RNA delivery vehicle

Abstract

This disclosure relates to an RNA delivery vehicle comprising lipid nanoparticles containing RNA internally and RNA-binding proteins or peptides coated on the outer surface of the lipid nanoparticles, and to the use thereof.

Description

[Technical Field]

[0001] Related application data This application claims priority to U.S. Patent Application No. 63 / 494,274, entitled “RNA Delivery Vehicle,” filed on April 5, 2023. The entire contents of that application are incorporated herein by reference.

[0002] Sequence List This application is filed together with an electronic sequence listing. The entire contents of the sequence listing are incorporated herein by reference.

[0003] This disclosure relates to an RNA delivery vehicle comprising lipid nanoparticles containing RNA internally and RNA-binding proteins or peptides coated on the outer surface of the lipid nanoparticles, and to the use thereof. [Background technology]

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

[0005] mRNA vaccines rely on the delivery of mRNA to the cytoplasm of host cells, where it is transcribed into antigen proteins and triggers the production of neutralizing antibodies. However, mRNA is large and negatively charged, which hinders its uptake by cells. Therefore, lipid delivery vehicles such as liposomes or lipid nanoparticles are used to encapsulate mRNA, preventing its degradation in plasma and promoting its uptake into cells, thereby enabling efficient mRNA delivery in the body. However, lipid nanoparticles do not always encapsulate all mRNA, and some mRNA may remain on the outer surface of the lipid nanoparticles.

[0006] Lipid delivery vehicles are typically composed of cationic lipids and other ionized lipid components such as neutral lipids, cholesterol, and PEGylated lipids. Cationic lipids are amphiphilic molecules having a lipophilic region containing one or more hydrocarbon groups and a hydrophilic region containing at least one positively charged polar head group. Cationic lipids and nucleic acids form a positively charged complex, making it easier for nucleic acids to pass through the cell membrane and enter the cytoplasm.

[0007] However, several harmful cytotoxic effects of cationic lipids are known, including the generation of reactive oxygen species and accumulation in plasma due to inadequate degradation in humans. Therefore, much effort has been devoted to 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 must be well-tolerated, provide an appropriate therapeutic index, and treatment with an effective amount of nucleic acid should not be associated with unacceptable toxicity and / or risk to the patient.

[0008] Therefore, it will be apparent to those skilled in the art that improved lipid nanoparticles for delivering oligonucleotides such as RNA will continue to be needed. [Overview of the project]

[0009] In developing this invention, the inventors have demonstrated that RNA-binding proteins or peptides coated on the outer surface of lipid nanoparticles can increase the stability of the associated RNA, and / or promote nucleation of the lipid nanoparticles, and / or reduce the toxicity and / or adverse side effects of the lipid nanoparticles. The inventors have also demonstrated that RNA-binding proteins or peptides coated on the outside of lipid nanoparticles can exhibit protective effects against stimulation / induction of Toll-like receptors (TLRs). Therefore, the inventors provide a basis for an RNA delivery vehicle comprising lipid nanoparticles containing RNA internally and RNA-binding proteins or peptides coated on the outer surface of the lipid nanoparticles, and a method for using the RNA delivery vehicle as a vaccine or therapeutic agent.

[0010] The inventors further sought to identify a method for determining the quality of RNA delivery vehicles (e.g., detecting exposed RNA on the RNA delivery vehicle that may contribute to RNA destabilization, degradation, and / or reduced efficiency). In particular, the inventors recognized the need for a method to distinguish between unencapsulated and / or partially encapsulated RNA on the outer surface of lipid nanoparticles and RNA inside. The inventors found that a combination of an RNA-binding protein or peptide and an antibody containing a detectable label that binds to it is particularly effective in detecting unencapsulated and / or partially encapsulated RNA on the outer surface of lipid nanoparticles.

[0011] The inventors' discovery provides an RNA delivery vehicle, and the delivery vehicle is (i) Lipid nanoparticles containing RNA inside, (ii) comprising an RNA-binding protein or peptide coated on the outer surface of a lipid nanoparticle.

[0012] In one example, an RNA-binding protein or peptide coated on the outer surface of a lipid nanoparticle encapsulates the lipid nanoparticle. For example, the RNA-binding protein or peptide completely or partially surrounds the outside of the lipid nanoparticle. For example, the RNA-binding protein or peptide completely encapsulates the lipid nanoparticle. In another example, the RNA-binding protein or peptide partially encapsulates the lipid nanoparticle. For example, the RNA-binding protein or peptide does not completely encapsulate the lipid nanoparticle.

[0013] In one example, an RNA-binding protein or peptide directly binds to unencapsulated RNA and / or partially encapsulated RNA on the outer surface of a lipid nanoparticle. In another example, an RNA-binding protein or peptide directly binds to unencapsulated RNA on the outer surface of a lipid nanoparticle. In yet another example, an RNA-binding protein or peptide directly binds to partially encapsulated RNA on the outer surface of a lipid nanoparticle. In yet another example, an RNA-binding protein or peptide directly binds to unencapsulated and partially encapsulated RNA on the outer surface of a lipid nanoparticle.

[0014] In one example, an RNA-binding protein or peptide non-covalently binds to unencapsulated RNA and / or partially encapsulated RNA on the outer surface of a lipid nanoparticle. In another example, an RNA-binding protein or peptide non-covalently binds to unencapsulated RNA on the outer surface of a lipid nanoparticle. In yet another example, an RNA-binding protein or peptide non-covalently binds to partially encapsulated RNA on the outer surface of a lipid nanoparticle. In yet another example, an RNA-binding protein or peptide non-covalently binds to unencapsulated and partially encapsulated RNA on the outer surface of a lipid nanoparticle.

[0015] In one example, the RNA-binding protein or peptide binds directly to the outer surface of the lipid nanoparticle. In another example, the RNA-binding protein or peptide binds directly to the lipid nanoparticle.

[0016] In one example, an RNA-binding protein or peptide non-covalently binds to the outer surface of a lipid nanoparticle. In another example, an RNA-binding protein or peptide non-covalently binds to a lipid nanoparticle.

[0017] In one example, an RNA-binding protein or peptide directly binds to unencapsulated and / or partially encapsulated RNA on the outer surface of a lipid nanoparticle, and also binds to the outer surface of the lipid nanoparticle. In another example, an RNA-binding protein or peptide directly binds to unencapsulated RNA on the outer surface of a lipid nanoparticle and to the outer surface of the lipid nanoparticle. In yet another example, an RNA-binding protein or peptide directly binds to partially encapsulated RNA on the outer surface of a lipid nanoparticle and to the outer surface of the lipid nanoparticle. In yet another example, an RNA-binding protein or peptide directly binds to unencapsulated and partially encapsulated RNA on the outer surface of a lipid nanoparticle, and also binds to the outer surface of the lipid nanoparticle.

[0018] In one example, an RNA-binding protein or peptide non-covalently binds to unencapsulated RNA and / or partially encapsulated RNA on the outer surface of lipid nanoparticles, as well as to the outer surface of lipid nanoparticles. In another example, an RNA-binding protein or peptide non-covalently binds to unencapsulated RNA and to the outer surface of lipid nanoparticles. In yet another example, an RNA-binding protein or peptide non-covalently binds to partially encapsulated RNA and to the outer surface of lipid nanoparticles. In yet another example, an RNA-binding protein or peptide non-covalently binds to unencapsulated RNA and partially encapsulated RNA on the outer surface of lipid nanoparticles, as well as to the outer surface of lipid nanoparticles.

[0019] In another example, an RNA-binding protein or peptide binds to the RNA within the lipid nanoparticle. In such a situation, the RNA-binding protein or peptide is present within the lipid nanoparticle and on the outer surface of the lipid nanoparticle. The RNA-binding protein or peptide within the lipid nanoparticle may not be the same as the RNA-binding protein or peptide coated on the outer surface of the lipid nanoparticle.

[0020] In one example, an 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, an RNA-binding protein or peptide binds to a nucleophilic side chain on the RNA. In one example, an RNA-binding protein or peptide binds to a nucleophilic side chain at the N-terminus of the RNA. In one example, an RNA-binding protein or peptide binds to a nucleophilic side chain at the C-terminus of the RNA. In one example, an RNA-binding protein or peptide binds to nucleophilic side chains at the N-terminus and C-terminus of the RNA.

[0021] In one example, an 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 is at the C-terminus of the RNA. In another example, it is at the N-terminus and C-terminus of the RNA.

[0022] For example, an RNA-binding protein or peptide does not encapsulate the RNA.

[0023] The RNA-binding protein or peptide is a lipidated RNA-binding protein or peptide.

[0024] In one example, an RNA-binding protein or peptide is lipidated before coating the outer surface of the lipid nanoparticle. In another example, an RNA-binding protein or peptide is lipidated after coating the outer surface of the lipid nanoparticle.

[0025] In one example, an RNA-binding protein or peptide is lipidated with a lipid moiety selected from the group consisting of fatty acids, isoprenoids, and combinations thereof.

[0026] In one example, an RNA-binding protein or peptide is lipidized with a fatty acid. For example, the fatty acid may be a triglyceride, phospholipid, or cholesterol ester. In one example, the fatty acid is a triglyceride. In another example, the fatty acid is a phospholipid. In yet another example, the fatty acid is a cholesterol ester.

[0027] For example, RNA-binding proteins or peptides are lipid-modified with isoprenoids. For instance, isoprenoids are a type of isoprenoid.

[0028] In one example, an RNA-binding protein or peptide is lipidized on its N-terminal and / or C-terminal nucleophilic side chains.

[0029] In one example, an RNA-binding protein or peptide is lipid-modified on its nucleophilic side chain. For example, it may be located on cysteine, serine, threonine, tyrosine, and / or lysine amino acid residues. In one example, the nucleophilic side chain is a cysteine ​​residue. In another example, the nucleophilic side chain is a serine residue. In yet another example, the nucleophilic side chain is a threonine residue. In one example, the nucleophilic side chain is a tyrosine residue. In yet another example, the nucleophilic side chain is a lysine residue.

[0030] In one example, RNA-binding proteins or peptides are lipid-modified at the N-terminus of the protein or peptide.

[0031] It will be apparent to those skilled in the art that the N-terminus of an RNA-binding protein or peptide contains a nuclear localization signal(s) (or sequence) and / or nuclear export signal. In one example, an RNA-binding protein or peptide is modified to remove a nuclear localization signal(s) and / or introduce a nuclear export signal(s).

[0032] In one example, an RNA-binding protein or peptide is modified to remove a nuclear localization signal. In another example, an RNA-binding protein or peptide is modified to inactivate or remove a nuclear localization signal(s). For example, an RNA-binding protein or peptide does not contain a nuclear localization signal(s) (or sequence). Those skilled in the art will understand that a nuclear localization signal is one or more positively charged sequences of lysine or arginine exposed on the protein surface, tagging the protein for translocation into the cell nucleus by nuclear transport. Methods for modifying nuclear localization signals are obvious to those skilled in the art and / or are described herein. For example, a nuclear localization signal is lipidized, removed, or inactivated. In one example, the nuclear localization signal at the N-terminus of an RNA-binding protein or peptide is lipidized. In another example, the nuclear localization signal at the N-terminus of an RNA-binding protein or peptide is removed. In yet another example, the nuclear localization signal at the N-terminus of an RNA-binding protein or peptide is inactivated.

[0033] In one example, an 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 nuclear export signals are obvious to those skilled in the art and / or are described herein.

[0034] In one example, RNA-binding proteins or peptides are lipid-modified at the C-terminus of the protein or peptide.

