Cationic lipids are amphiphilic molecules that have a lipophilic region that contains one or more hydrocarbon groups and a hydrophilic region that contains at least one positively charged polar head group.Cationic lipids and nucleic acids form a positively charged complex, which facilitates nucleic acid to pass through the cell membrane and enter the cytoplasm of cells.

[0007] However, some adverse cytotoxic effects of cationic lipids are known, including the generation of reactive oxygen species and accumulation in plasma due to insufficient degradation in humans. Therefore, much effort has been put into identifying novel lipids or specific lipid compositions that can protect nucleic acids from degradation and removal and provide effective intracellular delivery and / or mRNA expression / translation. Furthermore, these lipid-nucleic acid particles should be well tolerated and provide an appropriate therapeutic index so that treatment with an effective amount of nucleic acid is not accompanied by unacceptable toxicity and / or risk to patients.

[0008] Thus, it will be apparent to those skilled in the art that there is a continuing need for improved lipid nanoparticles for delivery of oligonucleotides, such as RNA. Summary of the Invention

[0009] The present disclosure is based on the inventors' discovery that incorporating RNA-binding proteins or peptides, or lipidated RNA-binding proteins or peptides, into lipid nanoparticles increases the stability of the associated RNA and / or promotes the nucleation of lipid nanoparticles and / or reduces the toxicity and / or harmful side effects of lipid nanoparticles. The inventors have also confirmed that incorporating RNA-binding proteins or peptides into lipid nanoparticles can prevent toll-like receptor (TLR) stimulation / induction. The inventors have further confirmed that incorporating nucleic acid-binding proteins or peptides (i.e., RNA-binding proteins and DNA-binding proteins) into lipid nanoparticles is protective.

[0010] In general, the inventors' discoveries provide the basis for lipid nanoparticles that contain nucleic acid binding proteins or peptides. The inventors' discoveries also provide the basis for lipid nanoparticles that contain lipidated nucleic acid binding proteins or peptides.

[0011] In one example, the nucleic acid binding protein or peptide is an RNA binding protein or peptide. In one example, the nucleic acid binding protein or peptide is an RNA binding protein or a DNA binding protein.

[0012] The inventors' discovery provides the basis for lipid nanoparticles containing RNA-binding proteins or peptides. The inventors' discovery further provides the basis for lipid nanoparticles containing lipidated nucleic acid-binding proteins or peptides. The inventors' discovery also provides the basis for lipid nanoparticles containing lipidated RNA-binding proteins or peptides.

[0013] Furthermore, the inventors' discoveries provide the basis for methods of using lipid nanoparticles as vaccines or therapeutic agents.

[0014] Thus, the present disclosure provides lipid nanoparticles for delivering RNA (eg, mRNA), which contain a nucleic acid binding protein or peptide therein that is bound to the RNA.

[0015] Thus, the present disclosure provides lipid nanoparticles for delivering RNA (eg, mRNA), which contain an RNA-binding protein or peptide therein that is bound to the RNA.

[0016] In one example, the nucleic acid binding protein or peptide is a lipidated nucleic acid binding protein or peptide. In one example, the RNA binding protein or peptide is a lipidated RNA binding protein or peptide.

[0017] Thus, the present disclosure provides lipid nanoparticles for delivering RNA (eg, mRNA), which contain a lipidated nucleic acid binding protein or peptide therein that is bound to the RNA.

[0018] Thus, the present disclosure provides lipid nanoparticles for delivering RNA (eg, mRNA), which contain a lipidated RNA-binding protein or peptide therein that is bound to the RNA.

[0019] In one example, the RNA binding protein or peptide is lipidated prior to binding to the RNA, hi another example, the RNA binding protein or peptide is lipidated after binding to the RNA.

[0020] In one example, the RNA binding protein or peptide is lipidated with a lipid moiety selected from the group consisting of fatty acids, isoprenoids, and combinations thereof.

[0021] In one example, the RNA binding protein or peptide is lipidated with fatty acid.For example, the fatty acid is triglyceride, phospholipid, or cholesteryl ester.In one example, the fatty acid is triglyceride.In another example, the fatty acid is phospholipid.In a further example, the fatty acid is cholesteryl ester.

[0022] In one example, the RNA binding protein or peptide is lipidated with an isoprenoid, for example, the isoprenoid is isoprene.

[0023] In one example, the RNA binding protein or peptide is lipidated on a nucleophilic side chain at the N-terminus and / or C-terminus.

[0024] In one example, the RNA binding protein or peptide is lipidated on a nucleophilic side chain. For example, it is lipidated on a cysteine, serine, threonine, tyrosine and / or lysine amino acid residue. In one example, the nucleophilic side chain is a cysteine ​​residue. In another example, the nucleophilic side chain is a serine residue. In a further example, the nucleophilic side chain is a threonine residue. In one example, the nucleophilic side chain is a tyrosine residue. In another example, the nucleophilic side chain is a lysine residue.

[0025] In one example, the RNA binding protein or peptide is lipidated at the N-terminus of the protein or peptide.

[0026] It will be apparent to one of skill in the art that the N-terminus of an RNA binding protein or peptide comprises a nuclear localization signal(s) (or sequence) and / or a nuclear export signal. In one example, the RNA binding protein or peptide is modified to remove the nuclear localization signal(s) and / or introduce a nuclear export signal(s).

[0027] In one example, the RNA binding protein or peptide is modified to remove the nuclear localization signal. In another example, the RNA binding protein or peptide is modified to inactivate or remove the nuclear localization signal(s). For example, the RNA binding protein or peptide does not include the nuclear localization signal(s) (or sequence). Those skilled in the art will understand that a nuclear localization signal is one or more sequences of positively charged lysines or arginines exposed on the protein surface, tagging the protein for import into the cell nucleus by nuclear transport. Methods for modifying the nuclear localization signal will be apparent to those skilled in the art and / or described herein. For example, the nuclear localization signal is lipidated, removed, or inactivated. In one example, the nuclear localization signal at the N-terminus of the RNA binding protein or peptide is lipidated. In another example, the nuclear localization signal at the N-terminus of the RNA binding protein or peptide is removed. In another example, the nuclear localization signal at the N-terminus of the RNA binding protein or peptide is inactivated.

[0028] In one example, RNA binding protein or peptide is modified to introduce a nuclear export signal. Those skilled in the art will understand that a nuclear export signal is a short leucine-rich motif that targets a protein for export from the cell nucleus to the cytoplasm through the nuclear pore complex. Methods for introducing a nuclear export signal are clear to those skilled in the art and / or described herein.

[0029] In one example, the RNA binding protein or peptide is lipidated at the C-terminus of the protein or peptide.

[0030] In one example, the RNA binding protein or peptide is lipidated by palmitoylation, myristoylation, fatty acid acylation, esterification, prenylation, or a combination thereof.

[0031] In one example, the RNA binding protein or peptide is lipidated by palmitoylation, e.g., by N-terminal cysteine ​​palmitoylation.

[0032] In one example, the RNA binding protein or peptide is lipidated by myristoylation, e.g., by N-terminal glycine myristoylation.

[0033] In one example, the RNA-binding protein or peptide is lipidated by fatty acid acylation, for example, by lysine N-acylation, or in another example, by serine O-acylation.

[0034] In one example, the RNA binding protein or peptide is lipidated by esterification, e.g., by C-terminal cholesterol esterification.

[0035] In one example, the RNA-binding protein or peptide is lipidated by prenylation. For example, the prenylation is farnesylation or geranylgeranylation. In one example, the prenylation is cysteine ​​prenylation.

[0036] In one example, the RNA binding protein or peptide is lipidated by N-terminal cysteine ​​palmitoylation, N-terminal glycine myristoylation, lysine N-acylation, C-terminal cholesterol esterification, cysteine ​​prenylation, serine O-acylation, or a combination thereof.

[0037] In one example, the lipid moiety is linked to the RNA binding protein or peptide by a thioether bond, an ester bond, a thioester bond and / or an amide bond.

[0038] In one example, the lipid moiety is linked to the RNA-binding protein or peptide by a thioether bond.

[0039] In one example, the lipid moiety is linked to the RNA-binding protein or peptide by an ester bond.

[0040] In one example, the lipid moiety is linked to the RNA-binding protein or peptide by a thioester bond.

[0041] In one example, the lipid moiety is linked to the RNA-binding protein or peptide by an amide bond.

[0042] In one example, the RNA-binding protein or peptide is lipidated using chemical lipidation or enzymatic lipidation. For example, the RNA-binding protein or peptide is lipidated using chemical lipidation. In one example, the chemical lipidation is selected from the group consisting of chemical ligation, click chemistry, expressed protein ligation, and combinations thereof. In another example, the RNA-binding protein or peptide is lipidated using enzymatic lipidation. For example, the enzymatic lipidation is selected from the group consisting of sortase A-mediated lipidation, transglutaminase-mediated lipidation, and combinations thereof. In one example, the enzymatic lipidation is performed in vivo or in vitro. For example, the enzymatic lipidation is performed in vivo. In another example, the enzymatic lipidation is performed in vitro.

[0043] In one example, nucleic acid binding protein or peptide binds directly to RNA.In another example, nucleic acid binding protein or peptide binds to RNA before RNA is incorporated into lipid nanoparticle.In a further example, nucleic acid binding protein or peptide binds to RNA in lipid nanoparticle after RNA is incorporated into lipid nanoparticle, where nucleic acid binding protein or peptide is in lipid nanoparticle.For example, nucleic acid binding protein or peptide binds to RNA encapsulated in lipid nanoparticle.

[0044] In one example, the nucleic acid binding protein or peptide further binds to RNA on the surface of lipid nanoparticle.In this situation, the nucleic acid binding protein or peptide will be present in lipid nanoparticle and on the surface of lipid nanoparticle.The nucleic acid binding protein or peptide on the surface of lipid nanoparticle does not have to be the same as the RNA binding protein or peptide in lipid nanoparticle.

[0045] For example, lipid nanoparticles can be formed with a nucleic acid binding protein or peptide that binds to RNA therein, and the formed lipid nanoparticles can then be coated with a nucleic acid binding protein or peptide to bind to any unencapsulated and / or partially encapsulated RNA.

[0046] In one example, the nucleic acid binding protein or peptide encapsulates the RNA. In another example, the nucleic acid binding protein or peptide binds to a nucleophilic side chain at the N-terminus and / or C-terminus of the RNA. In one example, the nucleic acid binding protein or peptide binds to a nucleophilic side chain at the RNA. In another example, the nucleic acid binding protein or peptide binds at the N-terminus and / or C-terminus of the RNA. For example, it binds at the N-terminus of the RNA. In another example, it binds at the C-terminus of the RNA. For example, the nucleic acid binding protein or peptide does not encapsulate the RNA.

[0047] In one example, RNA binding protein or peptide binds directly to RNA.In another example, RNA binding protein or peptide binds to RNA before RNA is incorporated into lipid nanoparticle.In a further example, RNA binding protein or peptide binds to RNA in lipid nanoparticle after RNA is incorporated into lipid nanoparticle, where RNA binding protein or peptide is in lipid nanoparticle.For example, RNA binding protein or peptide binds to RNA encapsulated in lipid nanoparticle.

[0048] In one example, RNA binding protein or peptide further binds to RNA on the surface of lipid nanoparticle.In this situation, RNA binding protein or peptide will be present in lipid nanoparticle and on the surface of lipid nanoparticle.RNA binding protein or peptide on the surface of lipid nanoparticle does not need to be the same as RNA binding protein or peptide in lipid nanoparticle.

[0049] For example, lipid nanoparticles can be formed with an RNA binding protein or peptide that binds to RNA therein, and the formed lipid nanoparticles can then be coated with an RNA binding protein or peptide to bind to any unencapsulated and / or partially encapsulated RNA.

[0050] In one example, the RNA binding protein or peptide encapsulates the RNA. In another example, the RNA binding protein or peptide binds to a nucleophilic side chain at the N-terminus and / or C-terminus of the RNA. In one example, the RNA binding protein or peptide binds to a nucleophilic side chain at the RNA. In another example, the RNA binding protein or peptide binds at the N-terminus and / or C-terminus of the RNA. For example, it binds at the N-terminus of the RNA. In another example, it binds at the C-terminus of the RNA. For example, the RNA binding protein or peptide does not encapsulate the RNA.