[0035] For example, RNA-binding proteins or peptides can be lipid-modified by palmitoylation, myristoylation, fatty acid acylation, esterification, prenylation, or a combination thereof.

[0036] For example, RNA-binding proteins or peptides are lipid-modified by palmitoylation. For instance, lipid modification occurs via N-terminal cysteine ​​palmitoylation.

[0037] For example, RNA-binding proteins or peptides are lipid-modified by myristoylation. For instance, they are lipid-modified by N-terminal glycine myristoylation.

[0038] In one example, RNA-binding proteins or peptides are lipid-modified by fatty acid acylation. For example, they are lipid-modified by lysine N-acylation. In another example, they are lipid-modified by serine O-acylation.

[0039] For example, RNA-binding proteins or peptides are lipid-modified by esterification. For instance, they are lipid-modified by C-terminal cholesterol esterification.

[0040] For example, RNA-binding proteins or peptides are lipid-modified by prenylation. Prenylation can be farnesylation or geranylgeranylation. Another example is cysteine ​​prenylation.

[0041] In one example, RNA-binding proteins or peptides are lipid-modified 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.

[0042] In one example, the lipid portion is linked to an RNA-binding protein or peptide by thioether bonds, ester bonds, thioester bonds, and / or amide bonds.

[0043] In one example, the lipid portion is linked to an RNA-binding protein or peptide via a thioether bond.

[0044] In one example, the lipid portion is linked to an RNA-binding protein or peptide by an ester bond.

[0045] In one example, the lipid portion is linked to an RNA-binding protein or peptide via a thioester bond.

[0046] In one example, the lipid portion is linked to an RNA-binding protein or peptide via an amide bond.

[0047] In one example, RNA-binding proteins or peptides are lipid-treated using either chemical or enzymatic lipid treatment. For example, RNA-binding proteins or peptides are lipid-treated using chemical lipid treatment. In one example, chemical lipid treatment is selected from the group consisting of chemical ligation, click chemistry, expression protein ligation, and combinations thereof. In one example, chemical lipid treatment is chemical ligation. In another example, chemical lipid treatment is click chemistry. In yet another example, RNA-binding proteins or peptides are lipid-treated using enzymatic lipid treatment. For example, enzymatic lipid treatment is selected from the group consisting of saltase A-mediated lipid treatment, transglutaminase-mediated lipid treatment, and combinations thereof. In one example, enzymatic lipid treatment is saltase A-mediated lipid treatment. In another example, enzymatic lipid treatment is transglutaminase-mediated lipid treatment.

[0048] In one example, enzymatic lipidization can occur in vivo or extracorporeally. For instance, enzymatic lipidization can occur in vivo. In another example, enzymatic lipidization can occur extracorporeally.

[0049] For example, RNA-binding proteins or peptides are a) Reduce the toxicity of lipid nanoparticles, b) Stabilize the RNA, c) Protect RNA from degradation, d) Promote nucleation of lipid nanoparticles, and / or e) Inhibit the induction of signal transduction by one or more Toll-like receptors.

[0050] For example, RNA-binding proteins or peptides can mitigate the toxicity of lipid nanoparticles.

[0051] In one example, RNA-binding proteins or peptides stabilize RNA. In another example, RNA-binding proteins or peptides stabilize unencapsulated RNA and / or partially encapsulated RNA. In yet another example, RNA-binding proteins or peptides stabilize internal RNA.

[0052] In one example, RNA-binding proteins or peptides protect RNA from degradation. In another example, RNA-binding proteins or peptides protect unencapsulated RNA and / or partially encapsulated RNA from degradation. In yet another example, RNA-binding proteins or peptides protect internal RNA from degradation.

[0053] In one example, RNA-binding proteins or peptides promote the nucleation of lipid nanoparticles.

[0054] In one example, an RNA-binding protein or peptide inhibits the induction of signaling by one or more Toll-like receptors. In another example, an RNA-binding protein or peptide does not inhibit the induction of signaling by one or more Toll-like receptors.

[0055] Those skilled in the art will understand the existence of endosomal Toll-like receptors, namely TLR-3, TLR-7, TLR-8, and TLR-9, which recognize and bind to nucleic acids such as RNA. When these receptors are activated, inflammatory cytokines and type I interferons (interferon (type I)) are produced.

[0056] In one example, an RNA-binding protein or peptide inhibits the induction of signaling by one or more endosomal Toll-like receptors. For example, an RNA-binding protein or peptide inhibits the induction of signaling by one or more Toll-like receptors selected from the group consisting of TLR-3, TLR-7, TLR-8, and TLR-9. In one example, an RNA-binding protein or peptide inhibits the induction of signaling by TLR-3. In another example, an RNA-binding protein or peptide inhibits the induction of signaling by TLR-7. In yet another example, an RNA-binding protein or peptide inhibits the induction of signaling by TLR-9. In yet another example, an RNA-binding protein or peptide inhibits the induction of signaling by TLR-8.

[0057] In one example, an RNA-binding protein or peptide is two RNA-binding proteins or peptides linked by a linker (i.e., a first RNA-binding protein or peptide and a second RNA-binding protein or peptide). For example, the first and second RNA-binding proteins or peptides are covalently linked by an amide bond. This disclosure also encompasses other forms of covalent and non-covalent bonding. For example, RNA-binding proteins or peptides can be linked by a chemical linker. In one example, the first RNA-binding protein or peptide is the same as the second RNA-binding protein or peptide. In another example, the first RNA-binding protein or peptide is different from the second RNA-binding protein or peptide.

[0058] In one example, the linker is a flexible linker, such as a flexible peptide linker. For instance, a first RNA-binding protein or peptide is linked to a second RNA-binding protein via a flexible linker.

[0059] 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 with a length of 2 to 31 amino acids. In one example, the linker is (Gly4Ser) n The linker contains the array (Ala) where n is 1 to 6. For example, the linker contains the array SGGGGS(GS6) or the array SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(GS31). In another example, the linker contains the array (Ala) n This includes n, where n is between 2 and 31.

[0060] For example, the linker is a rigid linker. For example, a rigid linker is arranged in (EAAAK) n This includes n, where n is 1 to 3. In one example, the rigid linker is (EAAAK) n This includes n being between 1 and 10 or approximately 1 and 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 approximately less than 80, or approximately less than 60, or approximately less than 50, or approximately less than 40, or approximately less than 30, or approximately less than 20, or approximately less than 10.

[0061] For example, RNA-binding proteins or peptides are viral or nonviral RNA-binding proteins or peptides.

[0062] In one example, the RNA-binding protein or peptide is a viral RNA-binding protein. The viral RNA-binding protein or peptide is derived from a Class III, Class IV, Class V, and / or Class VI 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 yet another 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.

[0063] In one example, the viral RNA-binding protein or peptide is derived from a respiratory virus selected from the group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, and bocavirus.

[0064] In one example, the viral RNA-binding protein or peptide is derived from the influenza virus. For instance, the influenza virus is influenza A. In another example, the influenza virus is influenza B.

[0065] In one example, the viral RNA-binding protein or peptide is derived from respiratory syncytial virus.

[0066] In one example, the viral RNA-binding protein or peptide is derived from the parainfluenza virus.

[0067] In one example, the viral RNA-binding protein or peptide is derived from metapneumovirus.

[0068] In one example, the viral RNA-binding protein or peptide is derived from rhinovirus.

[0069] In one example, the viral RNA-binding protein or peptide is derived from a coronavirus. For instance, the coronavirus is Severe Acute Respiratory Disease 2 (SARS-CoV-2).

[0070] In one example, the viral RNA-binding protein or peptide is derived from adenovirus.

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

[0072] In one example, a viral RNA-binding protein or peptide is a nucleoprotein, a non-structural protein, a substrate protein, and / or a nucleocapsid protein. For example, a viral RNA-binding protein or peptide is a nucleoprotein. In one example, a viral RNA-binding protein or peptide is a substrate protein. In a further example, a viral RNA-binding protein or peptide is a nucleocapsid protein. In another example, a viral RNA-binding protein or peptide is a non-structural protein.

[0073] For example, a viral RNA-binding protein or peptide contains the sequence described in one of sequence numbers 9-11.

[0074] In one example, the viral RNA-binding protein or peptide is a non-structural (NS) protein derived from the influenza B virus. For example, the viral RNA-binding protein or peptide is the influenza B NS1 RNA-binding domain. 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 cleaved binding domain. In yet another example, the influenza B NS1 RNA-binding domain is a modified binding domain. In one example, the influenza B NS1 RNA-binding domain is described in Sequence ID No. 9. In yet another example, the influenza B NS1 RNA-binding domain is described in Sequence ID No. 10. In yet another example, the influenza B NS1 RNA-binding domain is described in Sequence 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 described in Sequence ID No. 11 and a second influenza B NS1 RNA-binding domain described in Sequence ID No. 10, where the first and second RNA-binding domains are linked by an appropriate linker. For example, the 3' end of the first influenza B NS1 RNA-binding domain is ligated to the 5' end of the second influenza B NS1 RNA-binding domain.

[0075] For example, a viral RNA-binding protein or peptide is a nucleoprotein that encapsulates and stabilizes RNA, thereby inhibiting the induction of signaling by one or more endosomal Toll-like receptors (e.g., TLR-3, TLR-7, TLR-8, and / or TLR-9).

[0076] For example, a viral RNA-binding protein or peptide is a nucleocapsid that encapsulates and stabilizes RNA, thereby inhibiting the induction of signaling by one or more endosomal Toll-like receptors (e.g., TLR-3, TLR-7, TLR-8, and / or TLR-9).

[0077] For example, a viral RNA-binding protein or peptide is a substrate protein that binds to RNA and stabilizes it, but does not inhibit the induction of signaling by one or more endosomal Toll-like receptors (e.g., TLR-3, TLR-7, TLR-8, and / or TLR-9).

[0078] For example, RNA-binding proteins or peptides are nonviral RNA-binding proteins or peptides. For instance, RNA-binding proteins or peptides are nonviral proteins or peptides derived from cellular proteins. For example, RNA-binding proteins or peptides are derived from cellular proteins involved in cell proliferation, cell signaling, and / or antiviral pathways.

[0079] For example, the cellular protein is selected from a 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.

[0080] For example, a cell protein contains the sequence described in one of sequence numbers 1 through 8.

[0081] In one example, the cell protein is a TAR RNA-binding protein (TRBP). For example, the cell protein is TRBP RNA-binding domain 2. In one example, TRBP RNA-binding domain 2 is full-length. For example, full-length TRBP RNA-binding domain 2 is described in Sequence ID No. 1. In another example, TRBP RNA-binding domain 2 is a cleaved binding domain. For example, cleaved TRBP RNA-binding domain 2 is described in Sequence ID No. 2.

[0082] In one example, the cell protein is a protein kinase R (PKR) RNA-binding protein. For example, the cell protein is PKR RNA-binding motif 2. In another example, PKR RNA-binding motif 2 is a full-length binding motif. For example, the full-length PKR RNA-binding motif 2 is described in SEQ ID NO: 3. In yet another example, PKR RNA-binding motif 2 is a cleaved binding motif. For example, the cleaved PKR RNA-binding motif 2 is described in SEQ ID NO: 4. In yet another example, the cleaved PKR RNA-binding motif 2 is described in SEQ ID NO: 5.

[0083] In one example, the cell protein is TLR-3 dsRNA-binding domain 1. For example, the cell protein is TLR-3 dsRNA-binding domain 1 (leucine-rich repeats 1-3). In another example, the cell protein is TLR-3 dsRNA-binding domain 1 (leucine-rich repeats 17-18). In one example, TLR-3 dsRNA-binding domain 1 is described in SEQ ID NO: 6. In yet another example, TLR-3 dsRNA-binding domain 1 is described in SEQ ID NO: 7.

[0084] In one example, the cell protein is the TLR-7 RNA binding site. For example, the cell protein is the TLR-7 RNA binding site (leucine-rich repeat 14-15). In one example, the TLR-7 RNA binding site is described in SEQ ID NO: 8.

[0085] In one example, the lipid nanoparticles further comprise PEG lipids, structural lipids, and / or neutral lipids. For example, the lipid nanoparticles further comprise PEG lipids, structural lipids, and neutral lipids. In another example, the lipid nanoparticles further comprise PEG lipids, structural lipids, or neutral lipids.

[0086] For example, the lipid nanoparticles further contain PEG lipids. For instance, the PEG lipids are selected from the group consisting of PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0087] For example, lipid nanoparticles may further contain structural lipids. For instance, the structural lipids may be selected from the group consisting of cholesterol, campesterol, and combinations thereof.

[0088] For example, lipid nanoparticles further contain neutral lipids. For instance, the neutral lipids are selected from the group consisting of DSPC, DOPE, DLPC, DMPC, DOPC, DPPC, and combinations thereof.

[0089] For example, lipid nanoparticles do not contain cationic lipids. A cationic lipid is 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP).

[0090] In one example, RNA is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), and antisense RNA.

[0091] In one example, RNA is mRNA. For example, mRNA can be self-amplified mRNA (sa-mRNA) or conventional mRNA (cRNA). In one example, mRNA is sa-mRNA. In another example, mRNA is cRNA.

[0092] For example, RNA is siRNA.

[0093] For example, RNA is miRNA.

[0094] In one example, the RNA is antisense RNA.

[0095] This disclosure also provides immunogenic compositions comprising the RNA delivery vehicle of this disclosure. For example, the compositions of this 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-mediated immune response includes the activation of antigen-specific cytotoxic T cells.

[0096] This disclosure also provides pharmaceutical compositions comprising the RNA delivery vehicle, the immunogenic composition, and pharmaceutically acceptable carriers of this disclosure. Pharmacochemically acceptable carriers suitable for use in this disclosure are apparent to those skilled in the art and / or are described herein.

[0097] This disclosure also provides immunogenic compositions or pharmaceutical compositions of this disclosure for use in therapeutic purposes. For example, the immunogenic compositions or pharmaceutical compositions of this disclosure are suitable for use as vaccines.

[0098] In one example, the immunogenic composition or pharmaceutical composition of this disclosure is supplied in a vial. In another example, the immunogenic composition or pharmaceutical composition of this disclosure is supplied in a syringe.

[0099] This disclosure also provides a method for detecting unencapsulated RNA and / or partially encapsulated RNA on the outer surface of lipid nanoparticles, the method being, (i) Forming a complex by contacting a sample containing lipid nanoparticles with a detection protein containing an antibody variable region that binds to unencapsulated RNA and / or partially encapsulated RNA on the outer surface of the lipid nanoparticles, wherein the detection protein contains a detectable label. (ii) detecting a detectable label, the presence of which indicates the presence of unencapsulated RNA and / or partially encapsulated RNA on the outer surface of a lipid nanoparticle.

[0100] This disclosure further provides a method for detecting unencapsulated RNA and / or partially encapsulated RNA on the outer surface of lipid nanoparticles, the method being: (i) Contacting a sample containing lipid nanoparticles with RNA-binding proteins or peptides that bind to unencapsulated RNA and / or partially encapsulated RNA on the outer surface of the lipid nanoparticles to form a complex, (ii) Contacting the complex with a detection protein containing an antibody variable region, wherein the detection protein is bound to an RNA-binding protein or peptide, and the detection protein contains a detectable label, (iii) detecting a detectable label, the presence of which indicates the presence of unencapsulated RNA and / or partially encapsulated RNA on the outer surface of lipid nanoparticles in the sample.

[0101] In one example, this method further includes immobilizing an RNA-binding protein or peptide that binds to unencapsulated RNA and / or partially encapsulated RNA onto a solid surface.

[0102] In one example, this method further includes washing the solid surface to remove unbound lipid nanoparticles. For instance, after contacting the sample with an RNA-binding protein or peptide immobilized on a solid surface that binds to unencapsulated RNA and / or partially encapsulated RNA, the solid surface is washed to remove the unbound lipid nanoparticles.

[0103] In one example, this method further includes washing a solid surface to remove unbound lipid nanoparticles. For instance, the complex is brought into contact with a detection protein containing an antibody variable region, and then the solid surface is washed to remove unbound lipid nanoparticles.

[0104] In one example, an RNA-binding protein or peptide is a nucleoprotein. For example, a nucleoprotein is a recombinant nucleoprotein. In another example, an RNA-binding protein or peptide is a substrate protein. In yet another example, an RNA-binding protein or peptide is a nucleocapsid protein. In yet another example, an RNA-binding protein or peptide is a non-structural protein.

[0105] In one example, the detected protein is an antibody. For instance, the antibody may be an anti-RNA binding protein or a peptide antibody. In another example, the detected protein is a monoclonal antibody. In yet another example, the detected protein is a polyclonal antibody.

[0106] In one example, detectable labels are selected from the group consisting of magnetic labels, radioactive labels, enzymes, fluorescent labels, luminescent labels, bioluminescent labels, prosthetic groups, and contrast agents.

[0107] In one example, the detectable label is a magnetic label. For example, a magnetic label is a magnetic bead. In another example, the detectable label is a radioactive label. For another example, the detectable label is an enzyme. In yet another example, the detectable label is a fluorescent label. In yet another example, the detectable label is a luminescent label. For another example, the detectable label is a bioluminescent label. In yet another example, the detectable label is a prosthetic group. For another example, the detectable label is a contrast agent.

[0108] In one example, this method involves determining the amount of unencapsulated RNA and / or partially encapsulated RNA in a sample. For example, the amount of unencapsulated RNA and / or partially encapsulated RNA is determined directly by determining the amount of bound unencapsulated RNA and / or partially encapsulated RNA in the sample. In another example, the amount of unencapsulated RNA and / or partially encapsulated RNA is determined indirectly by determining the amount of unbound lipid nanoparticles in the sample.

[0109] In one example, this method involves performing an enzyme-linked immunosorbent assay (ELISA). In another example, this method involves performing an enzyme-linked immunosorbent assay (ELISA). In yet another example, this method involves performing a fluorescence-linked immunosorbent assay (FLISA). In one example, this method involves performing chromatography, for example, affinity chromatography.

[0110] In one example, the present disclosure provides a method for determining the amount of encapsulated RNA, the method comprising determining the amount of RNA in a sample, determining the amount of unencapsulated RNA and / or partially encapsulated RNA in the sample using the method described herein, and obtaining the amount of encapsulated RNA in the sample by subtracting the amount of unencapsulated RNA and / or partially encapsulated RNA in the sample from the amount of RNA in the sample. [Table 1] [Brief explanation of the drawing]

[0111] [Figure 1] This is a graph of a rabbit reticulocyte lysate assay, showing the amount of RNA measured by the amount of luciferase produced, which is evaluated by measuring the relative luminescence (RLU) of nanoluciferase RNA (nLuc RNA) alone or in combination with influenza virus RNA-free nucleoprotein (NP:nLuc RNA). [Figure 2] This graph shows the amount of RNA in samples of (A) NP:nLucRNA and (B) nLucRNA alone, after treatment with or without the thermally unstable protease K (PK) and / or RNase. [Figure 3] This is a series of graphs showing the stability of nLuc RNA after incubation of nLuc RNA alone and NP:nLuc RNA samples at (A) 4°C, (B) 24°C, and (C) 37°C for up to 96 hours. [Figure 4] This is a series of graphs showing the levels of (AB)TLR3 and (CD)TLR8 induction in nLuc RNA alone and in NP:nLuc RNA samples at 2 and 4 hours after incubation. [Figure 5] (A) Protection against RNA degradation by SARS-CoV-2 nucleocapsid as measured by the amount of luciferase produced, which was assessed by measuring the luminescence of nanoluciferase RNA (RNA) alone or in combination with COVID RNA-free nucleocapsid (RNA+NP(SCov2)) using RLU, and (B) Dye exclusion assay of nLuc RNA with or without COVID RNA-free nucleocapsid. [Figure 6] This is a series of graphs showing mRNA immunoprecipitation with (A) recombinant NPs absent and (B) recombinant NPs present. [Figure 7] This is a series of graphs showing the immunoprecipitation of mRNA outside and inside LNPs, with (A) no recombinant NPs and (B) recombinant NPs. Legend: (mRNA)-LNP = inclusion mRNA, mRNA-LNP = mRNA ligated to the surface. [Figure 8] This is a series of graphs showing the immunoprecipitation of NP-binding mRNA outside and inside LNPs, with (A) no recombinant NPs and (B) recombinant NPs. Legend: (NP+mRNA)-LNP = encapsulated NP-binding mRNA, NP+mRNA-LNP = NP-binding mRNA ligated to the surface. [Figure 9] This is a series of graphs showing the effect on immunoprecipitation due to a decrease in the amount of ionized lipids, with (A) no recombinant NPs and (B) recombinant NPs. [Figure 10] (A) A series of graphs showing the effect of NP-binding mRNA on immunoprecipitation with and without recombinant NPs. [Figure 11] This is a series of graphs showing that LNPs containing ionized lipids, namely (A) mRNA-LNP and (B) NP+mRNA-LNP, do not bind to magnets used in immunoprecipitation assays. [Modes for carrying out the invention]

[0112] General Throughout this specification, unless otherwise specifically stated or the context should indicate otherwise, any reference to a single step, event configuration, step group, or event configuration shall be interpreted as encompassing one or more (i.e., one or more) of these steps, event configurations, step groups, or event configurations.

[0113] Those skilled in the art will understand that variations and modifications are likely to occur in addition to those specifically described herein. It should be understood that this disclosure includes all such variations and modifications. Furthermore, this disclosure includes all steps, features, compositions, and compounds mentioned or shown herein individually or comprehensively, as well as any combination or any two or more of such steps or features.

[0114] This disclosure is not limited in scope by the specific examples described herein, which are intended to be illustrative only. It is evident that functionally equivalent products, compositions, and methods fall within the scope of this disclosure.

[0115] Any example in this disclosure shall apply mutatis mutandis to any other example in this disclosure unless otherwise specified.

[0116] Unless otherwise defined, all technical and scientific terms used herein are to be considered to have the same meaning as those commonly understood by those skilled in the art (e.g., cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0117] Unless otherwise stated, the recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. These 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), DMGlover and BDHames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JEColigan et al. (editors), Current Protocols in Immunology, John This is described in literature such as Wiley & Sons (including all updates to date).

[0118] The term "and / or," for example "X and / or Y," is understood to mean either "X and Y" or "X or Y," and is considered to explicitly support both meanings or either one of them.

[0119] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” shall be understood to mean that the specified elements, integers, steps, or groups of elements, integers, or steps are included, but not that other elements, integers, steps, or groups of elements, integers, or steps are excluded.

[0120] Where used herein, the term “derived from” should be interpreted as indicating that the specified whole may be obtained from a particular source (but not necessarily directly from that source).