[0051] In one example, the nucleic acid binding protein or peptide is a) reduce the toxicity of lipid nanoparticles; b) stabilizing RNA; c) protects RNA from degradation; d) promoting the nucleation of lipid nanoparticles; and / or e) inhibiting the induction of signal transduction by one or more Toll-like receptors.

[0052] In one example, the RNA binding protein or peptide is a) reduce the toxicity of lipid nanoparticles; b) stabilizing RNA; c) protects RNA from degradation; d) promoting the nucleation of lipid nanoparticles; and / or e) inhibiting the induction of signal transduction by one or more Toll-like receptors.

[0053] In one example, the RNA binding protein or peptide reduces the toxicity of the lipid nanoparticle.

[0054] In one example, the RNA binding protein or peptide stabilizes the RNA.

[0055] In one example, the RNA binding protein or peptide protects the RNA from degradation.

[0056] In one example, an RNA-binding protein or peptide promotes the nucleation of lipid nanoparticles.

[0057] In one example, the RNA binding protein or peptide inhibits the induction of signaling by one or more Toll-like receptors. In one example, the RNA binding protein or peptide does not inhibit the induction of signaling by one or more Toll-like receptors.

[0058] Those skilled in the art will appreciate that there is a series of Toll-like receptors that recognize and bind nucleic acids, such as RNA, including endosomal Toll-like receptors, including TLR3, TLR7, TLR8 and TLR9. Activation of these receptors results in the production of inflammatory cytokines and type I interferons (interferon type I).

[0059] In one example, the RNA binding protein or peptide inhibits induction of signaling by one or more endosomal Toll-like receptors. For example, the RNA binding protein or peptide inhibits induction of signaling by one or more Toll-like receptors selected from the group consisting of TLR3, TLR7, TLR8 and TLR9. In one example, the RNA binding protein or peptide inhibits induction of signaling by TLR3. In another example, the RNA binding protein or peptide inhibits induction of signaling by TLR7 / 9. In a further example, the RNA binding protein or peptide inhibits induction of signaling by TLR8.

[0060] In one example, the RNA binding protein or peptide is two RNA binding proteins or peptides (i.e., a first and a second RNA binding protein or peptide) linked by a linker. For example, the first and second RNA binding proteins or peptides are covalently linked by an amide bond. The present disclosure encompasses other forms of covalent and non-covalent bonds. For example, the RNA binding proteins or peptides can be linked by a chemical linker.

[0061] In one example, the linker is a flexible linker, such as a flexible peptide linker, for example, a first RNA binding protein or peptide is linked to a second RNA binding protein via a flexible linker.

[0062] In one example, the linker is a peptide linker. For example, a first RNA binding protein or peptide is linked to a second RNA binding protein or peptide via a linker, where the linker is a peptide linker of 2 to 31 amino acids in length. In one example, the linker has the sequence (Gly4Ser) n where n is 1 to 6. For example, the linker comprises the sequence SGGGGS (GS6) or the sequence SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGGS (GS31). In another example, the linker comprises the sequence (Ala) n where n is 2 to 31.

[0063] In one example, the linker is a rigid linker. For example, the rigid linker has the sequence (EAAAK) n where n is 1 to 3. In one example, the rigid linker comprises (EAAAK) n where n is 1 to 10 or about 1 to 100. For example, n is at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10. In one example, n is less than 100. For example, n is less than 90, or less than about 80, or less than about 60, or less than about 50, or less than about 40, or less than about 30, or less than about 20, or less than about 10.

[0064] In one example, the nucleic acid binding protein or peptide is a viral or non-viral nucleic acid binding protein or peptide. For example, the nucleic acid binding protein is a viral nucleic acid binding protein. In another example, the nucleic acid binding protein is a non-viral nucleic acid binding protein.

[0065] In one example, the RNA binding protein or peptide is a viral or non-viral RNA binding protein or peptide.

[0066] In one example, the RNA binding protein or peptide is a viral RNA binding protein. For example, the viral RNA binding protein or peptide is derived from a class III, class IV, class V, and / or class IV virus. In one example, the viral RNA binding protein or peptide is derived from a class III virus. In another example, the viral RNA binding protein or peptide is derived from a class IV virus. In a further example, the viral RNA binding protein or peptide is derived from a class V virus. In one example, the RNA binding protein or peptide is derived from a class VI virus.

[0067] In one example, the viral RNA-binding protein or peptide is from a respiratory virus selected from the group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, and bocavirus.

[0068] In one example, the viral RNA-binding protein or peptide is derived from an influenza virus. For example, the influenza virus is influenza A. In another example, the influenza virus is influenza B.

[0069] In one example, the viral RNA-binding protein or peptide is from a respiratory syncytial virus.

[0070] In one example, the viral RNA-binding protein or peptide is from a parainfluenza virus.

[0071] In one example, the viral RNA-binding protein or peptide is from a metapneumovirus.

[0072] In one example, the viral RNA-binding protein or peptide is from a rhinovirus.

[0073] In one example, the viral RNA-binding protein or peptide is from a coronavirus, for example, the coronavirus is Severe Acute Respiratory Syndrome 2 (SARS-CoV 2).

[0074] In one example, the viral RNA-binding protein or peptide is derived from an adenovirus.

[0075] In one example, the viral RNA-binding protein or peptide is from a bocavirus.

[0076] In one example, the viral RNA binding protein or peptide is a nucleoprotein, a nonstructural protein, a matrix protein, and / or a nucleocapsid protein. For example, the viral RNA binding protein or peptide is a nucleoprotein. In another example, the viral RNA binding protein or peptide is a matrix protein. In a further example, the viral RNA binding protein or peptide is a nucleoprotein. In another example, the viral RNA binding protein or peptide is a nonstructural protein.

[0077] In one example, the viral RNA-binding protein or peptide comprises a sequence set forth in any one of SEQ ID NOs:9-11.

[0078] In one example, the viral RNA binding protein or peptide is a nonstructural (NS) protein from influenza B virus. For example, the viral RNA binding protein or peptide is influenza B NS1 RNA binding domain (RBD). In one example, the viral RNA binding protein or peptide is influenza B NS1 RBDA. In another example, the viral RNA binding protein or peptide is influenza B NS1 RBDB. In yet another example, the viral RNA binding protein or peptide is influenza B NS1 RBDC. In one example, the influenza B NS1 RNA binding domain is a full length binding domain. In another example, the influenza B NS1 RNA binding domain is a truncated binding domain. In a further example, the influenza B NS1 RNA binding domain is a modified binding domain. In one example, the influenza B NS1 RNA binding domain is set forth in SEQ ID NO: 9. In another example, the influenza B NS1 RNA binding domain is set forth in SEQ ID NO: 10. In a further example, the influenza B NS1 RNA binding domain is set forth in SEQ ID NO: 11. In one example, the influenza B NS1 RNA binding domain is a modified binding domain comprising a first influenza B NS1 RNA binding domain set forth in SEQ ID NO: 11 and a second influenza B NS1 RNA binding domain set forth in SEQ ID NO: 10, where the first and second RNA binding domains are linked by a suitable linker. For example, the 3' end of the first influenza B NS1 RNA binding domain is linked to the 5' end of the second influenza B NS1 RNA binding domain.

[0079] In one example, the viral nucleic acid binding protein is from a hepadnavirus, for example, the hepadnavirus is Hepatitis B virus (HBV).

[0080] In one example, the viral RNA-binding protein or peptide is a nuclear protein, where the RNA-binding protein or peptide encapsulates the RNA, stabilizes the RNA, and inhibits induction of signaling by one or more endosomal Toll-like receptors (e.g., TLR3, TLR7, TLR8, and / or TLR9).

[0081] In one example, the viral RNA-binding protein or peptide is a nucleocapsid, where the RNA-binding protein or peptide encapsulates the RNA, stabilizes the RNA, and inhibits induction of signaling by one or more endosomal Toll-like receptors (e.g., TLR3, TLR7, TLR8 and / or TLR9).

[0082] In one example, the viral RNA-binding protein or peptide is a substrate protein, where the RNA-binding protein or peptide binds to and stabilizes the RNA but does not inhibit induction of signaling by one or more endosomal Toll-like receptors (e.g., TLR3, TLR7, TLR8 and / or TLR9).

[0083] In one example, the RNA binding protein or peptide is a non-viral RNA binding protein or peptide. For example, the RNA binding protein or peptide is a non-viral protein or peptide derived from a cellular protein. In one example, the RNA binding protein or peptide is derived from a cellular protein associated with cell proliferation, cell signaling and / or antiviral pathways.

[0084] In one example, the cellular protein is selected from the group consisting of TAR RNA binding protein (TRBP), protein kinase R (PKR) RNA binding protein, Toll-like receptor 3 (TLR-3) binding protein, TLR-7 binding protein, and combinations thereof.

[0085] In one example, the cellular protein comprises a sequence set forth in any one of SEQ ID NOs:1-8.

[0086] In one example, the cellular protein is TAR RNA binding protein (TRBP). For example, the cellular protein is TRBP RNA binding domain (RBD) 2. In one example, the cellular protein is TRBP RBDA. In another example, the cellular protein is TRBP RBDB. In one example, the TRBP RNA binding domain 2 is full length. For example, the full length TRBP RNA binding domain 2 is set forth in SEQ ID NO: 1. In another example, the TRBP RNA binding domain 2 is a truncated binding domain. For example, the truncated TRBP RNA binding domain 2 is set forth in SEQ ID NO: 2.

[0087] In one example, the cellular protein is a protein kinase R (PKR) RNA binding protein. For example, the cellular protein is PKR RNA binding motif 2. In one example, the cellular protein is PKR RNA binding domain (RBD). In another example, the cellular protein is PKR RBDA. In a further example, the cellular protein is PKR RBDB. In one example, the PKR RNA binding motif 2 is a full-length binding motif. For example, the full-length PKR RNA binding motif 2 is set forth in SEQ ID NO:3. In another example, the PKR RNA binding motif 2 is a truncated binding motif. For example, the truncated PKR RNA binding motif 2 is set forth in SEQ ID NO:4. In another example, the truncated PKR RNA binding motif 2 is set forth in SEQ ID NO:5.

[0088] In one example, the cellular protein is TLR-3 dsRNA binding domain 1. For example, the cellular protein is TLR-3 dsRNA binding domain 1 (leucine-rich repeats 1-3). In another example, the cellular protein is TLR-3 dsRNA binding domain 1 (leucine-rich repeats 17-18). In one example, the TLR-3 dsRNA binding domain 1 is set forth in SEQ ID NO:6. In a further example, the TLR-3 dsRNA binding domain 1 is set forth in SEQ ID NO:7. In another example, the TLR-3 is TLR-3 leucine-rich repeat (LRR) A. In yet another example, the TLR-3 is TLR-3 LRRB.

[0089] In one example, the cellular protein is a TLR-7 RNA binding site. For example, the cellular protein is a TLR-7 RNA binding site (leucine-rich repeats 14-15). In one example, the TLR-7 RNA binding site is set forth in SEQ ID NO: 8. In another example, the TLR-7 is TLR-7 leucine-rich repeat (LRR) A.

[0090] In one example, the lipid nanoparticles further comprise PEG lipids, structured lipids, and / or neutral lipids. For example, the lipid nanoparticles further comprise PEG lipids, structured lipids, and neutral lipids. In another example, the lipid nanoparticles further comprise PEG lipids, structured lipids, or neutral lipids. In one example, the lipid nanoparticles further comprise PEG lipids, structured lipids, ionizable lipids, and / or neutral lipids. For example, the lipid nanoparticles further comprise PEG lipids, structured lipids, ionizable lipids, and neutral lipids. In another example, the lipid nanoparticles further comprise PEG lipids, structured lipids, ionizable lipids, or neutral lipids.

[0091] In one example, the lipid nanoparticle further comprises a PEG lipid, for example, the PEG lipid is selected from the group consisting of PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipid, and combinations thereof.

[0092] In one example, the lipid nanoparticle further comprises a structured lipid, for example, the structured lipid is selected from the group consisting of cholesterol, campesterol, and combinations thereof.

[0093] In one example, the lipid nanoparticle further comprises a neutral lipid, for example, the neutral lipid is selected from the group consisting of DSPC, DOPE, DLPC, DMPC, DOPC, DPPC, and combinations thereof.