[0121] Selected definition As used herein, the term “RNA delivery vehicle” refers to an RNA delivery system that enables the transport of RNA to a host cell.

[0122] As used herein, the terms “lipid nanoparticles” or “LNPs” are understood to refer to lipid-based particles having at least one dimension on the order of nanometers (e.g., 1 to 1,000 nm) and containing compounds of any of the formulas described herein. In some examples, LNPs are formulated as compositions for delivering oligonucleotides to desired targets such as cells, tissues, organs, and tumors. For example, lipid nanoparticles or LNPs may 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, monolayer, or multiple, multilayer), micelle-like lipid nanoparticles having a non-aqueous core, and solid lipid nanoparticles lacking a lipid bilayer.

[0123] As used herein, the term “outer surface” refers to the outer encapsulation wall of the LNP.

[0124] As used herein, “unencapsulated RNA” refers to RNA that is not encapsulated during the preparation of lipid nanoparticles and remains accessible on the outer surface of the lipid nanoparticles. It is clear from this that RNA does not need to be completely unencapsulated; rather, it may be “partially encapsulated” or “incompletely encapsulated” to, for example, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Methods for determining the level of unencapsulated RNA are known in the art and / or described herein.

[0125] As used herein, the terms “partially encapsulated RNA” or “incompletely encapsulated RNA” refer to RNA that is not completely encapsulated within a lipid nanoparticle, in which at least a portion of the RNA is accessible to or remains exposed on the outer surface of the lipid nanoparticle.

[0126] In some examples, the level of unencapsulated RNA on the outer surface of lipid nanoparticles is at least 50%, e.g., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In one example, the level of unencapsulated RNA is at least 80%. In another example, the level of unencapsulated RNA is at least 90%. In yet another example, the level of unencapsulated RNA is at least 95%. For example, at least 95% of the RNA is unencapsulated.

[0127] In some examples, the level of partially encapsulated RNA on the outer surface of lipid nanoparticles may be at least 50%, e.g., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In one example, the partially encapsulated level is at least 80%. In another example, the partially encapsulated level is at least 90%. In yet another example, the partially encapsulated level is at least 95%. For example, at least 95% of the RNA is partially encapsulated. In yet another example, at least 95% of the RNA is incompletely encapsulated.

[0128] As used herein, the term "coating" refers to RNA-binding proteins or peptides that bind to and adhere to the outer surface of lipid nanoparticles.

[0129] As used herein, the terms “lipidized” or “lipidized” refer to the process of covalently modifying a protein (i.e., an RNA-binding protein or peptide) with one or more lipids.

[0130] As used herein, the terms “RNA-binding protein or peptide” or “RBP” are understood to refer to proteins and peptides that bind to double-stranded or single-stranded RNA and are involved in the formation of ribonucleoprotein complexes.

[0131] As used herein, the term “recombinant” is understood to mean a product of which genes have been artificially modified.

[0132] As used herein, the term “self-replicating RNA” refers to structures based on RNA viruses designed to enable the expression of heterologous RNA and proteins. Self-replicating RNA (e.g., in the form of naked RNA) can be amplified in host cells, leading to the expression of desired gene products within the host cells.

[0133] As used herein, the terms “conventional RNA,” “cRNA,” or “non-amplified RNA” refer to RNA that enables the expression of heterologous RNA and proteins but cannot be amplified within a host cell.

[0134] As used herein, the terms “detect” or “detect” refer to identifying the presence of unencapsulated RNA and / or partially encapsulated RNA on the outer surface of lipid nanoparticles, or RNA inside lipid nanoparticles.

[0135] As used herein, the terms “contact” or “in contact” are used to refer to a direct or indirect interaction or binding between RNA and an RNA-binding protein or peptide. For example, an RNA-binding protein or peptide binds to RNA directly or indirectly.

[0136] As used herein, the term “binding” in relation to the interaction of two or more proteins means that the interaction depends on the presence of a specific structure on the protein (e.g., an antigenic determinant or epitope). For example, a protein (e.g., an RNA-binding protein or peptide) recognizes and binds to a specific protein structure, rather than to a general protein. If a protein binds to epitope “A”, the presence of a molecule containing epitope “A” (or free, unlabeled “A”) during a reaction involving label “A” and the protein will reduce the amount of label “A” bound to the protein.

[0137] As used herein, the terms “specifically binds” or “binds specifically” are interpreted to mean that a protein (e.g., an RNA-binding protein or peptide) reacts or binds to a particular antigen (e.g., unencapsulated RNA and / or partially encapsulated RNA on the outer surface of lipid nanoparticles) more frequently, more rapidly, for a longer period, and / or with higher affinity than other antigens. For example, an RNA-binding protein or peptide binds to RNA more readily, with substantially higher affinity (e.g., 20, 40, 60, 80–100, 150, or 200 times higher affinity) and for a longer period than it would to bind to other antigens. Generally, references to binding force mean a specific binding force, and each term is understood to explicitly support other terms, though not necessarily.

[0138] As used herein, the term “subject” is interpreted to mean any animal, including humans, such as mammals. Illustrative subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0139] RNA delivery vehicle This disclosure provides an RNA delivery vehicle comprising lipid nanoparticles containing RNA internally and RNA-binding proteins or peptides coated on the outer surface of the lipid nanoparticles. For example, the RNA delivery vehicle of this disclosure provides lipid nanoparticles containing RNA-binding proteins or peptides as an outer layer.

[0140] Those skilled in the art will understand that in the process of formulating lipid nanoparticles, not all RNA is encapsulated within the nanoparticles; some RNA may remain unencapsulated or be partially encapsulated within the lipid nanoparticles. These unencapsulated and / or partially encapsulated RNAs may contribute to the destabilization, degradation, or reduced efficiency of the RNA delivery vehicle. By coating the lipid nanoparticles with RNA-binding proteins or peptides to protect the unencapsulated and / or partially encapsulated RNAs, improved lipid nanoparticles for RNA delivery are provided.

[0141] RNA-binding proteins or peptides may non-covalently bind to the outer surface of lipid nanoparticles, further promoting nucleation of these nanoparticles.

[0142] RNA-binding proteins or peptides This disclosure provides RNA-binding proteins or peptides coated on the outer surface of lipid nanoparticles. For example, the RNA-binding proteins or peptides bind to the outer surface of lipid nanoparticles. This disclosure also provides RNA-binding proteins or peptides that bind to RNA within lipid nanoparticles. This disclosure provides RNA-binding proteins or peptides that bind to unencapsulated RNA and / or partially encapsulated RNA on the outside of lipid nanoparticles.

[0143] RNA-binding proteins or peptides regulate various aspects of cotranscription and posttranscriptional gene expression, including 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 are involved in the formation of ribonucleoprotein complexes.

[0144] Those skilled in the art will understand that RNA-binding proteins or peptides can be viral or nonviral proteins or peptides.

[0145] Nonviral RNA-binding proteins or peptides For example, RNA-binding proteins are non-viral proteins or peptides derived from cellular proteins. For instance, RNA-binding proteins or peptides may be derived from cellular proteins involved in cell proliferation, cell signaling, and / or antiviral pathways.

[0146] Nonviral RNA-binding proteins or peptides contain numerous 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 (ZnF), double-stranded RNA-binding motifs (dsRBDs), cold shock domains; Pumilio / FBF (PUF or Pum-HD) domains and Piwi / Argonaute / Zwille (PAZ) domains.

[0147] For example, an RNA-binding protein or peptide includes 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.

[0148] For example, RNA-binding proteins or peptides contain RNA recognition motifs. Examples of RNA-binding proteins or peptides containing RNA recognition motifs include 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, and 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 The group is selected from 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.

[0149] In one example, an RNA-binding protein or peptide contains 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, RNA-binding proteins or peptides containing a K homology domain are 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.

[0150] In one example, the RNA-binding domain includes a CCCH-type zinc finger domain.

[0151] Exemplary nonviral RNA-binding proteins or peptides will be apparent to those skilled 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).

[0152] Viral RNA binding protein For example, RNA-binding proteins or peptides are viral RNA-binding proteins or peptides. RNA-binding proteins are, for instance, nucleoproteins, substrate proteins, nucleocapsid proteins, and / or non-structural proteins derived from RNA viruses.

[0153] As shown in Table 1, it will be apparent to those skilled in the art that viruses are classified according to the Baltimore classification system, which is primarily based on the transcription of the viral genome. [Table 2]

[0154] For example, RNA-binding proteins or peptides are derived from RNA viruses. For instance, RNA-binding proteins or peptides are derived from Class III, Class IV, Class V, and / or Class IV viruses.

[0155] For example, RNA viruses are Class III viruses (i.e., double-stranded RNA viruses). Class III viruses include, for example, all viruses of the phylum Duplornaviricota and all viruses of the class Dulopiviricetes (of the phylum Pisuviricota). Exemplary Class III viruses include, but are not limited to, reoviruses (e.g., orthoreovirus, rotavirus, orbivirus, or cortivirus).

[0156] For example, RNA viruses are Class IV viruses (i.e., positive-sense single-stranded RNA viruses). Class IV viruses include, for example, viruses of the phyla Lenarviricota, Pisuviricota (excluding the class Duplopidiviricetes), and Kitrinoviricota. Examples of Class IV viruses include togaviruses (e.g., rubivirus, alphavirus, or arterivirus), flaviviruses (e.g., tick-borne encephalitis (TBE) virus, dengue (types 1, 2, 3, or 4) virus, yellow fever virus, Japanese encephalitis virus, Kasanul Forest disease virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Poissant encephalitis virus), picornaviruses (e.g., enterovirus, rhinovirus, heparnavirus, parechovirus, cardiovirus, and aftovirus), enteroviruses (e.g., poliovirus type 1, 2, or 3, coxsackie A virus types 1-22 and 24, coxsackie B virus types 1-6, echovirus (ECHO) type 1) Examples include, but are not limited to, types 9-9, 11-27, and 29-34, enterovirus types 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., Hawaiian 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), avian infectious bronchitis (IBV), mouse hepatitis virus (MHV), and porcine infectious gastroenteritis virus (TGEV)), and hepatitis E virus (HEV).

[0157] For example, RNA viruses are Class V viruses (i.e., negative-sense single-stranded RNA viruses). Class V viruses include, for example, viruses of phylum Negarnaviricota. Examples of 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, mouse pneumonia virus, turkey rhinotracheitis virus)), paramyxoviruses types 1-4 (PIV), mumps, Sendai virus, simian virus type 5, nipah virus, henipavirus, Newcastle disease virus, morbillivirus (e.g., measles), bunyavirus (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), becyclovirus (VSV)), hepatitis delta virus (HDV), and arenaviruses.

[0158] In one example, a class VRNA virus is an influenza virus. For instance, it is the influenza A virus. In another example, it is the influenza B virus.

[0159] For example, RNA viruses are Class VI viruses (i.e., single-stranded RNA viruses that have a DNA intermediate during their 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, hepadnaviruses (e.g., hepatitis A virus (HAV)), hepadnaviruses (e.g., hepatitis B virus, hepatitis C virus), and retroviruses (e.g., oncovirus, lentivirus, or supumavirus).

[0160] Linker For example, an RNA-binding protein or peptide may include a first RNA-binding protein or peptide and a second RNA-binding protein or peptide linked via a linker. For instance, the linker may be a linker peptide.

[0161] For example, a linker is a flexible linker.

[0162] A "flexible" linker is an amino acid sequence that does not have a fixed structure (secondary or tertiary structure) in solution. Therefore, such flexible linkers can freely adopt a variety of three-dimensional structures. Flexible linkers suitable for use in this disclosure are known in the art. An example of a flexible linker used in the present invention is the linker sequence SGGGGS / GGGGS / GGGGS or (Gly4Ser)3. Another example of a flexible linker is the alanine linker (e.g., Ala n )

[0163] The linker may contain any amino acid sequence that does not substantially hinder the interaction between the RNA-binding protein or peptide and RNA. Preferred amino acid residues for a flexible linker sequence include, but are not limited to, glycine, alanine, serine, threonine, proline, lysine, arginine, glutamine, and glutamic acid.

[0164] The linker sequence between RNA-binding proteins or peptides preferably comprises five or more amino acid residues. The flexible linker sequence according to this disclosure consists of five 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 very preferred embodiment of the present invention, the flexible linker sequence consists of 5, 7, 10, or 16 residues.

[0165] For example, a linker is a rigid linker. A "rigid linker" (including a "semi-rigid linker") refers to a linker with limited flexibility. For example, a relatively rigid linker is an array (EAAAK) nThe formula includes (where n is between 1 and 3). The value of n can be between 1 and about 10, or about 1 and 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 about less than 80, or about less than 70, or about less than 60, or about less than 50, or about less than 40, or about less than 30, or about less than 20, or about less than 10. A rigid linker does not need to be completely lacking in flexibility.

[0166] Lipidization of RNA-binding proteins This disclosure provides an RNA delivery vehicle comprising lipid nanoparticles and an RNA-binding protein or peptide coated on the outer surface of the lipid nanoparticles, wherein the RNA-binding protein or peptide is a lipidized RNA-binding protein or peptide.

[0167] It will be obvious to those skilled in the art that lipidization of a protein or peptide involves the covalent bonding of a lipid portion to a protein or peptide (i.e., an RNA-binding protein or peptide).

[0168] Lipids Suitable lipid portions for use in this disclosure will be apparent to those skilled in the art and include, for example, fatty acids, isoprenoids, and combinations thereof. In one example, the lipid portion is selected from the group consisting of isoprenoids, triglycerides, phospholipids, cholesteryl esters, and combinations thereof.

[0169] isoprenoids Isoprenoids, also known as terpenoid or prenol lipids, are branched lipids and a type of organic compound composed of two or more hydrocarbon units, with each unit consisting of five carbon atoms arranged in a specific pattern. These five-carbon units are called isoprene and are synthesized from a common intermediate known as mevalonic acid, which itself is synthesized from acetyl-CoA. Isoprenoids may have one or more functional chemical groups such as hydroxyl or carbonyl attached to their carbon skeletons, which contribute to the diversity of isoprenoids. Isoprenoids can be classified as 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 .

[0170] Isoprenoids suitable for use in the present disclosure will be apparent to those skilled in the art and / or are described herein.

[0171] In one example, the isoprenoid is a monoterpene. Exemplary monoterpenes include citronellol, citronellal, citral, geraniol, menthol, pseudoionone, and β-ionone.

[0172] In one example, the isoprenoid is a sesquiterpene. Exemplary sesquiterpenes include cadalene, eudesmol, cadinen, and β-selinene.

[0173] In one example, the isoprenoid is a diterpene. Exemplary diterpenes include phytol and abietic acid.

[0174] For example, isoprenoids are triterpenes. Exemplary triterpenes include squalene and β-amyrin.

[0175] For example, isoprenoids are tetraterpenes. Exemplary tetraterpenes include carotenoids (e.g., β-carotene) and lycopene.

[0176] fatty acid Fatty acids are lipids containing long-chain hydrocarbons terminated with carboxylic acid functional groups. Fatty acids can be saturated or unsaturated. For example, a fatty acid may contain a carbon chain with 6 to 22 carbon atoms. Exemplary fatty acids include palmitic acid, myristic acid, oleic acid, alpha-linolenic acid, and stearic acid.

[0177] Fatty acids rarely exist in their free form in nature, and generally exist as three main types of esters: triglycerides, phospholipids, and cholesteryl esters.

[0178] For example, a fatty acid is a triglyceride. A triglyceride is a triester consisting of glycerol bonded to three fatty acid molecules via ester bonds. The three fatty acids may be the same or different. An example triglyceride is tristearin.

[0179] For example, fatty acids are phospholipids. Phospholipids are complex lipids comprising a hydrophilic polar head group containing one or more phosphate groups and a hydrophobic tail group containing two fatty acyl chains. The polar head group is bonded to the hydrophobic portion by phosphodiester bonds via glycerol (i.e., phosphoglycerides) or sphingosine molecules (i.e., phosphosphingolipids). Phospholipids may be saturated or unsaturated. Exemplary phosphoglycerides include phosphatidic acid (phosphatidic acid salt), phosphatidylethanolamine (cephalin), phosphatidylcholine (lecithin), phosphatidylserine, phosphoinositides (e.g., phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidylinositol diphosphate (PIP2), and phosphatidylinositol triphosphate (PIP3)), phosphatidylglycerol, and cardiolipin. Examples of phosphosphingolipids include ceramide phosphorylcholine (sphingomyelin), ceramide phosphorylethanolamine (sphingomyelin), ceramide phosphoryl lipids, galactocerebroside, glucocerebroside, and lactosylceramide.

[0180] One example is a fatty acid, specifically a cholesteryl ester. Cholesteryl esters are formed by esterifying cholesterol with long-chain fatty acids. Exemplary cholesteryl esters include cholesteryl oleate, cholesteryl benzoate, and cholesteryl linoleate.

[0181] Lipidization Exemplary lipidization processes include palmitoylation, myristoylation, fatty acid acylation, esterification, prenylation, or combinations thereof.

[0182] Palmitoylation In one example, the lipid portion is bound to an RNA-binding protein or peptide by palmitoylation.

[0183] 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 in 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.

[0184] Myristoylation In one example, the lipid portion attaches to RNA-binding proteins or peptides through myristoylation.

[0185] For example, myristoylation is N-glycine myristoylation. Those skilled in the art will recognize that N-glycine myristoylation refers to the co-translation or post-translational attachment of myristoyl, a saturated 14-carbon alipid acyl group, to the N-terminal glycine of a protein via an amide bond.

[0186] For example, myristoylation is lysine myristoylation.

[0187] Fatty acid acylation In one example, the lipid portion is bound to an RNA-binding protein or peptide by fatty acid acylation.

[0188] Those skilled in the art will recognize that fatty acid acylation involves the covalent bonding of an acyl group to a protein.

[0189] One example is fatty acid acylation, which is lysine N-acylation. Those skilled in the art will understand that lysine N-acylation refers to the transfer of the acetyl moiety from acetyl-CoA to the epsilon (ε)-amino group of a lysine residue on a protein.

[0190] Esterification In one example, the lipid portion is bound to an RNA-binding protein or peptide through esterification.

[0191] One example of esterification is C-terminal sterol esterification, such as C-terminal cholesterol esterification. Those skilled in the art will understand that C-terminal cholesterol esterification involves substituting at least one hydroxyl (-OH) group with an alkoxy (-O-alkyl) group.

[0192] Prenylation In one example, the lipid portion attaches to RNA-binding proteins or peptides through prenylation.

[0193] For example, prenylation is cysteine ​​prenylation. Those skilled in the art will understand that cysteine ​​prenylation is the addition of multiple isoprene units to a cysteine ​​residue near the C-terminus of a protein.

[0194] For example, prenylation is farnesylation (i.e., the addition of three isoprene units), or prenylation is geranylgeranylation (i.e., the addition of four isoprene units).

[0195] 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 yet another example, the bond is a thioester bond.

[0196] Methods of lipidization Lipid modifications typically occur at the N-terminus and / or C-terminus of proteins or peptides, specifically in the nucleophilic side chains of proteins or peptides (e.g., cysteine, serine, and lysine).

[0197] Various methods of lipidization are apparent to those skilled in the art and / or are described herein. Suitable methods may include chemical lipidization or enzymatic lipidization.

[0198] chemical lipidation In one example, the lipid moiety is bound to an RNA-binding protein or peptide using chemical ligation. The lipid moiety may contain an amine, carboxylic acid, hydrazide, or maleimide group, and the lipid moiety can be chemically bound 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 contains a maleimide group, and the lipid moiety is bound 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 another example, the lipid moiety contains a carboxylic acid, which is activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS). The activated acylaminoester or sulfo-NHS ester then reacts with the primary amine of the lysine residue in the RNA-binding protein or peptide to form an amide bond.

[0199] In one example, the lipid portion contains a maleimide group. For instance, the lipid portion is a phospholipid coated with a maleimide group. In one example, the lipid portion is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-maleimide (DSPE-maleimide, DSPE-Mal).

[0200] In one example, the lipid portion attaches to an RNA-binding protein or peptide using various "click chemistry" strategies, such as those disclosed in Kolb et al. (2001), WO 2003 / 101972, and Malkoch et al. (2005).

[0201] In one example, the lipid moiety is bound to an RNA-binding protein or peptide using expression protein ligation. Expression protein ligation involves chemoselective ligation between a protein or peptide with a C-terminal thioester and a protein or peptide with an N-terminal cysteine ​​in an aqueous solution at physiological pH. In one example, the C-terminal thioester is inserted into an RNA-binding protein or peptide by genetic engineering, and the lipid moiety is fused to a peptide with an N-terminal cysteine ​​residue.

[0202] Other chemical lipidization methods known to those skilled in the art, such as the method disclosed in Takahara & Kamiya (2020), may also be used.

[0203] enzymatic lipidation In one example, the lipid portion is bound to an RNA-binding protein or peptide using enzymatic lipidization. Enzymatic lipidization can be performed in vivo or in vitro. In some examples, the RNA-binding protein or peptide is genetically engineered using techniques known to those skilled in the art to contain a consensus sequence recognized by the lipophilase.

[0204] In one example, the lipid portion attaches to RNA-binding proteins or peptides via saltase A-mediated lipidization. Saltase A (e.g., SrtA derived from Staphylococcus aureus) is Ca 2+ In the presence of saltase, secreted proteins are covalently bonded to bacterial cell wall peptidoglycans via a peptide transfer reaction. In this example, the RNA-binding protein or peptide is genetically engineered to contain an LPXTG motif (e.g., LPETG) at its C-terminus, and the lipid portion contains a nucleophile and an oligo-glycine motif (e.g., triglycine, tetraglycine, or pentaglysine). Upon addition of saltase, the RNA-binding protein or peptide is covalently bonded to the lipid via peptide bonds.

[0205] In one example, the lipid portion is bound to an RNA-binding protein or peptide using transglutaminase-mediated lipidization. Transglutaminase (e.g., microbial transglutaminase: MTG) is Ca 2+ It catalyzes the reaction between glutamine and lysine residues in a peptide or protein in the absence of MTG, forming irreversible crosslinks. For example, an RNA-binding protein or peptide is genetically engineered to include, for example, an MTG lysine recognition sequence (e.g., MRHKGS) at the N-terminus or C-terminus, and the lipid moiety includes an MTG glutamine recognition sequence (e.g., LLQG). For example, an RNA-binding protein or peptide is genetically engineered to include, for example, an MTG glutamine recognition sequence (e.g., LLQG or LQ) at the N-terminus or C-terminus, and the lipid moiety includes an MTG lysine recognition sequence (e.g., MRHKGS).

[0206] Other enzymatic lipidization methods known to those skilled in the art, such as the method disclosed in Takahara & Kamiya (2020), may be used.