[0094] In one example, the lipid nanoparticle further comprises an ionizable lipid.

[0095] In one example, the ionizable lipids are 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-di Methylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DM A), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (also called heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) (SM-102), 2-hexyldecanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexanediyl]ester (((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis( 2-Hexyldecanoate) (ALC-0315), 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (DLin-MC3-DMA or MC3), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester, and combinations thereof.

[0096] In one example, the lipid nanoparticles do not include ionizable lipids.

[0097] In one example, the lipid nanoparticles do not include cationic lipids.

[0098] In one example, the lipid nanoparticles have an average particle size of about 80 nm to 200 nm. For example, the lipid nanoparticles have an average particle size of about 100 nm to 200 nm. In one example, the lipid nanoparticles have an average particle size of about 100 nm to 190 nm, or about 100 nm to 180 nm, or about 110 nm to 180 nm, or about 110 nm to 150 nm, or about 110 nm to 140 nm, or about 110 nm to 130 nm. For example, the lipid nanoparticles have an average particle size of about 125 nm. In one example, the lipid nanoparticles have an average particle size of about 150 to 200 nm. In one example, the lipid nanoparticles have an average particle size of about 160 to 200 nm. For example, the lipid nanoparticles have an average particle size of about 160 nm, or about 165 nm, or about 170 nm, or about 175 nm, or about 180 nm, or about 185 nm, or about 190 nm, or about 200 nm. In one example, the average particle size is determined by measuring the Z-average diameter of the lipid nanoparticles.

[0099] In one example, the lipid nanoparticles have a nitrogen to phosphate ratio of about 2 to about 10. For example, the lipid nanoparticles have a nitrogen to phosphate ratio of about 2, or about 2.5, or about 3, or about 3.5, or about 4, or about 4.5, or about 5, or about 5.5, or about 6, or about 6.5, or about 7, or about 7.5, or about 8, or about 8.5, or about 9, or about 9.5, or about 10. In one example, the lipid nanoparticles have a nitrogen to phosphate ratio of about 3. In another example, the lipid nanoparticles have a nitrogen to phosphate ratio of about 4.5. In a further example, the lipid nanoparticles have a nitrogen to phosphate ratio of about 6.

[0100] In one example, at least 50% of the RNA is encapsulated in the lipid nanoparticle. For example, at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% of the RNA is encapsulated in the lipid nanoparticle. In one example, at least 80% of the RNA is encapsulated. In another example, at least 85% of the RNA is encapsulated. It will be clear to those skilled in the art that encapsulation efficiency (or encapsulation percentage) can be determined by measuring the escape or activity of the pharmaceutical composition or mRNA of the present disclosure using fluorescence (e.g., using RiboGreen) and / or electron microscopy.

[0101] In one example, the RNA is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA) and antisense RNA.

[0102] In one example, the RNA is mRNA. For example, the mRNA is a self-replicating mRNA (sa-mRNA) or a conventional mRNA (cRNA). In one example, the mRNA is a sa-mRNA. In another example, the mRNA is a cRNA.

[0103] In one example, the RNA is a siRNA.

[0104] In one example, the RNA is a miRNA.

[0105] In one example, the RNA is antisense RNA.

[0106] The present disclosure also provides an immunogenic composition comprising the lipid nanoparticles of the present disclosure. For example, the composition of the present disclosure can induce an immune response in a subject when administered. For example, administration of the composition induces a humoral immune response and / or a cellular immune response. In one example, the composition induces a humoral immune response in a subject. For example, the humoral immune response is an antibody-mediated immune response. In another example, the composition induces a cellular immune response. For example, the cellular immune response includes the activation of antigen-specific cytotoxic T cells.

[0107] The present disclosure also provides a pharmaceutical composition comprising an immunogenic composition of the present disclosure and a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers suitable for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0108] The present disclosure also provides an immunogenic or pharmaceutical composition of the present disclosure for use in therapy. For example, the immunogenic or pharmaceutical composition of the present disclosure is suitable for use as a vaccine.

[0109] In one example, the immunogenic or pharmaceutical composition of the disclosure is supplied in a vial, hi another example, the immunogenic or pharmaceutical composition of the disclosure is supplied in a syringe.

[0110] In one example, an immunogenic or pharmaceutical composition of the disclosure is stable for at least 60 days at 4° C. In another example, an immunogenic or pharmaceutical composition of the disclosure is stable for at least 90 days at 4° C.

[0111] Additional embodiments of the present disclosure are as follows.

[0112] 1. A lipid nanoparticle for delivering RNA, comprising an RNA-binding protein or peptide therein that is bound to said RNA.

[0113] 2. The lipid nanoparticle described in 1, wherein the RNA-binding protein or peptide is a lipidated RNA-binding protein or peptide.

[0114] 3. A lipid nanoparticle for delivering RNA, comprising a lipidated RNA-binding protein or peptide therein that is bound to said RNA.

[0115] 4. The lipid nanoparticle described in 2 or 3, wherein the RNA-binding protein or peptide is lipidated before binding to the RNA.

[0116] 5. The lipid nanoparticle according to any one of 2 to 4, wherein the RNA-binding protein or peptide is lipidated with a lipid moiety selected from the group consisting of fatty acids, isoprenoids, and combinations thereof.

[0117] 6. The lipid nanoparticle according to 5, wherein the fatty acid is a triglyceride, a phospholipid, or a cholesteryl ester.

[0118] 7. The lipid nanoparticle described in any one of 2 to 6, wherein the RNA-binding protein or peptide is lipidated on a nucleophilic side chain at the N-terminus and / or C-terminus.

[0119] 8. The lipid nanoparticle according to 7, wherein the nucleophilic side chain is a cysteine, serine, threonine, tyrosine and / or lysine amino acid residue.

[0120] 9. The lipid nanoparticle described in any one of 2 to 8, wherein the RNA-binding protein or peptide is lipidated by palmitoylation, myristoylation, fatty acid acylation, esterification, prenylation, or a combination thereof.

[0121] 10. The lipid nanoparticle according to 9, wherein the RNA-binding protein or peptide is lipidated by N-terminal cysteine ​​palmitoylation, N-terminal glycine myristoylation, lysine N-acylation, C-terminal cholesterol esterification, cysteine ​​prenylation, serine O-acylation, or a combination thereof.

[0122] 11. The lipid nanoparticle described in 9 or 10, wherein the prenylation is farnesylation or geranylgeranylation.

[0123] 12. The lipid nanoparticle according to any one of 5 to 11, wherein the lipid moiety is linked to the RNA-binding protein or peptide by a thioether bond, an ester bond, a thioester bond and / or an amide bond.

[0124] 13. The lipid nanoparticle according to any one of 2 to 12, wherein the RNA-binding protein or peptide is lipidated using chemical or enzymatic lipidation.

[0125] 14. The lipid nanoparticle described in 13, wherein the RNA binding protein or peptide is lipidated using a chemical lipidation method selected from the group consisting of chemical ligation, click chemistry, expressed protein ligation, and combinations thereof.

[0126] 15. The lipid nanoparticle according to 13, wherein the RNA binding protein or peptide is lipidated using enzymatic lipidation selected from the group consisting of sortase A-mediated lipidation, transglutaminase-mediated lipidation, and combinations thereof.

[0127] 16. The lipid nanoparticle according to 15, wherein the enzymatic lipidation is carried out in vivo or in vitro.

[0128] 17. The lipid nanoparticle described in any one of 1 to 16, wherein the RNA-binding protein or peptide encapsulates the RNA.

[0129] 18. The lipid nanoparticle described in any one of 1 to 17, wherein the RNA-binding protein or peptide directly binds to the RNA.

[0130] 19. The RNA-binding protein or peptide is a) reducing the toxicity of the lipid nanoparticles; and / or b) stabilizing the RNA; and / or c) protecting the RNA from degradation, and / or d) promoting nucleation of the lipid nanoparticles; and / or e) A lipid nanoparticle described in any one of 1 to 18, which inhibits the induction of signal transduction by one or more Toll-like receptors.

[0131] 20. The lipid nanoparticle described in any one of 1 to 19, wherein the RNA binding protein or peptide is modified to remove nuclear localization signal(s) and / or introduce nuclear export signal(s).

[0132] 21. The lipid nanoparticle described in any one of 1 to 20, wherein the RNA-binding protein or peptide is a viral or non-viral RNA-binding protein or peptide.

[0133] 22. The lipid nanoparticle described in 21, wherein the viral RNA-binding protein is derived from a class III, class IV, class V, and / or class VI virus.

[0134] 23. The lipid nanoparticle described in 22, wherein the viral RNA-binding protein or peptide is a respiratory virus selected from the group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus and bocavirus.

[0135] 24. The lipid nanoparticle described in 23, wherein the viral RNA-binding protein or peptide is a nucleoprotein, a nonstructural protein, a matrix protein, and / or a nucleocapsid protein.

[0136] 25. The lipid nanoparticle described in any one of 21 to 24, wherein the viral RNA-binding protein or peptide is a nonstructural (NS) protein derived from influenza B virus.

[0137] 26. The lipid nanoparticle described in any one of 21 to 25, wherein the viral RNA-binding protein or peptide comprises a sequence described in any one of SEQ ID NOs: 9 to 11.

[0138] 27. The lipid nanoparticle described in 21, wherein the non-viral RNA-binding protein or peptide is derived from a cellular protein associated with cell proliferation, cell signaling and / or antiviral pathways.

[0139] 28. The lipid nanoparticle described in 27, wherein the cellular protein is selected from the group consisting of TAR RNA binding protein (TRBP), protein kinase R (PKR) RNA binding protein, Toll-like receptor 3 (TLR-3) binding protein, TLR-7 binding protein, and combinations thereof.

[0140] 29. The lipid nanoparticle described in 27 or 28, wherein the cellular protein comprises an array set forth in any one of SEQ ID NOs: 1 to 8.

[0141] 30. The lipid nanoparticle described in any one of 1 to 29, wherein the lipid nanoparticle further comprises a PEG lipid, a structured lipid, and / or a neutral lipid.

[0142] 31. The lipid nanoparticle according to any one of 1 to 30, which does not contain a cationic lipid.

[0143] 32. The lipid nanoparticle described in any one of 1 to 31, wherein the RNA is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA) and antisense RNA.

[0144] 33. The lipid nanoparticle described in 29, wherein the mRNA is a self-replicating mRNA (sa-mRNA) or a conventional RNA (cRNA).

[0145] 34. An immunogenic composition comprising the lipid nanoparticles according to any one of 1 to 33.

[0146] 35. A pharmaceutical composition comprising the lipid nanoparticles according to any one of 1 to 33 or the immunogenic composition according to 34, and a pharma- ceutically acceptable carrier.

[0147] 36. A lipid nanoparticle according to any one of 1 to 33, an immunogenic composition according to 34, or a pharmaceutical composition according to 35, for use in therapy.