[0207] Additional lipids In one example, the lipid nanoparticles further comprise PEG lipids, sterol-structured lipids, and / or neutral lipids. In another example, the lipid nanoparticles do not contain cationic lipids.

[0208] PEG lipids For example, this disclosure provides lipid nanoparticles containing PEGylated lipids.

[0209] It will be apparent to those skilled in the art that PEGylated lipids are lipids modified with polyethylene glycol. Examples of 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.

[0210] neutral lipid In one example, this disclosure provides lipid nanoparticles containing neutral lipids.

[0211] Neutral or amphoteric lipids suitable for use in this disclosure will be apparent to those skilled in the art, 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-dipalmito Il-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 dietherPC), 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-difytanol-sn-glycero-3-phosphoethanolamine (ME 16.0 This includes 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.

[0212] structured lipids In one example, this disclosure provides lipid nanoparticles containing structural lipids.

[0213] Exemplary structural lipids include, but are not limited to, cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and alpha-tocopherol.

[0214] In one example, the structural lipid is a sterol. For example, the structural lipid is cholesterol. In another example, the structural lipid is campesterol.

[0215] Pharmacologically acceptable carriers Preferably, in a composition or method for administering the RNA delivery vehicle of the present disclosure to a target, the delivery vehicle is combined with a pharmaceutically acceptable carrier as understood in the art. Thus, an example of the present disclosure provides a composition (e.g., a pharmaceutical composition) combining an RNA delivery vehicle comprising lipid nanoparticles coated with the RNA-binding protein or peptide of the present disclosure with a pharmaceutically acceptable carrier.

[0216] Generally, "carrier" refers to any solid or liquid filler, binder, diluent, encapsulant, emulsifier, wetting agent, solvent, suspension agent, coating agent, or lubricant that can be safely administered to any target, such as humans. Depending on the specific route of administration, a variety of acceptable carriers known in the art may be used, such as those described in, for example, Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991).

[0217] The RNA delivery vehicles of this disclosure are useful for parenteral, topical, oral, or local, intramuscular, aerosol, or transdermal administration for prophylactic or therapeutic treatment. For example, the RNA delivery vehicle is administered parenterally, such as intramuscular, subcutaneous, or intravenously. For example, the RNA delivery vehicle is administered intramuscularly.

[0218] The formulation of the RNA delivery vehicle to be administered varies depending on the route of administration and the selected formulation (e.g., solution, emulsion, capsule). A suitable pharmaceutical composition containing the RNA delivery vehicle to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous solutions or alcohol / aqueous solutions, emulsions or suspensions (including saline and buffering media). Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixative oil. Suitable aqueous carriers are known to those skilled in the art and include a variety of options such as water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine. Vehicles for intravenous administration may contain various additives, preservatives, or water, nutrients, or electrolyte supplements (see Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980 for details). The composition may optionally contain pharmaceutically acceptable auxiliary substances such as pH adjusters, buffers, and toxicity modifiers, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate, if necessary to approximate physiological conditions. The RNA delivery vehicle may be stored in liquid form or lyophilized for storage and reconstituted with a suitable carrier before use according to lyophilization and reconstitution techniques known in the art.

[0219] The optimal concentration of the active ingredient(s) (i.e., RNA) in the selected vehicle can be determined empirically according to procedures known to those skilled in the art and depends on the desired final pharmaceutical formulation.

[0220] Once formulated, the compositions of this disclosure are administered in a therapeutically / prophylactically effective amount in a manner suitable for the administered formulation. The dosage range for the administration of the RNA delivery vehicle of this disclosure is a range large enough to produce the desired effect. For example, the composition contains an effective amount of RNA. In one example, the composition contains a therapeutically effective amount of RNA. In another example, the composition contains a prophylactically effective amount of RNA.

[0221] The dosage should not be so high as to cause side effects. Generally, the dosage varies depending on the patient's age, condition, sex, and the severity of the disease, and this can be determined by those skilled in the art. If complications occur, the individual physician may adjust the dosage.

[0222] RNA This disclosure provides an RNA delivery vehicle comprising lipid nanoparticles containing RNA internally and RNA-binding proteins or peptides coated on the outer surface of the lipid nanoparticles. For example, the RNA-binding proteins or peptides completely or partially encapsulate the lipid nanoparticles.

[0223] The RNAs in this disclosure may be natural or non-natural RNAs, or may contain one or more modified nucleic acid bases, nucleosides, or nucleotides. It will be apparent to those skilled in the art that RNAs suitable for use in this disclosure may also include a 5' untranslated region (5'-UTR), a 3' untranslated region (3'UTR), and / or coding or translating sequences. Furthermore, the RNAs may include a 5' cap structure, a strand termination nucleotide, a stem-loop (e.g., a histone stem-loop), and a 3' tailing sequence (e.g., a polyadenylation signal or one or more poly-A tails).

[0224] In one example of this disclosure, the RNA is self-replicating mRNA (sa-mRNA).

[0225] In one example of this disclosure, RNA is conventional mRNA (cRNA).

[0226] Preparation method Suitable methods for generating the RNA delivery vehicles of this disclosure are apparent to those skilled in the art and / or are described herein. For example, the RNA delivery vehicles of this disclosure can be prepared using techniques well known in the field of formulation. For example, a suitable LNP can be formed using microfluidics, including herringbone micromixing, and mixing processes such as T-junction mixing of two fluid streams, one of which typically contains messenger RNA as an aqueous solution, and the other typically contains various required lipid components as ethanol.

[0227] Next, LNP can be prepared by mixing phospholipids (such as DOPE or DSPC, which can be purchased from commercial sources including Avanti Polar Lipids, Alabaster, and AL), PEGylated lipids (such as 1,2-dimiristoyl-sn-glycerol methoxypolyethylene glycol, also known as PEG-DMG, which can be purchased from commercial sources including Avanti Polar Lipids, Alabaster, and AL), and structural lipids / sterols (such as cholesterol, which can be purchased from commercial sources including Sigma-Aldrich) at a concentration of approximately 50 mM in ethanol. The solution needs to be refrigerated during storage, for example, at -20°C. 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, approximately 5.5 mM to approximately 25 mM.

[0228] LNP compositions containing RNA (including, but not limited to, sa-mRNA or cRNA) can be prepared by mixing the above-mentioned lipid solution with the RNA-containing solution, for example, in a weight-to-weight ratio of lipid component 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 produce a suspension with a water-to-ethanol ratio of about 1:1 to about 4:1.

[0229] For LNP compositions containing sa-mRNA or cRNA, a stock solution can be formed by diluting a 1.0 mg / ml RNA solution in deionized water with 50 mM sodium citrate buffer at pH 3-6.

[0230] The LNP composition can be further processed, as is known in the art, by diluting it 10-fold in 50 mM citrate buffer at pH 6 and subjecting it to tangential flow filtration (TFF) using a 300 kM cutoff membrane (mPES) until concentrated to the original volume. Then, in one example, the citrate buffer may be replaced with a buffer containing 20 mM Tris buffer at pH 7.5, 80 mM sodium chloride, and 3% sucrose, using dialysfiltration at 10 times the volume of the new buffer. The LNP solution is concentrated to, for example, 5–10 mL, filtered using a 0.2 micron PES syringe filter, dispensed into vials, and frozen at 1°C / min until the sample reaches -80°C using a Corning® CoolCell® LX cell freezing vessel. The sample can be stored at -80°C until needed.

[0231] The above method induces nanoprecipitation and particle formation. The same nanoprecipitation can be achieved using alternative processes, including but not limited to T-junctions and direct injection.

[0232] In some examples, the lipid component of LNP formulations contains approximately 2 mol% to 25 mol% phospholipids (neutral lipids), approximately 18.5 mol% to 60 mol% structural lipids (sterols), and approximately 0.2 mol% to 10 mol% PEGylated lipids, with the total mol% not exceeding 100%. In some examples, the lipid component of LNP formulations contains approximately 5 mol% to 20 mol% phospholipids, approximately 30 mol% to 55 mol% structural lipids, and approximately 1 mol% to 5 mol% PEGylated lipids. In specific examples, the lipid component contains approximately 10 mol% phospholipids, approximately 48 mol% structural lipids, and approximately 2.0 mol% PEG lipids. In some examples, the phospholipids may be DOPE or DSPC. In other examples, the PEG lipids may be PEG-DMG, and / or the structural lipids may be cholesterol. In some examples, the PEG lipids may be PEG-DMG, and the structural lipids may be cholesterol. In some examples, the PEG lipids may be PEG-DMG. In some cases, structural lipids can be cholesterol.

[0233] Next, RNA delivery vehicles can be formed by combining LNPs with RNA-binding proteins or peptides in solution (typically aqueous solutions). The RNA-binding proteins or peptides bind to unencapsulated RNA and / or partially encapsulated RNA and / or to the outer surface of lipid nanoparticles. In one example, the RNA-binding proteins or peptides bind to unencapsulated and partially encapsulated RNA on the outer surface of lipid nanoparticles, and also to the outer surface of lipid nanoparticles. In another example, the RNA-binding proteins or peptides bind to unencapsulated and partially encapsulated RNA on the outer surface of lipid nanoparticles. In another example, the RNA-binding proteins or peptides bind to unencapsulated RNA on the outer surface of lipid nanoparticles. In another example, the RNA-binding proteins or peptides bind to partially encapsulated RNA on the outer surface of lipid nanoparticles. In another example, the RNA-binding proteins or peptides bind to the outer surface of lipid nanoparticles.

[0234] In some examples, the bond is a non-covalent bond. For instance, the bond is a reversible ionic non-covalent bond.

[0235] The encapsulation efficiency of RNA into LNPs is 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 cases, the encapsulation efficiency can be at least 80%. In certain cases, the encapsulation efficiency can be at least 90%.

[0236] Assay of lipid nanoparticles in this disclosure Lipid nanoparticles of the present disclosure can be easily screened for physical and biological activity and / or stability using methods known in the art and / or the methods described below.

[0237] Evaluation of RNA degradation In one example, the level of RNA degradation by RNase is evaluated. For instance, RNA is treated with RNase alone or in combination with an RNA-binding protein or peptide.

[0238] In one example, real-time PCR is used to evaluate RNA levels in RNase-treated and untreated samples. In this example, the cycle threshold (CT) value of RNA samples without RNA-binding proteins or peptides is increased compared to RNA samples containing RNA-binding proteins or peptides, indicating RNA degradation.

[0239] Evaluation of RNA translation In one example, RNA translation is evaluated using an in vitro translation system. Suitable systems for use in this disclosure will be apparent to those skilled in the art and include, for example, rabbit reticulocyte lysate assays.

[0240] One example is the rabbit reticulocyte lysate assay.

[0241] In one example, RNA is evaluated in the presence or absence of RNA-binding proteins or peptides.

[0242] In one example, the RNA is nanoluciferase RNA (nLuc RNA), and the amount of RNA translation is measured by the amount of luciferase produced, which is assessed by measuring the luminescence in relative light units (RLU). In one example, the assay is performed at 4°C, 24°C, and / or 37°C. For example, the assay is performed at 4°C. In one example, the assay is performed at 24°C. In another example, the assay is performed at 37°C. In yet another example, the assay is performed after incubating the sample for 0 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, or 96 hours.

[0243] Evaluation 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 combined with an RNA-binding protein or peptide is evaluated. In one example, TLR3 and / or TLR8 induction is evaluated using a TLR-inducing NfKB reporter assay. In this assay, NfKB is operationally linked to secreted alkaline phosphatase (SEAP). The RNA is introduced into either a cell type (conditionally transduced TLR3 or conditionally transduced TLR8). Binding to the TLR receptor induces NfKB activation, which in turn induces SEAP. In one example, SEAP levels are determined by reading the chemical reaction and heat energy.