[0148] Key to sequence table SEQ ID NO: 1: Amino acid sequence of full-length TAR RNA-binding protein domain 2 SEQ ID NO:2 Amino acid sequence of truncated TAR RNA binding protein domain 2 SEQ ID NO:3 Amino acid sequence of protein kinase R RNA binding motif 2 SEQ ID NO: 4 Truncated protein kinase R RNA binding motif 2 (#1) The amino acid sequence of SEQ ID NO:5 Truncated protein kinase R RNA binding motif 2 (#2) The amino acid sequence of SEQ ID NO:6 Toll-like receptor 3 dsRNA binding domain 1 (Leucine-rich Amino acid sequence of repeats 1-3 SEQ ID NO: 7 Toll-like receptor 3 dsRNA binding domain 1 (Leucine-rich Amino acid sequence of repeats 17-18 SEQ ID NO:8 Toll-like receptor 7 RNA binding site (leucine-rich repeat 14-15) Amino acid sequence SEQ ID NO: 9 Amino acid sequence of influenza B NS1 RNA binding domain SEQ ID NO: 10 Amino acid sequence of truncated influenza B NS1 RNA binding domain array SEQ ID NO: 11 Amino acid sequence of modified influenza B NS1 RNA binding domain array [Brief description of the drawings]

[0149] [Figure 1]FIG. 1 is a graphical representation of a rabbit reticulocyte lysate assay showing the amount of RNA measured by luciferase production assessed by measuring luminescence in relative light units (RLU) for nanoluciferase RNA (nLuc RNA) alone or in combination with influenza virus RNA-free nucleoprotein (NP:nLuc RNA). [Diagram 2] 1 is a graphical representation of the amount of RNA in (A) NP:nLuc RNA samples and (B) nLuc RNA alone samples after treatment with or without heat labile proteinase K (PK) and / or RNase. [Diagram 3] 1 is a series of graphical representations showing the stability of nLuc RNA in nLuc RNA alone and NP:nLuc RNA samples at (A) 4° C., (B) 24° C., and (C) 37° C. after incubation for up to 96 hours. [Figure 4A] 1 is a graphical representation showing the levels of TLR3 induction in nLuc RNA alone and NP:nLuc RNA samples at 2 and 4 hours after incubation. [Figure 4B] 1 is a graphical representation showing the levels of TLR3 induction in nLuc RNA alone and NP:nLuc RNA samples at 2 and 4 hours after incubation. [Figure 4C] 1 is a graphical representation showing the levels of TLR8 induction in nLuc RNA alone and NP:nLuc RNA samples at 2 and 4 hours after incubation. [Figure 4D] 1 is a graphical representation showing the levels of TLR8 induction in nLuc RNA alone and NP:nLuc RNA samples at 2 and 4 hours after incubation. [Diagram 5](A) A series of graphical representations showing the protection of RNA degradation by SARS-CoV-2 nucleocapsids as measured by luciferase production assessed by measuring luminescence in RLU in nanoluciferase RNA (RNA) alone or in combination with COVID RNA-free nucleocapsid (RNA+NP(SCov2)), and (B) dye exclusion essay of nLuc RNA with and without COVID RNA-free nucleocapsid. [Figure 6] 1 is a graphical representation showing the level of protection afforded by an RNA-binding peptide reacted with RNA compared to free RNA. The dashed line represents the level of protection in the absence of peptide (LHS), which is equivalent to free RNA and 100% protection. [Figure 7A] 13 is a graphical representation showing expression of nLuc RNA in NP:nLuc RNA samples without RNase inhibitors. [Figure 7B] 13 is a graphical representation showing expression of nLuc RNA in NP:nLuc RNA samples with and without RNase inhibitors. [Figure 7C] 13 is a graphical representation showing expression of nLuc RNA in NP:nLuc RNA samples without RNase inhibitors. [Figure 7D] 13 is a graphical representation showing expression of nLuc RNA in NP:nLuc RNA samples with and without RNase inhibitors. [Figure 8] 1 is a graphical representation showing nLuc expression (measured as relative light units, RLU) in Hela cells transfected with NP:nLuc RNA and nLuc RNA. [Figure 9] 1 is a graphical representation showing nLuc RNA expression in the spleen and liver. [Figure 10] 1 is a graphical representation showing the stability over time of LNPs formulated with nLuc mRNA or NP-nLuc mRNA at 4° C. after incubation for up to 90 days. [Figure 11A]1 is a graphical representation showing the biodistribution of LNPs formulated with nLuc mRNA and LNPs formulated with nLuc mRNA and nucleoprotein (NP) at an MC3:PEG ratio of 0:1.5. [Figure 11B] 1 is a graphical representation showing the biodistribution of LNPs formulated with nLuc mRNA and LNPs formulated with nLuc mRNA and nucleoprotein (NP) at a MC3:PEG ratio of 5:1.5. [Figure 11C] 1 is a graphical representation showing the biodistribution of LNPs formulated with nLuc mRNA and LNPs formulated with nLuc mRNA and nucleoprotein (NP) at an MC3:PEG ratio of 10:1.5. [Figure 11D] 1 is a graphical representation showing the biodistribution of LNPs formulated with nLuc mRNA and LNPs formulated with nLuc mRNA and nucleoprotein (NP) at a MC3:PEG ratio of 15:15. [Figure 11E] 1 is a graphical representation showing the biodistribution of LNPs formulated with nLuc mRNA and LNPs formulated with nLuc mRNA and nucleoprotein (NP) at an MC3:PEG ratio of 20:1.5. [Figure 11F] 1 is a graphical representation showing the biodistribution of LNPs formulated with nLuc mRNA and LNPs formulated with nLuc mRNA and nucleoprotein (NP) at a MC3:PEG ratio of 50:1.5. [Figure 12A] 1 is a graphical representation of LNPs formulated with mRNA or mRNA NuPs showing LNP size with varying nitrogen to phosphate ratios. [Figure 12B] 1 is a graphical representation of LNPs formulated with mRNA or mRNA NuPs showing encapsulation efficiency at varying nitrogen to phosphate ratios. [Figure 12C] 1 is a graphical representation of LNPs formulated with mRNA or mRNA NuPs showing biodistribution at varying nitrogen to phosphate ratios. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0150] overview Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps, or group of compositions of matter shall be interpreted as encompassing one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0151] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present invention also includes all of the steps, features, compositions and compounds referred to or shown herein, individually or collectively, as well as any combination or any two or more of the steps or features described above.

[0152] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for illustrative purposes only, and functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0153] Any example of the present disclosure shall apply mutatis mutandis to any other example of the present disclosure, unless specifically stated otherwise.

[0154] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., of cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0155] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques used in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMG lover and BD Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), FM Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all revisions to date), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JE Coligan et al. (editors) Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1998). These techniques are described and explained throughout the literature in such sources as Protocols in Immunology, John Wiley & Sons (including all current editions).

[0156] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by a discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901-917, 1987, Chothia et al. Nature 342, 877-883, 1989, and / or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[0157] Any discussion of a protein or antibody herein will be understood to include any variants of the protein or antibody produced during production and / or storage. For example, during production and / or storage, an antibody may be deamidated (e.g., at asparagine or glutamine residues), and / or glycosylation may be altered, and / or glutamine residues may be converted to pyroglutamic acid, and / or N- or C-terminal residues may be removed or "clipped," and / or some or all of the signal sequence may be incompletely processed, resulting in a residue at the end of the antibody. It is understood that a composition comprising a particular amino acid sequence may be a heterogeneous mixture of the described or encoded sequence and / or variants of the described or encoded sequence.

[0158] The term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as providing clear support for both meanings or either meaning.

[0159] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0160] As used herein, the term "derived from" shall be interpreted as indicating that a particular entity is not necessarily obtained directly from a particular source, but may be obtained from that source.

[0161] Selected Definitions As used herein, the term "lipid nanoparticle" or "LNP" shall be understood to refer to a lipid-based particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) and comprising a compound of any formula described herein. In embodiments, LNPs are formulated in compositions for delivering polynucleotides to desired targets, such as cells, tissues, organs, tumors, etc. For example, lipid nanoparticles or LNPs can be selected from, but are not limited to, liposomes or vesicles in which the aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single, unilamellar or multiple, multilamellar), micelle-like lipid nanoparticles with a non-aqueous core, and solid lipid nanoparticles (solid lipid nanoparticles lack a lipid bilayer).

[0162] As used herein, the term "lipidated" or "lipidation" refers to the process of covalently modifying a protein (i.e., an RNA-binding protein or peptide) with one or more lipids.

[0163] As used herein, the term "RNA binding protein or peptide" or "RBP" shall be understood to refer to proteins and peptides that bind to double-stranded or single-stranded RNA and participate in the formation of ribonucleoprotein complexes.

[0164] The term "protein" shall be taken to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains that are covalently or non-covalently bound to one another (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently linked using suitable chemicals or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0165] The term "peptide" as used herein is intended to include compounds composed of amino acid residues linked by amide bonds. Peptides may be natural or non-natural, ribosomally encoded or synthetically derived. Typically, peptides consist of 2-200 amino acids. For example, peptides may have a length ranging from 10-20 amino acids or 10-30 amino acids or 10-40 amino acids or 10-50 amino acids or 10-60 amino acids or 10-70 amino acids or 10-80 amino acids or 10-90 amino acids or 10-100 amino acids, including any length within the above range(s).

[0166] As used herein, the term "recombinant" shall be understood to mean the product of artificial genetic recombination.

[0167] As used herein, the term "self-replicating RNA" refers to an RNA virus-based construct that has been engineered to allow expression of heterologous RNA and proteins. Self-replicating RNA (e.g., in the form of naked RNA) can be amplified in a host cell, leading to expression of a desired gene product within the host cell.

[0168] As used herein, the term "conventional RNA" or "cRNA" or "non-amplified RNA" refers to a construct that allows for the expression of heterologous RNA and proteins, but the RNA cannot be amplified in a host cell.

[0169] As used herein, the term "subject" shall be taken to mean any animal, e.g., a mammal, including a human. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0170] Lipid Nanoparticles The present disclosure provides lipid nanoparticles for delivering RNA, the lipid nanoparticles comprising a nucleic acid binding protein or peptide bound to RNA. For example, the lipid nanoparticles comprise a lipidated nucleic acid binding protein or peptide bound to RNA.

[0171] The present disclosure provides lipid nanoparticles for delivering RNA, the lipid nanoparticles comprising a lipidated RNA-binding protein or peptide bound to RNA.

[0172] Nucleic acid binding proteins or peptides The present disclosure provides lipid nanoparticles comprising a nucleic acid binding protein or peptide. For example, the present disclosure provides lipid nanoparticles comprising a lipidated nucleic acid binding protein or peptide.

[0173] In one example, the nucleic acid binding protein is an RNA binding protein or peptide, hi another example, the nucleic acid binding protein is an RNA binding protein or peptide and a DNA binding protein or peptide.

[0174] RNA-binding proteins or peptides The present disclosure provides lipid nanoparticles that include an RNA-binding protein or peptide. For example, the present disclosure provides lipid nanoparticles that include a lipidated RNA-binding protein or peptide.

[0175] RNA binding proteins regulate many aspects of co-transcriptional and post-transcriptional gene expression, including, for example, RNA splicing, RNA editing, polyadenylation, export, mRNA stabilization, mRNA localization and translation. RNA binding proteins or peptides bind to double-stranded or single-stranded RNA and participate in the formation of ribonucleoprotein complexes. Those skilled in the art will understand that RNA binding proteins or peptides can be viral or non-viral proteins or peptides.

[0176] Non-viral RNA-binding proteins or peptides In one example, the RNA binding protein is a non-viral protein or peptide derived from a cellular protein, for example, the RNA binding protein or peptide is derived from a cellular protein associated with cell proliferation, cell signaling and / or anti-viral pathways.

[0177] Non-viral RNA-binding proteins or peptides contain a number of structural motifs or RNA-binding domains that facilitate RNA binding, including, for example, RNA recognition motifs (RRMs), K homology (KH) domains (type I and type II), RGG (Arg-Gly-Gly) boxes, Sm domains; DEAD / DEAH boxes, CCCH-type zinc fingers (ZnFs), double-stranded RNA-binding motifs (dsRBDs), cold shock domains; Pumilio / FBF (PUF or Pum-HD) domains, and Piwi / Argonaute / Zwille (PAZ) domains.

[0178] In one example, the RNA binding protein or peptide comprises an RNA binding domain selected from the group consisting of an RNA recognition motif, a K homology domain (type I or type II), and a CCCH-type zinc finger.

[0179] In one example, the RNA binding protein or peptide comprises an RNA recognition motif. For example, the RNA binding protein or peptide comprising an RNA recognition motif includes A2BP1, ACF, BOLL, BRUNOL4, BRUNOL5, BRUNOL6, CCBL2, CGI-96, CIRBP, CNOT4, CPEB2, CPEB3, CPEB4, CPSF7, CSTF2, CSTF2T, CUGBP1, CUGBP2, D10S102, DAZ1, DAZ2, DAZ3, DAZ4, DAZAP1, DAZL, DNAJC17, DND1, EIF3S4, EIF3S9, EIF4B, EIF4H, ELAVL1, ELA VL2, ELAVL3, ELAVL4, ENOX1, ENOX2, EWSR1, FUS, FUSIP1, G3BP, G3BP1, G3BP2, GRSF1, HNRNPL, HNRPA0, HNRPA1, HNRPA2B1, HNRPA3, HNRPAB, HNRPC, HNRRPCL1, HNRPD, HNRPDL, HNRPF, HNRPH1, HNRPH2, HNRPH3, HNRPL, HNRPLL, HNRPM, HNRPR, HRNBP1, HSU53209, HTATSF1, IGF2BP1, IGF2BP2, IGF2BP3 , LARP7, MKI67IP, MSI1, MSI2, MSSP-2, MTHFSD, MYEF2, NCBP2, NCL, NOL8, NONO, P14, PABPC1, PABPC1L, PABPC3, PABPC4, PABPC5, PABPN1, PKR, POLD IP3, PPARGC1, PPARGC1A, PPARGC1B, PPIE, PPIL4, PPRC1, PSPC1, PTBP1, PTBP2, PUF60, RALY, RALYL, RAVER1, RAVER2, RBM10, RBM11, RBM12, RBM12B , RBM14, RBM15, RBM15B, RBM16, RBM17, RBM18, RBM19, RBM22, RBM23, RBM24, RBM25, RBM26, RBM27, RBM28, RBM3, RBM32B, RBM33, RBM34, RBM35A, RBM 35B, RBM38, RBM39, RBM4, RBM41, RBM42, RBM44, RBM45, RBM46, RBM47, RBM4B, RBM5, RBM7, RBM8A, RBM9, RBMS1, RBMS2, RBMS3, RBMX, RBMX2, RBMXL2,RBMY1A1, RBMY1B, RBMY1E, RBMY1F, RBMY2FP, RBPMS, RBPMS2, RDBP, RNPC3, RNPC4, RNPS1, ROD1, SAFB, SAFB2, SART3, SERB P1, SETD1A, SF3B14, SF3B4, SFPQ, SFRS1, SFRS10, SFRS11, SFRS12, SFRS15, SFRS2, SFRS2B, SFRS3, SFRS4, SFRS5, SFRS6, S Selected from the group consisting of FRS7, SFRS9, SLIRP, SLTM, SNRP70, SNRPA, SNRPB2, SPEN, SR140, SRRP35, SSB, SYNCRIP, TAF15, TRBP, THOC4, TIA1, TIAL1, TNRC4, TNRC6C, TRA2A, TRSPAP1, TUT1, U1SNRNPBP, U2AF1, U2AF2, UHMK1, ZCRB1, ZNF638, ZRSR1, and ZRSR2.

[0180] In one example, the RNA binding protein or peptide comprises a K homology domain. For example, the K homology domain is a type I domain. In another example, the K homology domain is a type II domain. In one example, the RNA binding protein or peptide comprising a K homology domain is selected from the group consisting of AKAP1, ANKHD1, ANKRD17, ASCC1, BICC1, DDX43, DDX53, DPPA5, FMR1, FUBP1, FUBP3, FXR1, FXR2, GLD1, HDLBP, HNRPK, IGF2BP1, IGF2BP2, IGF2BP3, KHDRBS1, KHDRBS2, KHDRBS3, KHSRP, KRR1, MEX3A, MEX3B, MEX3C, MEX3D, NOVA1, NOVA2, PCBP1, PCBP2, PCBP3, PCBP4, PNO1, PNPT1, QKI, SF1, and TDRKH.

[0181] In one example, the RNA binding domain comprises a CCCH-type zinc finger domain.

[0182] Exemplary non-viral RNA-binding proteins or peptides will be apparent to those of skill in the art and include, for example, TAR RNA-binding protein (TRBP), protein kinase R (PKR), Toll-like receptor 3 (TLR-3) and Toll-like receptor 7 (TLR).

[0183] Viral RNA-binding proteins In one example, the RNA binding protein or peptide is a viral RNA binding protein or peptide, e.g., the RNA binding protein is a nucleoprotein, matrix protein, nucleocapsid protein, and / or a nonstructural protein from an RNA virus.

[0184] As shown in Table 1, those skilled in the art will appreciate that viruses are classified according to the Baltimore classification system, which is primarily based on the transcription of the viral genome. [Table 1]

[0185] In one example, the RNA binding protein or peptide is from an RNA virus, e.g., the RNA binding protein or peptide is from a class III, class IV, class V, and / or class IV virus.

[0186] In one example, the RNA virus is a class III virus (i.e., a double-stranded RNA virus). Class III viruses include, for example, all viruses in the phylum Duplonaviricota and all viruses in the class Duplopiviricetes (of the phylum Pisuviricota). Exemplary class III viruses include, but are not limited to, reoviruses (e.g., orthoreoviruses, rotaviruses, orbiviruses, or coltiviruses).

[0187] In one example, the RNA virus is a Class IV virus (i.e., a positive-sense single-stranded RNA virus). Class IV viruses include, for example, viruses of the phyla Lenarviricota, Pisuviricota (excluding the class Duplopidiviricetes), and Kitrinoviricota. Exemplary Class IV viruses include togaviruses (e.g., rubiviruses, alphaviruses, or arteriviruses), flaviviruses (e.g., tick-borne encephalitis (TBE) virus, dengue (types 1, 2, 3, or 4) virus, yellow fever virus, Japanese encephalitis virus, Kyasanur Forest disease virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Powassan encephalitis virus), picornaviruses (e.g., enteroviruses, rhinoviruses, heparnaviruses, parechoviruses, cardioviruses, and aphthoviruses), enteroviruses (e.g., polioviruses types 1, 2, or 3, coxsackie A viruses types 1-22 and 24, coxsackie B viruses types 1-6, echovirus (ECHO) type 1, Examples of viruses that may be present include, but are not limited to, viruses such as enteroviruses 68-71, pestiviruses (e.g., bovine viral diarrhea (BVDV), classical swine fever (CSFV) or border disease (BDV)), caliciviruses (e.g., Norwalk virus, and Norwalk-like viruses (e.g., Hawaii virus and Snow Mountain virus)), coronaviruses (e.g., severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), SARS coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), infectious bronchitis virus (IBV), mouse hepatitis virus (MHV), and transmissible gastroenteritis virus (TGEV)), and hepatitis E virus (HEV).

[0188] In one example, the RNA virus is a class V virus (i.e., a negative-sense single-stranded RNA virus). Class V viruses include, for example, viruses of the phylum Negarnaviricota. Exemplary class V viruses include, but are not limited to, orthomyxoviruses (e.g., influenza A, B, and C), Paramyxoviridae viruses (Pneumoviruses (e.g., respiratory syncytial virus (RSV), bovine respiratory syncytial virus, pneumonia virus of mice, and turkey rhinotracheitis virus), paramyxoviruses types 1-4 (PIV), mumps, Sendai virus, Simian virus 5, Nipah virus, Henipavirus, Newcastle disease virus, morbilliviruses (e.g., measles), Bunyaviruses (e.g., California encephalitis virus), Phleboviruses (e.g., Rift Valley fever virus), Nairoviruses (e.g., Crimean-Congo hemorrhagic fever virus), Rhabdoviruses (e.g., Lyssavirus (rabies virus) and Vesiculovirus (VSV)), Delta hepatitis virus (HDV), and Arenaviruses.

[0189] In one example, the class V RNA virus is an influenza virus, such as influenza A virus. In another example, it is an influenza B virus.

[0190] In one example, the RNA virus is a class VI virus (i.e., a single-stranded RNA virus with a DNA intermediate during its life cycle). Class VI viruses include, for example, viruses of the class Revtraviricetes (phylum Artverviricota, excluding Caulimoviridae). Exemplary class VI viruses include, but are not limited to, heparnaviruses (e.g., hepatitis A virus (HAV)), hepadnaviruses (e.g., hepatitis B virus, hepatitis C virus), and retroviruses (e.g., oncoviruses, lentiviruses, or spumaviruses). In one example, the RNA virus is a hepadnavirus. For example, the hepadnavirus is hepatitis B virus (HBV).

[0191] Linker In one example, the RNA binding protein or peptide comprises a first RNA binding protein or peptide and a second RNA binding protein or peptide linked via a linker, e.g., the linker is a linker peptide.

[0192] In one example, the linker is a flexible linker.

[0193] A "flexible" linker is an amino acid sequence that does not have a fixed structure (secondary or tertiary structure) in solution. Thus, such a flexible linker is free to adopt various conformations. Flexible linkers suitable for use in the present disclosure are known in the art. One example of a flexible linker for use in the present invention is the linker sequence SGGGGS / GGGGS / GGGGS or (Gly4Ser)3. Another example of a flexible linker is an alanine linker (e.g., Ala n ).

[0194] The linker may comprise any amino acid sequence that does not substantially interfere with the interaction of the RNA-binding protein or peptide with the RNA. Preferred amino acid residues for flexible linker sequences include, but are not limited to, glycine, alanine, serine, threonine, proline, lysine, arginine, glutamine and glutamic acid.

[0195] The linker sequence between the RNA-binding proteins or peptides preferably comprises 5 or more amino acid residues. The flexible linker sequence according to the present disclosure consists of 5 or more residues, preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more residues. In a highly preferred embodiment of the present invention, the flexible linker sequence consists of 5, 7, 10, or 16 residues.

[0196] In one example, the linker is a rigid linker. A "rigid linker" (including a "semi-rigid linker") refers to a linker that has limited flexibility. For example, a relatively rigid linker may have the sequence (EAAAK) n where n is 1 to 3. The value of n can be 1 to about 10, or about 1 to 100. For example, n is at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10. In one example, n is less than 100. For example, n is less than 90, or less than about 80, or less than about 70, or less than about 60, or less than about 50, or less than about 40, or less than about 30, or less than about 20, or less than about 10. A rigid linker need not be completely devoid of flexibility.

[0197] Lipidation of binding proteins The present disclosure provides lipid nanoparticles comprising lipidated nucleic acid binding proteins or peptides.

[0198] The present disclosure provides lipid nanoparticles comprising lipidated RNA-binding proteins or peptides.

[0199] It will be apparent to one of skill in the art that lipidation of a protein or peptide is the covalent attachment of a lipid moiety to a protein or peptide (ie, an RNA binding protein or peptide).

[0200] Lipids Lipid moieties suitable for use in the present disclosure will be apparent to one of skill in the art and include, for example, fatty acids, isoprenoids, and combinations thereof. In one example, the lipid moiety is selected from the group consisting of isoprenoids, triglycerides, phospholipids, cholesteryl esters, and combinations thereof.

[0201] Isoprenoids Isoprenoids, also known as terpenoids or prenol lipids, are branched lipids, a class of organic compounds composed of two or more hydrocarbon units, each of which consists of five carbon atoms arranged in a specific pattern. These five carbon units are called isoprenes, which are synthesized from a common intermediate known as mevalonic acid, which is itself synthesized from acetyl-CoA. Isoprenoids may have one or more functional chemical groups, such as hydroxyl or carbonyl, attached to their carbon skeleton, which constitute the diversity of isoprenoids. Isoprenoids are classified into monoterpenes (C 10 H 16 ), sesquiterpenes (C 15 H 24 ), diterpenes (C 20 H 32 ), triterpenes (C 30 H 48 ), tetraterpenes (C 40 H 64 ), or other polyterpenes (C5H8) n It can be classified as:

[0202] Suitable isoprenoids for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0203] In one example, the isoprenoid is a monoterpene. Exemplary monoterpenes include citronellol, citronellal, citral, geraniol, metol, pseudoionone, and β-ionone.

[0204] In one example, the isoprenoid is a sesquiterpene. Exemplary sesquiterpenes include cadalene, eudalene, cadinene, and β-selinene.

[0205] In one example, the isoprenoid is a diterpene. Exemplary diterpenes include phytol and abietic acid.

[0206] In one example, the isoprenoid is a triterpene. Exemplary triterpenes include squalene and β-amyrin.

[0207] In one example, the isoprenoid is a tetraterpene. Exemplary tetraterpenes include carotenoids (e.g., β-carotene) and lycopene.

[0208] fatty acid Fatty acids are lipids that contain a long chain hydrocarbon terminated with a carboxylic acid functional group. Fatty acids can be saturated or unsaturated. In one example, a fatty acid contains a carbon chain having 6 to 22 carbons. Exemplary fatty acids include palmitic acid, myristic acid, oleic acid, α-linolenic acid, and stearic acid.

[0209] Fatty acids are rarely found in free form in nature, but generally exist as three major types of esters: triglycerides, phospholipids, and cholesteryl esters.

[0210] In one example, the fatty acid is a triglyceride. A triglyceride is a triester consisting of glycerol linked to three fatty acid molecules via ester bonds. The three fatty acids may be the same or different. An exemplary triglyceride is tristearin.