[0244] Evaluation of unencapsulated RNA and / or partially encapsulated RNA This disclosure provides a method for detecting unencapsulated RNA and / or partially encapsulated RNA on the outer surface of lipid nanoparticles.

[0245] Appropriate detection methods are obvious to those skilled in the art and / or are described herein. Exemplary methods for detecting unencapsulated RNA and / or partially encapsulated RNA on the outer surface of lipid nanoparticles include ELISA, FLISA, lateral flow assay, or affinity chromatography.

[0246] Standard ELISA, FLISA, lateral flow, or affinity chromatography methods are useful for determining the concentration of unencapsulated and / or partially encapsulated RNA on the outer surface of lipid nanoparticles. In one example, this method involves immobilizing an RNA-binding protein or peptide that binds to unencapsulated and / or partially encapsulated RNA onto a solid support.

[0247] The term “solid support” is interpreted to mean a substrate to which molecules can be directly or indirectly bound. A solid support may include any substrate material capable of providing physical support to the compositions described herein. The material may be natural, synthetic, or a modified natural material. Suitable substrate materials include glass fibers, polyester, cellulose, rayon, silicon, silicon wafer chips, graphite, mirrors, laminates, films, ceramics, plastics (including polymers such as polyvinyl chloride, cycloolefin copolymers, agarose gels or beads, polyacrylamide, polyacrylate, polyethylene, polypropylene, poly-4-methylbutene, polystyrene, polymethacrylate, polyethylene terephthalate, polytetrafluoroethylene (PTFE or Teflon®), nylon, and polyvinyl butyrate), germanium, gallium arsenide, gold, silver, Langmuir / Bludget films, flow-through chips, etc., used alone or in combination with other materials. Additional rigid materials, such as silica-containing glass and even glass available as bioglass, may also be considered. Other usable materials include, for example, porous materials such as controlled-pore glass beads, cross-linked beads Sepharose® or agarose resin, or copolymers of cross-linked bisacrylamide and azalactone.

[0248] An RNA-binding protein or peptide that specifically binds to unencapsulated RNA and / or partially encapsulated RNA on the outer surface of lipid nanoparticles is brought into contact with a sample containing lipid nanoparticles, so that the RNA-binding protein or peptide forms a direct bond with either the unencapsulated RNA and / or partially encapsulated RNA of its target present in the sample. In the case of FLISA, this RNA-binding protein or peptide is brought into contact with a detection protein containing an antibody variable region that is generally labeled with a detectable reporter molecule such as magnetically labeled (e.g., magnetic beads), fluorescently labeled (e.g., FITC or Texas Red), or fluorescent semiconductor nanocrystal (as described in U.S. Patent No. 6,306,610), or in the case of ELISA, an enzyme (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), or β-galactosidase), or a labeled protein or antibody that binds to the RNA-binding protein or peptide is used. After washing to remove unbound lipid nanoparticles, the label is detected directly in the case of magnetic or fluorescent labeling, or, in the case of enzyme labeling, by adding a substrate such as hydrogen peroxide, TMB, toluidine, or 5-bromo-4-chloro-3-indole-beta-D-galaotopyranoside (x-gal). For example, magnetic labeling is detected by a magnetic reader that measures the change in the magnetic field induced by the label. The signal measured by the magnetic reader is proportional to the bound, unencapsulated RNA and / or partially encapsulated RNA in the sample.

[0249] As used herein, “detectable label” is a tag or marker of a molecule or atom that can produce or be induced to produce an optical signal or other signal or product that can be detected visually or using a suitable detector. Detectable labels are well known in the art and include, for example, magnetic labels, radioactive labels, enzymes, fluorescent labels, luminescent labels, bioluminescent labels, prosthetic groups, contrast agents, and sonicants.

[0250] This method is particularly suitable for highly sensitive quantification of unencapsulated and / or partially encapsulated RNA in a sample by calibrating the detection system to a known amount of a standard substance to which RNA proteins or peptides, such as RNA, bind.

[0251] Those skilled in the art will understand that the methods of this disclosure are suitable for use in capture enzyme-linked immunosorbent assays (ELISA) or enzyme-mediated immunoassays (EIA). As used herein, the terms “ELISA,” “sandwich ELISA,” “capture ELISA,” or “EIA” refer to immobilizing RNA-binding proteins or peptides onto a matrix such as a membrane, polystyrene or polycarbonate microwells, polystyrene or polycarbonate dipsticks, or a glass support, followed by the addition of a certain amount of sample containing lipid nanoparticles. Unencapsulated RNA and / or partially encapsulated RNA are “bound” or “captured.” Binded or captured, unencapsulated and / or partially encapsulated RNA can be detected by a detection protein that can covalently bind to a magnetic label, or by the addition of a magnetically labeled secondary protein or antibody.

[0252] Lateral flow assays, also known as "immunochromatography strip tests," operate on the same principle as ELISA. Essentially, these tests involve flowing a liquid sample along the surface of a membrane or filter paper containing reactive molecules that produce a visually positive or negative result depending on the presence of a specific analyte (e.g., unencapsulated RNA and / or partially encapsulated RNA).

[0253] Those skilled in the art will understand that the method of this disclosure is also suitable for use in chromatography columns (e.g., affinity chromatography columns).

[0254] Microarrays, BIAcore assays, fractionation centrifugation, chromatography, electrophoresis, immunoprecipitation, photobiosensors, and other surface plasmon resonance assays can be used as described in WO 2011 / 061351, which is incorporated herein by reference. Methods for using magnetic labeling are described in US 2007 / 0254375, which is incorporated herein by reference.

[0255] It will be apparent to those skilled in the art that the methods described herein are suitable for, for example, the automation of screening processes or high-throughput methods such as the microarray format described in Mendoza et al., 1999. Furthermore, variations of the above methods, such as competitive ELISA and serial affinity chromatography, will be apparent to those skilled in the art.

[0256] The present invention is further disclosed in the following numbered paragraphs.

[0257] 1. RNA delivery vehicle, wherein the delivery vehicle is (i) Lipid nanoparticles containing the RNA inside, (ii) The RNA delivery vehicle comprising an RNA-binding protein or peptide coated on the outer surface of the lipid nanoparticles.

[0258] 2. The RNA-binding protein or peptide is an RNA delivery vehicle as described in paragraph 1, which encapsulates the lipid nanoparticles.

[0259] 3. The RNA delivery vehicle according to paragraph 1 or 2, wherein the RNA-binding protein or peptide directly binds to unencapsulated RNA and / or partially encapsulated RNA on the outer surface of the lipid nanoparticles.

[0260] 4. The RNA delivery vehicle according to any one of paragraphs 1 to 3, wherein the RNA-binding protein or peptide is a lipidized RNA-binding protein or peptide.

[0261] 5. 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) inhibiting the induction of signaling by one or more Toll-like receptors, the RNA delivery vehicle according to any one of paragraphs 1 to 4.

[0262] 6. The RNA-binding protein or peptide is modified to remove a nuclear localization signal(s) and / or introduce a nuclear export signal(s), the RNA delivery vehicle according to any one of paragraphs 1 to 5.

[0263] 7. The RNA-binding protein or peptide is a viral or non-viral RNA-binding protein or peptide, the RNA delivery vehicle according to any one of paragraphs 1 to 6.

[0264] 8. The viral RNA-binding protein is derived from class III, class IV, class V, and / or class VI viruses, the RNA delivery vehicle according to paragraph 7.

[0265] 9. 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, the RNA delivery vehicle according to paragraph 8.

[0266] 10. The viral RNA-binding protein or peptide is a nucleoprotein, non-structural protein, matrix protein, and / or nucleocapsid protein, the RNA delivery vehicle according to paragraph 9.

[0267] 11. The RNA delivery vehicle according to any one of paragraphs 7 to 10, wherein the viral RNA-binding protein or peptide is a non-structural (NS) protein derived from influenza B virus.

[0268] 12. The viral RNA-binding protein or peptide comprises the sequence described in SEQ ID NOs: 9-11, and is an RNA delivery vehicle as described in any one of paragraphs 7-11.

[0269] 13. The RNA delivery vehicle described in paragraph 7, wherein the nonviral RNA-binding protein or peptide is derived from a cellular protein associated with cell proliferation, cell signaling, and / or antiviral pathways.

[0270] 14. The RNA delivery vehicle described in paragraph 13, wherein the cell 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.

[0271] 15. The cellular protein comprises the sequence described in any one of Sequence IDs 1 to 8, as described in the RNA delivery vehicle described in paragraph 13 or 14.

[0272] 16. The RNA delivery vehicle according to any one of paragraphs 1 to 15, wherein the lipid nanoparticles include PEG lipids, structural lipids, neutral lipids and / or cationic lipids.

[0273] 17. The lipid nanoparticles are RNA delivery vehicles according to any one of paragraphs 1 to 16, and do not contain cationic lipids.

[0274] 18. The RNA is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), and antisense RNA, and is an RNA delivery vehicle as described in any one of paragraphs 1 to 17.

[0275] 19. The RNA delivery vehicle described in paragraph 18, wherein the mRNA is either self-replicating mRNA (sa-mRNA) or conventional RNA (cRNA).

[0276] 20. An immunogenic composition comprising an RNA delivery vehicle as described in any one of paragraphs 1 to 19.

[0277] 21. A pharmaceutical composition comprising an RNA delivery vehicle described in any one of paragraphs 1 to 19 or an immunogenic composition described in paragraph 20 and a pharmaceutically acceptable carrier.

[0278] 22. An RNA delivery vehicle as described in any one of paragraphs 1 to 19, an immunogenic composition as described in paragraph 20, or a pharmaceutical composition as described in paragraph 21, for use in therapeutic purposes.

[0279] 23. A method for detecting unencapsulated RNA and / or partially encapsulated RNA on the outer surface of the lipid nanoparticles, wherein the method is: (i) Contacting the sample containing the lipid nanoparticles with an RNA-binding protein or peptide that binds to unencapsulated RNA and / or partially encapsulated RNA on the outer surface of the lipid nanoparticles to form a complex; (ii) Contacting the complex with a detection protein containing an antibody variable region, wherein the detection protein is bound to the RNA-binding protein or peptide, and the detection protein contains a detectable label, (iii) The method comprising detecting a detectable label, wherein the presence of the detectable label indicates the presence of unencapsulated RNA and / or partially encapsulated RNA on the outer surface of the lipid nanoparticles in the sample.

[0280] 24. The method according to paragraph 23, wherein the detectable sign is a magnetic sign.

[0281] 25. The method according to paragraph 23 or 24, wherein the sample comprises the RNA delivery vehicle according to any one of paragraphs 1 to 19.

[0282] This disclosure includes the following non-limiting examples.

Example

[0283] Example 1: Protection of RNA from degradation by RNase RNA-free monovalent pooled harvest (MPH) was prepared by treating MPH with RNase.

[0284] MPH (B / Malaysia / 2506 / 2004) was treated twice with RNase A (Promega) at 37 °C for 1 hour. RNA was extracted from 140 μl of treated and untreated MPH (eluted with 60 μl) and tested for hemagglutinin (HA) and neuraminidase (NA) RNA using real-time PCR. The results showed that the CT value of HA RNA did not increase and the CT value of NA RNA increased slightly (Table 2, Experiment 1A), indicating minimal degradation of viral RNA.