[0211] In one example, the fatty acid is a phospholipid. A phospholipid is a complex lipid that includes a hydrophilic polar head group containing one or more phosphate groups and a hydrophobic tail containing two fatty acyl chains. The polar head group is linked to the hydrophobic portion by a phosphodiester bond via a glycerol (i.e., phosphoglyceride) or sphingosine molecule (i.e., phosphosphingolipid). Phospholipids can be saturated or unsaturated. Exemplary phosphoglycerides include phosphatidic acid (phosphatidate), phosphatidylethanolamine (cephalin), phosphatidylcholine (lecithin), phosphatidylserine, phosphoinositides (e.g., phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidylinositol diphosphate (PIP2) and phosphatidylinositol triphosphate (PIP3)), phosphatidylglycerol, and cardiolipin. Exemplary phosphosphingolipids include ceramide phosphorylcholine (sphingomyelin), ceramide phosphorylethanolamine (sphingomyelin), ceramide phosphoryl lipid, galactocerebroside, glucocerebroside, and lactosylceramide.

[0212] In one example, the fatty acid is a cholesteryl ester. Cholesteryl esters are cholesterol esterified with long chain fatty acids. Exemplary cholesteryl esters include cholesteryl oleate, cholesteryl benzoate, and cholesteryl linoleate.

[0213] Lipidization Exemplary lipidations include palmitoylation, myristoylation, fatty acid acylation, esterification, prenylation, or a combination thereof.

[0214] Palmitoylation In one example, a lipid moiety is attached to an RNA-binding protein or peptide by palmitoylation.

[0215] In one example, palmitoylation is cysteine ​​palmitoylation (also known as S-palmitoylation). Those skilled in the art will understand that cysteine ​​palmitoylation is the addition of a 16-carbon palmitoyl group to a cysteine ​​residue of a protein. In one example, the palmitoyl group is added via a thioester bond. In another example, the palmitoyl group is added via an amide bond.

[0216] Myristoylation In one example, the lipid moiety is attached to the RNA-binding protein or peptide by myristoylation.

[0217] In one example, myristoylation is N-glycine myristoylation. Those skilled in the art will recognize that N-glycine myristoylation refers to the co- or post-translational attachment of myristoyl, a saturated 14-carbon fatty acyl group, to the N-terminal glycine of a protein via an amide bond.

[0218] In one example, the myristoylation is lysine myristoylation.

[0219] Fatty acid acylation In one example, a lipid moiety is attached to an RNA-binding protein or peptide by fatty acid acylation.

[0220] Those of skill in the art will recognize that fatty acid acylation involves the covalent attachment of an acyl group to a protein.

[0221] In one example, fatty acid acylation is lysine N-acylation, which one of skill in the art will understand to refer to the transfer of an acetyl moiety from acetyl-CoA to the epsilon (ε)-amino group of a lysine residue on a protein.

[0222] Esterification In one example, the lipid moiety is attached to the RNA-binding protein or peptide by esterification.

[0223] In one example, the esterification is a C-terminal sterol esterification, such as a C-terminal cholesterol esterification. Those skilled in the art will understand that a C-terminal cholesterol esterification is the replacement of at least one hydroxyl (-OH) group with an alkoxy (-O-alkyl) group.

[0224] Prenylation In one example, a lipid moiety is attached to an RNA-binding protein or peptide by prenylation.

[0225] In one example, the prenylation is cysteine ​​prenylation, which one of skill in the art will appreciate is the addition of multiple isoprene units to a cysteine ​​residue near the C-terminus of a protein.

[0226] In one example, the prenylation is farnesylation (ie, the addition of three isoprene units) or the prenylation is geranylgeranylation (ie, the addition of four isoprene units).

[0227] In one example, the bond between the farnesyl or geranylgeranyl group and the cysteine ​​residue is a thioether bond. In another example, the bond is an ester bond. In a further example, the bond is a thioester bond.

[0228] Method of lipidation Lipid modifications typically occur at nucleophilic side chains of the protein or peptide (eg, cysteine, serine, and lysine) at the N-terminus and / or C-terminus of the protein or peptide.

[0229] Various methods of lipidation will be apparent to those skilled in the art and / or are described herein. Suitable methods may include chemical lipidation or enzymatic lipidation.

[0230] chemical lipidation In one example, the lipid moiety is attached to the RNA-binding protein or peptide using chemical ligation. The lipid moiety can include an amine, a carboxylic acid, a hydrazide, or a maleimide group, and the lipid moiety can be chemically attached to the RNA-binding protein or peptide via a primary amine group of lysine or a thiol group of cysteine. In one example, the lipid moiety includes a maleimide group, and the lipid moiety is attached to the RNA-binding protein or peptide via the formation of a thioether bond with a sulfhydryl group in the RNA-binding protein or peptide. In one example, the lipid moiety includes a carboxylic acid, and the carboxylic acid is activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS). The activated acylamino ester or sulfo-NHS ester is then reacted with the primary amine of a lysine residue in the RNA-binding protein or peptide to form an amide bond.

[0231] In one example, the lipid moiety includes a maleimide group. For example, the lipid moiety is a phospholipid capped with a maleimide group. In one example, the lipid moiety is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-maleimide (DSPE-maleimide, DSPE-Mal).

[0232] In one example, lipid moieties are attached to RNA-binding proteins or peptides using various "click chemistry" strategies such as those disclosed in Kolb et al. (2001), WO2003 / 101972 and Malkoch et al. (2005).

[0233] In one example, lipid moiety is linked to RNA-binding protein or peptide by using expressed protein ligation.Expressed protein ligation involves the chemoselective ligation between a protein or peptide with a C-terminal thioester and a protein or peptide with an N-terminal cysteine ​​in aqueous solution at physiological pH.In one example, C-terminal thioester is inserted into RNA-binding protein or peptide by genetic engineering, and lipid moiety is fused to a peptide with an N-terminal cysteine ​​residue.

[0234] Other chemical lipidation methods known to those skilled in the art may also be used, such as the methods disclosed in Takahara & Kamiya (2020).

[0235] enzymatic lipidation In one example, lipid moieties are attached to RNA-binding proteins or peptides using enzymatic lipidation.Enzymatic lipidation can be carried out in vivo or in vitro.In some examples, RNA-binding proteins or peptides are genetically engineered using techniques known to those skilled in the art to include consensus sequences that are recognized by lipidation enzymes.

[0236] In one example, lipid moieties are attached to RNA-binding proteins or peptides using sortase A-mediated lipidation. Sortase A (e.g., SrtA from Staphylococcus aureus) binds Ca 2+ In the presence of a sortase, secreted proteins are covalently attached to bacterial cell wall peptidoglycan via a transpeptidation reaction. In this example, the RNA-binding protein or peptide is engineered to contain an LPXTG motif (e.g., LPETG) at the C-terminus, and the lipid moiety contains a nucleophile and an oligo-glycine motif (e.g., triglycine, tetraglycine, or pentaglycine). Upon addition of a sortase, the RNA-binding protein or peptide is covalently attached to the lipid via a peptide bond.

[0237] In one example, lipid moieties are attached to RNA-binding proteins or peptides using transglutaminase-mediated lipidation. Transglutaminase (e.g., microbial transglutaminase: MTG) binds Ca 2+ In the absence of , the RNA-binding protein or peptide is engineered to contain an MTG lysine recognition sequence (e.g., MRHKGS) at, for example, the N-terminus or C-terminus, and the lipid portion contains an MTG glutamine recognition sequence (e.g., LLQG). In one example, the RNA-binding protein or peptide is engineered to contain an MTG glutamine recognition sequence (e.g., LLQG or LQ) at, for example, the N-terminus or C-terminus, and the lipid portion contains an MTG lysine recognition sequence (e.g., MRHKGS).

[0238] Other enzymatic lipidation methods known to those skilled in the art may also be used, such as the method disclosed in Takahara & Kamiya (2020).

[0239] Additional lipids In one example, the lipid nanoparticles further comprise PEG lipids, sterol structure lipids and / or neutral lipids. In one example, the lipid nanoparticles further comprise PEG lipids, sterol structure lipids, ionizable lipids and / or neutral lipids. In one example, the lipid nanoparticles do not comprise cationic lipids.

[0240] PEGylated lipids In one example, the present disclosure provides a lipid nanoparticle comprising a PEGylated lipid.

[0241] It will be clear to those skilled in the art that the reference to PEGylated lipid refers to lipid modified with polyethylene glycol.Exemplary PEGylated lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.For example, PEG lipids include PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0242] neutral lipid In one example, the present disclosure provides a lipid nanoparticle comprising a neutral lipid.

[0243] Neutral or zwitterionic lipids suitable for use in the present disclosure will be apparent to those of skill in the art and include, for example, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DO ... 1-Oleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. The lipids may be saturated or unsaturated.

[0244] structural lipids In one example, the present disclosure provides lipid nanoparticles comprising structured lipids.

[0245] Exemplary structural lipids include, but are not limited to, cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and α-tocopherol.

[0246] In one example, the structured lipid is a sterol. For example, the structured lipid is cholesterol. In another example, the structured lipid is campesterol.

[0247] Ionizable lipids In one example, the present disclosure provides lipid nanoparticles comprising an ionizable lipid.

[0248] Ionizable lipids suitable for use in the present disclosure will be apparent to those of skill in the art and include, for example, 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DL-DMA ... ), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-Distearyloxy-N,N-Dimethyl-3-aminopropane (DSDMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (D Lin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), ,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 2,5-bis((9z,12z)-octadeca-9,12,Dien-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (also called heptadecane-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) (SM-102), 2-hexyldecanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexanediyl] ester (((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl) Bis(2-hexyldecanoate) (ALC-0315), 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (DLin-MC3-DMA or MC3), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester, and combinations thereof.

[0249] Pharmaceutically acceptable carriers Suitably, in the composition or method for administering the lipid nanoparticles of the present disclosure to a subject, the lipid nanoparticles are combined with a pharma- ceutically acceptable carrier, as understood in the art.Thus, one example of the present disclosure provides a composition (e.g., a pharmaceutical composition) that includes the lipid nanoparticles of the present disclosure in combination with a pharma- ceutically acceptable carrier.

[0250] Generally, a "carrier" refers to a solid or liquid filler, binder, diluent, encapsulating substance, emulsifier, wetting agent, solvent, suspending agent, coating, or lubricant that can be safely administered to any subject, such as a human. Depending on the particular route of administration, a variety of acceptable carriers known in the art can be used, for example, as described in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991).

[0251] The lipid nanoparticles of the present disclosure are useful for parenteral, topical, oral or topical, intramuscular, aerosol, or transdermal administration for preventive or therapeutic treatment.In one example, the lipid nanoparticles are administered parenterally, such as intramuscularly, subcutaneously, or intravenously.For example, the lipid nanoparticles are administered intramuscularly.

[0252] The formulation of lipid nanoparticles to be administered varies depending on the route of administration and the formulation selected (e.g., solution, emulsion, capsule). A suitable pharmaceutical composition containing lipid nanoparticles to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate or fixed oils. A variety of suitable aqueous carriers are known to those skilled in the art, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives, fluid, nutrient or electrolyte replenishers (see generally Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The composition may optionally contain pharma- ceutically acceptable auxiliary substances such as pH adjusting and buffering agents and toxicity adjusting agents (e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate) as necessary to approximate physiological conditions. The lipid nanoparticles may be stored in a liquid phase or lyophilized for storage and reconstituted in a suitable carrier prior to use according to lyophilization and reconstitution techniques known in the art.

[0253] The optimal concentration of the active ingredient(s) (i.e., RNA) in the selected medium can be determined empirically according to procedures known to those skilled in the art and will depend on the desired final pharmaceutical formulation.

[0254] When formulated, the composition of the present disclosure is administered in a manner compatible with the dosage formulation and in a therapeutically / prophylactically effective amount.The dosage range of the lipid nanoparticles of the present disclosure is an amount sufficient to produce the desired effect.For example, the composition comprises an effective amount of encapsulated RNA.In one example, the composition comprises a therapeutically effective amount of RNA.In another example, the composition comprises a prophylactically effective amount of RNA.

[0255] The dosage should not be so large as to cause side effects. In general, the dosage varies according to the age, condition, sex and degree of disease of the patient, which can be determined by those skilled in the art. In case of complications, the dosage can be adjusted by the individual physician.

[0256] RNA The present disclosure provides lipid nanoparticles for delivering RNA, wherein nucleic acid binding protein or peptide is bound to RNA.For example, the present disclosure provides lipid nanoparticles for delivering RNA, wherein lipidated nucleic acid binding protein or peptide is bound to RNA.

[0257] The present disclosure provides lipid nanoparticles for delivering RNA, wherein RNA-binding protein or peptide is bound to RNA.For example, the present disclosure provides lipid nanoparticles for delivering RNA, wherein RNA-binding protein or peptide is bound to RNA.

[0258] The RNA of the present disclosure may be natural or non-naturally occurring RNA, or may contain one or more modified nucleic acid bases, nucleosides, or nucleotides. It will be apparent to those skilled in the art that the RNA suitable for use in the present disclosure may also contain a 5' untranslated region (5'-UTR), a 3' untranslated region (3'UTR), and / or a coding or translation sequence. In addition, the RNA may contain a 5' cap structure, a chain terminating nucleotide, a stem loop (e.g., a histone stem loop), a 3' tailing sequence (e.g., a polyadenylation signal or one or more polyA tails).

[0259] In one example of the present disclosure, the RNA is a self-replicating mRNA (sa-mRNA).

[0260] In one example of the present disclosure, the RNA is conventional mRNA (cRNA).

[0261] Preparation method Suitable methods for preparing the lipid nanoparticles of the present disclosure are clear to those skilled in the art and / or described herein.For example, the lipid nanoparticles of the present disclosure can be made using approaches well known in the formulation field.For example, suitable LNPs can be formed using mixing processes such as microfluidics, including herringbone micromixing and T-junction mixing of two fluid streams, one of which contains messenger RNA, typically in aqueous solution, and the other of which has various required lipid components, typically in ethanol.

[0262] The LNPs can then be prepared by mixing phospholipids (such as DOPE or DSPC, which can be purchased from commercial sources including Avanti Polar Lipids, Alabaster, AL), PEGylated lipids (such as 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol, also known as PEG-DMG, which can be purchased from commercial sources including Avanti Polar Lipids, Alabaster, AL), and structured lipids / sterols (such as cholesterol, which can be purchased from commercial sources including Sigma-Aldrich) at a concentration of, for example, about 50 mM in ethanol. The solution should be refrigerated, for example at -20°C, during storage. The various lipids can be combined to obtain the desired molar ratio and then diluted with water and ethanol to the final desired lipid concentration, for example, about 5.5 mM to about 25 mM.

[0263] LNP compositions containing RNA (including, but not limited to, sa-mRNA or cRNA) can be prepared by mixing the lipid solution described above with a solution containing RNA, for example, at a weight:weight ratio of lipid components to RNA of about 5:1 to about 50:1. Using a NanoAssemblr microfluidic system, the lipid solution can be rapidly injected into the RNA solution at a flow rate of about 3 ml / min to about 18 ml / min to generate a suspension with a water to ethanol ratio of about 1:1 to about 4:1.

[0264] For LNP compositions containing sa-mRNA or cRNA, a 1.0 mg / ml concentration of RNA solution in deionized water can be diluted with 50 mM sodium citrate buffer at pH 3-6 to form a stock solution.

[0265] As known in the art, the LNP composition can be further processed by diluting 10-fold in 50 mM citrate buffer at pH 6 and subjected to tangential flow filtration (TFF) using a 300,000 molecular weight cut-off membrane (mPES) until concentrated to the original volume. In one example, the citrate buffer can then be replaced with a buffer containing 20 mM Tris buffer at pH 7.5, 80 mM sodium chloride, and 3% sucrose using diafiltration with 10-fold volume of new buffer. The LNP solution can be concentrated, for example, to a volume of 5-10 mL, filtered using a 0.2 micron PES syringe filter, aliquoted into vials, and frozen at 1° C. / min using a Corning® CoolCell® LX cell freezing container until the sample reaches −80° C. Samples may be stored at −80° C. until needed.

[0266] The above method results in nanoprecipitation and particle formation. The same nanoprecipitation can be achieved using alternative processes including, but not limited to, T-junction and direct injection.

[0267] In some embodiments, the lipid component of the LNP formulation comprises about 2 mol% to about 25 mol% phospholipids (neutral lipids), about 18.5 mol% to about 60 mol% structured lipids (sterols), and about 0.2 mol% to about 10 mol% PEGylated lipids, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the LNP formulation comprises about 5 mol% to about 20 mol% phospholipids, about 30 mol% to about 55 mol% structured lipids, and about 1 mol% to about 5 mol% PEGylated lipids. In certain embodiments, the lipid component comprises about 10 mol% phospholipids, about 48 mol% structured lipids, and about 2.0 mol% PEG lipids. In some embodiments, the phospholipids can be DOPE or DSPC. In other embodiments, the PEG lipid can be PEG-DMG and / or the structured lipid can be cholesterol.

[0268] The encapsulation efficiency of RNA in the LNP can be at least 50%, for example, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0269] Assays for lipid nanoparticles of the present disclosure The lipid nanoparticles of the present disclosure are readily screened for physical and biological activity and / or stability using methods known in the art and / or described below.

[0270] Assessment of RNA degradation In one example, the level of RNA degradation by RNase is assessed, e.g., RNA is treated with RNase, either alone or in combination with an RNA-binding protein or peptide.

[0271] In one example, the level of RNA is assessed in RNAse treated and untreated samples using real-time PCR. In one example, the cycle threshold (CT) value in the RNA sample without RNA binding protein or peptide is increased compared to the RNA sample with RNA binding protein or peptide, indicating RNA degradation.

[0272] Assessment of RNA translation In one example, RNA translation is assessed using an in vitro translation system. Suitable systems for use in the present disclosure will be apparent to those of skill in the art and include, for example, the rabbit reticulocyte lysate assay.

[0273] In one example, a rabbit reticulocyte lysate assay is used.

[0274] In one example, RNA is assessed in the presence or absence of an RNA binding protein or peptide.

[0275] In one example, the RNA is nanoluciferase RNA (nLuc RNA) and the amount of RNA translation is measured by the amount of luciferase produced, assessed by measuring luminescence in relative light units (RLU). In one example, the assay is performed at 4° C., 24° C. and / or 37° C. In another example, the assay is performed after incubating the samples for 0 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours or 96 hours.

[0276] Assessment of TLR induction In one example, the level of TLR3 and / or TLR8 induction is evaluated. For example, the level of TLR3 and / or TLR8 induction by RNA alone or RNA in combination with an RNA-binding protein or peptide is evaluated. In one example, TLR3 and / or TLR8 induction is evaluated using a TLR-induced NfKB reporter assay. In this assay, NfKB is operatively linked to secreted alkaline phosphatase (SEAP). RNA is introduced into either cell type (conditionally transduced TLR3 or conditionally transduced TLR8). Binding of the TLR receptor induces NfKB activation, which in turn induces SEAP. In one example, SEAP levels are determined by chemical reaction and calorific readout. EXAMPLES

[0277] Example 1: Protection of RNA from degradation by RNases RNA-free monovalent pool harvest (MPH) was prepared by treating MPH with RNase.

[0278] MPH (B / Malaysia / 2506 / 2004) was treated twice with RNase A (Promega) for 1 h at 37°C. RNA was extracted from 140 μl of treated and untreated MPH (eluted in 60 μl) and tested for hemagglutinin (HA) and neuraminidase (NA) RNA using real-time PCR. Results showed that the CT value of HA RNA did not increase and that of NA RNA increased slightly (Table 2, experiment 1A), indicating minimal degradation of viral RNA.

[0279] In the case where RNase was inactive, MPH was treated twice with a different RNase (RNase ONE, Promega M4261) prior to RNA extraction and analysis. The C values ​​of both HA and NA RNA increased (Table 2, experiment 1B), indicating a reduction (<83-fold) in the amount of viral RNA present.

[0280] An additional sample of MPH (180 μl) was treated twice with a reduced dose of RNase ONE in the presence of RNase ONE Sample Buffer. Analysis of the treated samples showed a dose-dependent decrease in RNA concentration and an increase in C values ​​(Table 2, experiment 1C), indicating that treatment of MPH with RNase ONE resulted in low levels of RNA degradation (<35-fold). Overall, the levels of RNA degradation in treated MPH were unexpectedly low.

[0281] MPH (B / Malaysia / 2506 / 2004) was then treated with RNase ONE in the presence or absence of disruption buffer. The use of disruption buffer did not increase the degradation of viral RNA in RNase-treated MPH (Table 2, experiment 2).

[0282] When MPH was treated with RNase, only viral RNA was slightly decreased, suggesting that the RNA may be protected from degradation by RNase. [Table 2]

[0283] Example 2: Purification of RNA-free nucleoproteins from influenza viruses RNA-free nucleoprotein (NP) was isolated from influenza virus by dialysis, concentration, and detergent treatment followed by glycerol step gradient centrifugation to isolate NP:RNA particles. Additional glycerol and cesium chloride gradient centrifugation steps were performed to isolate RNA-free NP.

[0284] Example 3: RNA-free NPs bind to RNA To assess whether RNA-free NPs protect RNA, NPs were combined with nanoluciferase mRNA and samples were heated to 40°C or incubated at room temperature and then analyzed by MOPS-agarose gel electrophoresis. Increasing the concentration of NPs in the reaction (from 0 to 4000 ng) while maintaining the concentration of mRNA (250 ng) shifted the level of detectable mRNA to higher molecular weights.

[0285] At a ratio of 16:1 (NP 4000ng, mRNA 250ng), the nanoluciferase mRNA was shifted so that it could not enter the gel. These results indicate that increasing the NP concentration increases the amount of NPs that can form a complex with RNA.

[0286] Using rabbit reticulocyte lysate as an in vitro translation system, we showed that at a ratio of 16:1 (4000 ng NP, 250 ng mRNA), luciferase production, assessed by measuring luminescence in relative light units (RLU), was comparable to luciferase RNA alone, but 4000 ng of NP alone reduced the signal by 2-3 fold (Figure 1). These results indicate that as NP concentrations are increased, RNA binds to NP without inhibiting in vitro translation.

[0287] Example 4: RNA-free NPs protect RNA from degradation To assess whether RNA-free NPs protect RNA, NP:RNA and RNA alone were evaluated in an RNase assay. Briefly, NP:RNA or RNA was treated with RNase and incubated at 30°C for 5-10 min. Samples were further treated with or without 1 μl of heat-labile proteinase K (PK, NEB P8111S). Reactions were incubated at 37°C for 15-30 min, followed by incubation at 60°C for 10-20 min to inactivate PK. When required, 1-2 μl of RNasine (Promega N2611) was added. Real-time PCR was used to assess the levels of RNA present in treated and untreated samples.

[0288] As shown in Figure 2, there was no increase in the CT value for NP:RNA treated with RNase, indicating no degradation of viral RNA. In contrast, the CT value for RNA treated with RNase alone increased significantly (p=0.0005), indicating a decrease in the amount of viral RNA present. These results further confirm that in the presence of NP, RNA is protected from degradation by RNase.

[0289] The addition of PK, which degrades NP, to NP:RNA showed a slight increase in the CT value, indicating minimal degradation of the RNA. The addition of PK in combination with RNase showed a significant increase in the CT value (<0.0001), indicating a reduction in the amount of viral RNA present. These results further confirm that in the presence of NP, RNA is protected from degradation by RNase.

[0290] Example 5: RNA-free NPs protect RNA from degradation at 4°C, 24°C, and 37°C To assess whether the ability of NPs to protect nanoluciferase RNA (nLuc RNA) was temperature dependent, NP:nLuc RNA and nLuc RNA were incubated at 4°C, 24°C, and 37°C for up to 96 h and luciferase production was assessed by measuring luminescence in RLU.

[0291] As shown in Figure 3, there was no significant difference in RLU between NP:nLuc RNA and nLuc RNA alone at 4°C. At 24°C, there was a significant decrease in RLU for nLuc RNA compared to NP:nLuc RNA after 96 hours of incubation (p=0.0034), indicating that NP protected the RNA from degradation. After 8 hours at 37°C, there was a significant decrease in RLU for nLuc RNA compared to NP:nLuc RNA, demonstrating further long-term protection of RNA by NP.

[0292] Example 6: RNA-free NPs protect against TLR induction To assess whether the presence of NPs inhibits RNA induction of dsRNA or ss RNA, TLR3 and / or TLR8 induction, a TLR-induced NfKB reporter assay was used. In this assay, NfKB is operatively linked to secreted alkaline phosphatase (SEAP). RNA is introduced into either cell type (conditionally transduced TLR3 or conditionally transduced TLR8). TLR receptor binding induces NfKB activation, which in turn induces SEAP. SEAP levels can be determined by chemical reaction and calorific readout.

[0293] As shown in FIG. 4, RNA alone stimulates the induction of both TLR3 and TLR8, while the presence of NP reduces the induction of TLR3 and TLR8 at both 2 and 4 hours after incubation.

[0294] Example 7: Conjugation of RNA-free NPs with maleimide-DSPE lipids NPs were conjugated with maleimide-DSPE by incubating NPs (0.85 mg / ml) with DSPE (1,2-distearoyl-sn-glycero-3-phosphorylethanolamine, 8 mM) in the presence of 10% ethanol. Labeled and unlabeled NPs were run on a non-reducing gel to assess protein molecular weight and confirm protein conjugation with the molecular weight shift of NPs. Additionally, because DSPE forms a suspension (i.e., liposomes), the compositions were centrifuged and the supernatants were evaluated.

[0295] It was subsequently shown that lipidated NP:RNA could be pulled down by centrifugation, whereas RNA alone could not be pulled down in the presence of lipids (i.e., not in the presence of NPs).

[0296] Example 8: COVID nucleocapsid protects RNA RNA-free nucleocapsids from SARS-CoV-2 (NP(SCoV2)) were purified as described above for influenza virus. The ability of NP(SCoV2) to protect nLuc RNA from degradation was evaluated in an in vitro translation system by measuring luciferase production, assessed by measuring luminescence in RLU for up to 168 h.

[0297] The RLU of nLuc RNA alone decreased over time, indicating degradation of nLuc RNA, as shown in Figure 5. There appeared to be a slight decrease in RLU of the NP(SCoV2):nLuc RNA compositions at 16:1 and 8:1 (w / w), suggesting RNA degradation, however, PCR analysis of the NP(SCoV2):nLuc RNA material at each time point confirmed that the RNA was intact and not degraded.

[0298] Furthermore, the accessibility of RNA encapsulated in NP(SCoV2) was assessed using a RiboGreen dye exclusion assay. As shown in Figure 5B, increasing concentrations of NP(SCoV2) prevent the binding of the dye to nLuc RNA, but based on the fluorescent signal, the dye is still accessible to the RNA at a ratio of 16:1 (NP 4000ng, mRNA 250ng). This suggests that NP(SCoV2) coats and protects the RNA but does not completely encapsulate the RNA.

[0299] Example 9: Preparation of RNA-binding peptides Cellular and viral protein sequences were examined to identify protein domains and peptide sequences with potential to bind RNA. Sequences from cellular proteins correlate with proteins involved in cell proliferation, cell signaling, and / or antiviral pathways, whereas sequences from viral proteins are derived from nonstructural and nuclear proteins.

[0300] Peptides were designed from sequences derived from cellular proteins including TAR RNA-binding protein (TRBP), protein kinase R (PKR), Toll-like receptor 3 (TLR-3), and TLR-7 (Table 3). Viral RNA-binding proteins included nucleoproteins and nonstructural proteins from influenza (Table 4).

[0301] The RNA-binding peptide sequences were modified to either exclude known nuclear localization signals or to include nuclear export signals to facilitate precise localization of the peptide-binding RNA when it was introduced into cells (Table 3). [Table 3] [Table 4]

[0302] Example 10: Protection of RNA from Degradation in the Presence of RNA-Binding Peptides To assess whether RNA-binding peptides (RBPs) protect RNA, real-time PCR was used to assess RBP:RNA and nanoluciferase (nLuc) RNA alone and the levels of nLuc RNA remaining after RNase treatment. The RBPs assessed were TAR RNA-binding protein (TRBP) domains A and B, protein kinase R (PKR) domains A and B, influenza nonstructural protein (NS RBD) domains A, B, and C, and Toll-like receptor 7 (TLR-7).

[0303] Briefly, RBP:RNA samples were prepared by mixing RBP and NLuc RNA and incubating at 37° C. for 1 hour. RBP:RNA and NLuc RNA samples were then prepared with and without RNase (Promega M426A). RNase-treated samples were incubated at room temperature for 30 minutes. Real-time PCR was used to assess the levels of RNA present in treated and untreated samples.

[0304] Real-time PCR was performed in the presence of SYBR-green (QuantiNova SYBR green RT-PCR kit #208152).

[0305] The C values ​​were compared (Table 5) and the protection rate was calculated. As shown in Figure 6, NS RBDC, TRBP RBDA, and TRBP RBDB showed a level of protecting RNA from RNase degradation. The remaining binding domains showed that the residual RNA was below the level of non-peptide-bound RNA. [Table 5]

[0306] HBV, dengue, RSV, and influenza A were also evaluated (Figure 7). The data show that HBV binding protects the RNA when RNase is added. However, recombinant dengue does not allow transcription similar to RSV, which is non-protective. Recombinant influenza A and B NP are only partially protective.

[0307] Example 11: In vivo expression of RNA:NP To evaluate whether NP:RNA can be translated in cells, Hela cells were transfected with NP:RNA or nLuc RNA alone. Briefly, 10 ng / well of NP:RNA or nLuc RNA was added to cells along with Lipofectamine 3000. Cells were washed 2 hours after transfection, and nLuc production was evaluated by measuring luminescence in RLU 24 hours after transfection. As shown in Figure 8, NP:RNA is expressed in Hela cells.

[0308] As shown in FIG. 9, nLuc RNA expression was also measured in the spleen and liver.

[0309] Example 12: Formulation of NP:mRNA LNPs LNPs were prepared with and without RNA bound to nucleoprotein. To determine whether nucleoprotein was fully encapsulated, various LNP formulations were developed (Table 6) and analyzed for average size (Z-Ave) and polydispersity index (PDI) (Table 7). LNPs were developed with or without nucleoprotein, and RNA was placed inside (internal) or on the surface (external) of the LNP. [Table 6] [Table 7]

[0310] Example 13: NP:LNP Thermal Stability To assess the stability of the NP:LNP formulations over time, the LNPs were evaluated at 60 and 90 days after injection by measuring the Z-average diameter of the LNPs at 4° C. As shown in FIG 10, the LNPs containing NPs demonstrated thermal stability over time at 4° C.

[0311] Example 14: In vivo biodistribution of LNP mRNA To evaluate the biodistribution of 0:1.5, 15:1.5, and 50:1, LNP formulations with and without NPs were injected intramuscularly into mice, and then the biodistribution in organs was evaluated 6 days after injection by measuring the amount of luminescence signal (RLU) per mg of each organ.

[0312] LNPs formulated with NP:mRNA or nLuc mRNA alone were detected in the draining and non-draining lymph nodes, spleen, liver, and muscle, as shown in Figure 11. Increased RLUs were observed in the draining and non-draining lymph nodes and liver of mice administered the 15:1.5 NP-mRNA-LNP formulation when compared to mRNA-LNPs.

[0313] The effect of altering the nitrogen (N) to phosphate (P) ratio (N / P) in the LNPs was also evaluated. As shown in FIG. 12, the effect of N / P ratios of 3, 4.5, and 6 on LNP size (A) and encapsulation efficiency (B) was evaluated. The effect on biodistribution was also evaluated, and the results are shown in FIG. 12C. Similar biodistribution of LNP-mRNA and NuP-LNP-mRNA in all other organs was observed at all N / P ratios (3, 4.5, and 6).

Claims

1. A lipid nanoparticle for delivering RNA, comprising: i) a nucleic acid binding protein or peptide bound to said RNA, optionally a nucleic acid binding protein or peptide bound to said lipidated RNA, optionally wherein said nucleic acid binding protein or peptide is an RNA binding protein or peptide; optionally a lipidated RNA-binding protein or peptide bound to said RNA; or ii) an RNA-binding protein or peptide bound to the RNA; The lipid nanoparticles comprising therein.

2. The lipid nanoparticle of claim 1ii), wherein the RNA-binding protein or peptide is a lipidated RNA-binding protein or peptide.

3. the RNA-binding protein or peptide a) lipidated before binding to the RNA; b) lipidated with a lipid moiety selected from the group consisting of fatty acids, isoprenoids, and combinations thereof, optionally wherein: b, i) the fatty acid is a triglyceride, a phospholipid, or a cholesteryl ester; or b,ii) the lipid moiety is linked to the RNA-binding protein or peptide by a thioether bond, an ester bond, a thioester bond, and / or an amide bond; c) lipidated on a nucleophilic side chain at the N-terminus and / or C-terminus, optionally wherein said nucleophilic side chain is a cysteine, serine, threonine, tyrosine and / or lysine amino acid residue; d) lipidated by palmitoylation, myristoylation, fatty acid acylation, esterification, prenylation, or a combination thereof, optionally wherein said RNA-binding protein or peptide is lipidated by N-terminal cysteine ​​palmitoylation, N-terminal glycine myristoylation, lysine N-acylation, C-terminal cholesterol esterification, cysteine ​​prenylation, serine O-acylation, or a combination thereof, optionally wherein said prenylation is farnesylation or geranylgeranylation; e) said RNA binding protein or peptide is lipidated using chemical or enzymatic lipidation, optionally wherein said RNA binding protein or peptide is: e,i) lipidated using a chemical lipidation method selected from the group consisting of chemical ligation, click chemistry, expressed protein ligation, and combinations thereof; e,ii) lipidated using an enzymatic lipidation selected from the group consisting of sortase A-mediated lipidation, transglutaminase-mediated lipidation, and combinations thereof, optionally wherein the enzymatic lipidation is performed in vivo or in vitro.

4. the RNA-binding protein or peptide i) encapsulating the RNA; ii) directly binds to the RNA; iii) wherein said RNA binding protein or peptide is a) reducing the toxicity of said lipid nanoparticles; b) stabilizing the RNA; c) protecting the RNA from degradation; d) promoting the nucleation of said lipid nanoparticles; and / or e) inhibiting the induction of signaling by one or more Toll-like receptors; and / or iv) The lipid nanoparticle of claim 1ii), which is modified to remove nuclear localization signal(s) and / or introduce nuclear export signal(s).

5. the RNA-binding protein or peptide is a viral or non-viral RNA-binding protein or peptide; Optionally, wherein said viral RNA binding protein is from a class III, class IV, class V, and / or class VI virus; Optionally, wherein said viral RNA binding protein or peptide is a respiratory virus selected from the group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, and bocavirus; Optionally, wherein said viral RNA-binding protein or peptide is a nucleoprotein, a nonstructural protein, a matrix protein, and / or a nucleocapsid protein. Lipid nanoparticles according to claim 1ii).

6. the viral RNA-binding protein or peptide a) a nonstructural (NS) protein from influenza B virus; b) comprising a sequence as set forth in any one of SEQ ID NOs: 9 to 11; c) Lipid nanoparticles according to claim 5, derived from cellular proteins associated with cell proliferation, cell signaling and / or antiviral pathways.

7. The lipid nanoparticle of claim 6c), wherein the cellular protein is selected from the group consisting of TAR RNA binding protein (TRBP), protein kinase R (PKR) RNA binding protein, Toll-like receptor 3 (TLR-3) binding protein, TLR-7 binding protein, and combinations thereof.

8. The lipid nanoparticle of claim 6c), wherein the cellular protein comprises a sequence set forth in any one of SEQ ID NOs: 1 to 8.

9. The lipid nanoparticles are a) further comprising PEG lipids, structural lipids, ionizable lipids and / or neutral lipids; b) does not contain cationic lipids; Lipid nanoparticles according to claim 1ii).

10. The lipid nanoparticle of claim 1ii), wherein the RNA is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA) and antisense RNA, optionally wherein the mRNA is self-replicating mRNA (sa-mRNA) or conventional RNA (cRNA).

11. An immunogenic composition comprising the lipid nanoparticles of claim 1ii).

12. A pharmaceutical composition comprising the lipid nanoparticles of claim 1ii) or the immunogenic composition of claim 11 and a pharmaceutically acceptable carrier.

13. A lipid nanoparticle according to claim 1ii), an immunogenic composition according to claim 11 or a pharmaceutical composition according to claim 12 for use in therapy.

Images