[0285] When RNase was inactive, MPH was treated twice with a different RNase (RNase ONE, Promega M4261) before RNA extraction and analysis. The CT values of both HA and NA RNA increased (Table 2, Experiment 1B), indicating a decrease in the amount of viral RNA present (<83-fold).

[0286] A further sample of MPH (180 μl) was treated twice using 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 CT value (Table 2, Experiment 1C), indicating a low level of RNA degradation (<35-fold) when MPH was treated with RNase ONE. Overall, the level of RNA degradation in treated MPH was unexpectedly low.

[0287] Next, MPH (B / Malaysia / 2506 / 2004) was 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).

[0288] Treatment of MPH with RNase resulted in a slight reduction in viral RNA only, suggesting that RNA may be protected from degradation by RNase. [Table 3]

[0289] Example 2: Purification of RNA-free nucleoprotein from influenza virus RNA-free nucleoproteins (NPs) were isolated from influenza viruses by dialysis, concentration, and surfactant treatment, followed by glycerol gradient centrifugation to isolate NP:RNA particles. Further glycerol and cesium chloride gradient centrifugation steps were performed to isolate RNA-free NPs.

[0290] Example 3: RNA-free NPs bind to RNA. To evaluate whether RNA-free NPs protect RNA, NPs were combined with nanoluciferase mRNA, and the samples were analyzed by MOPS agarose gel electrophoresis after heating to 40°C or incubation at room temperature. As the concentration of NPs during the reaction was increased (from 0 to 4000 ng) while maintaining the mRNA concentration (250 ng), the level of detectable mRNA shifted to higher molecular weights.

[0291] At a ratio of 16:1 (4000 ng NP, 250 ng mRNA), the nanoluciferase mRNA shifted away from the gel. These results suggest that as NP concentration increases, the amount of RNA that complexes with NP may also increase.

[0292] Using rabbit reticulocyte lysates as an in vitro translation system, the amount of luciferase produced, evaluated by luminescence measurements in relative light units (RLU) at a ratio of 16:1 (4000 ng NP, 250 ng mRNA), was equivalent to that produced by luciferase RNA alone, but the signal was reduced 2-3 times with 4000 ng NP alone (Figure 1). These results indicate that when the NP concentration is increased, RNA binds to NP without inhibiting in vitro translation.

[0293] Example 4: RNA-free NPs protect RNA from degradation. To evaluate whether RNA-free NPs protect RNA, NP:RNA and RNA alone were assessed using RNase assays. Briefly, NP:RNA or RNA was treated with RNase and incubated at 30°C for 5–10 minutes. Samples were further treated with or without 1 μl of heat-stable protease K (PK, NEB P8111S). The reaction mixture was incubated at 37°C for 15–30 minutes, followed by incubation at 60°C for 10–20 minutes to inactivate PK. If necessary, 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.

[0294] As shown in Figure 2, there was no increase in CT values ​​for NP:RNA treated with RNase, indicating no degradation of viral RNA. In contrast, the CT value of 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 RNA is protected from degradation by RNase in the presence of NP.

[0295] Adding a PK that degrades NPs to NP:RNA resulted in a slight increase in the CT value, indicating minimal RNA degradation. Adding the PK in combination with RNase resulted in a significant increase in the CT value (<0.0001), indicating a reduction in the amount of viral RNA present. These results further confirm that RNA is protected from RNase degradation in the presence of NPs.

[0296] 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) is temperature-dependent, the amount of luciferase produced was evaluated by incubating NPs:nLuc RNA and nLuc RNA at 4°C, 24°C, and 37°C for up to 96 hours and measuring luminescence in the RLU.

[0297] As shown in Figure 3, no significant difference in RLU was observed between NP:nLuc RNA and nLuc RNA alone at 4°C. At 24°C, the RLU of nLuc RNA decreased significantly compared to NP:nLuc RNA at 96 hours post-incubation (p=0.0034). This indicates protection of RNA from degradation by NP. At 37°C, after 8 hours, the RLU of nLuc RNA decreased significantly compared to NP:nLuc RNA, demonstrating further long-term protection of RNA by NP.

[0298] Example 6: RNA-free NPs protect TLR induction To assess whether the presence of NPs inhibits RNA induction of dsRNA or ssRNA, TLR3 and / or TLR8 induction was evaluated using a TLR-inducing NfKB reporter assay. In this assay, NfKB is operationally ligated to secreted alkaline phosphatase (SEAP). RNA is introduced into either cell type (conditionally transduced TLR3 or conditionally transduced TLR8). Binding to the TLR receptor induces NfKB activation, and consequently SEAP. SEAP levels can be determined by reading out the chemical reaction and heat output.

[0299] As shown in Figure 4, RNA alone stimulates the induction of both TLR3 and TLR8, while the presence of NPs reduces the induction of both TLR3 and TLR8 at both 2 and 4 hours post-incubation.

[0300] Example 7: Binding of RNA-free NPs to maleimide-DSPE lipids NP (0.85 mg / ml) was incubated with DSPE (1,2-distearoyl-sn-glycero-3-phosphorylethanolamine, 8 mM) in the presence of 10% ethanol to conjugate NP to maleimide-DSPE. Labeled and unlabeled NP were electrophoresed on a non-reducing gel to evaluate the molecular weight of the protein and confirm the binding of the molecular weight shift between the protein and NP. Furthermore, since DSPE forms a suspension (i.e., liposomes), the composition was centrifuged and the supernatant was evaluated.

[0301] Subsequently, it was shown that lipid-conjugated NP:RNA could be pulled down by centrifugation, but RNA alone could not be pulled down in the presence of lipids (i.e., not in the presence of NPs).

[0302] Example 8: COVID nucleocapsid protects RNA RNA-free nucleocapsids derived from SARS-CoV-2 (NP(SCoV2)) were purified in the same manner as those for influenza viruses. The ability of NP(SCoV2) to protect nLucRNA from degradation was assessed in an in vitro translation system by measuring the amount of luciferase produced by measuring luminescence at RLU for up to 168 hours.

[0303] As shown in Figure 5, the RLU of nLucRNA alone decreases over time, indicating that the nLucRNA has been degraded. When the NP(SCoV2):nLucRNA composition was 16:1 and 8:1 (w / w), the RLU appeared to decrease slightly, suggesting that the RNA was being degraded. However, PCR analysis of the NP(SCoV2):nLucRNA material at each time point confirmed that the RNA was intact and not degraded.

[0304] Furthermore, the accessibility of RNA encapsulated with NP(SCoV2) was evaluated using a ribogreen dye exclusion assay. As shown in Figure 5B, increasing the concentration of NP(SCoV2) hinders dye binding to nLucRNA, but based on the fluorescence signal, RNA is still accessible to the dye in a 16:1 ratio (4000 ng NP, 250 ng mRNA). This suggests that NP(SCoV2) covers and protects the RNA, but does not completely encapsulate it.

[0305] Example 9: Preparation of RNA-binding peptide We examined the protein sequences of cells and viruses to identify protein domains and peptide sequences that may bind to RNA. Sequences from cell proteins correlate with proteins involved in cell proliferation, cell signaling, and / or antiviral pathways, while sequences from viral proteins originate from non-structural and nuclear proteins.

[0306] 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). The viral RNA-binding proteins included nucleoproteins and non-structural proteins derived from influenza (Table 4).

[0307] The RNA-binding peptide sequences were modified to either exclude known nuclear localization signals or to include nuclear export signals to facilitate the correct localization of peptide-binding RNA when the substance is introduced into cells (Table 3).

[0308] Example 10: Inclusion assay for detecting RNA on the outside of LNPs To develop an inclusion assay for detecting and differentiating internal / external mRNA on LNPs, mRNA immunoprecipitation was evaluated with and without magnetic beads, anti-NP monoclonal antibodies (mAbs), and recombinant NPs.

[0309] Immunoprecipitation of mRNA in the absence of recombinant NPs Magnetic beads (Dynabeads) washed with PBS were coated with 10 μg of anti-NP mAb and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound anti-NP mAb was removed. Then, the magnetic beads were washed with PBS to remove any unbound residual anti-NP mAb, and the magnetic beads were coated with various concentrations of luciferase mRNA (500, 100, 50, 10, or 0 ng) and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant was collected (eluted), and the magnetic beads were resuspended in PBS (bound). The eluted and bound supernatants were diluted 1:10 with 0.05% Triton-X 100 and incubated at 95°C for 10 minutes. The supernatant was cooled to room temperature and the RNA concentration was measured using the Ribogreen RNA assay kit. [Table 4] [Table 5]

[0310] Immunoprecipitation of mRNA in the presence of recombinant NPs Magnetic beads (Dynabeads) washed with PBS were coated with 10 μg of anti-NP mAb and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound anti-NP mAb was removed. Next, the magnetic beads were washed with PBS to remove any unbound residual anti-NP mAb, coated with 20 μg of recombinant NP protein, and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound recombinant NP was removed. The magnetic beads were washed with PBS to remove any further unbound recombinant NP protein. Next, the magnetic beads were coated with luciferase mRNA at various concentrations (500, 100, 50, 10, or 0 ng) and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant was collected (eluted), and the magnetic beads were resuspended in PBS (bound). The eluted and bound supernatant was diluted 1:10 with 0.05% Triton-X 100 and incubated at 95°C for 10 minutes. The supernatant was cooled to room temperature, and the RNA concentration was measured using the Ribogreen RNA assay kit.

[0311] As shown in Figure 6, mRNA did not immunoprecipitate in the absence of recombinant NPs, but it did immunoprecipitate in the presence of recombinant NPs.

[0312] Preparation of LNPs mRNA found on the outer surface (mRNA-LNP) or inner surface ((mRNA)-LNP) of LNPs was prepared using DOTAP, DSPC, cholesterol, and DMG-PEG. Lipids were prepared to standard molar concentrations in ethanol, and then the lipids were mixed. Luciferase mRNA was prepared with or without RNA bound to the NP. The ratio of NP to mRNA was 2:1. Subsequently, the mRNA was encapsulated in the lipid mixture using a nanoassembler.

[0313] Immunoprecipitation of mRNA present on the outer or inner surface of LNPs in the presence of recombinant NP proteins. Magnetic beads (Dynabeads) washed with PBS were coated with 10 μg of anti-NP mAb and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound anti-NP mAb was removed. Next, the magnetic beads were washed with PBS to remove any unbound residual anti-NP mAb, coated with 20 μg of recombinant NP protein, and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound recombinant NP was removed. The magnetic beads were washed with PBS to remove any further unbound recombinant NP protein. Next, the magnetic beads were coated with LNPs containing mRNA on either the outer surface (mRNA-LNP) or inner surface ((mRNA)-LNP) and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant was collected (eluted), and the magnetic beads were resuspended in PBS (bound). The eluted and bound supernatant was diluted 1:10 with 0.05% Triton-X 100 and incubated at 95°C for 10 minutes. The supernatant was cooled to room temperature, and the RNA concentration was measured using the Ribogreen RNA assay kit.

[0314] Immunoprecipitation of mRNA present on the outer or inner surface of LNPs in the absence of recombinant NP proteins. Magnetic beads (Dynabeads) washed with PBS were coated with 10 μg of anti-NP mAb and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound anti-NP mAb was removed. Next, the magnetic beads were washed with PBS to remove any unbound residual anti-NP mAb. Next, the magnetic beads were coated with LNPs containing mRNA on either the outer surface (mRNA-LNP) or the inner surface ((mRNA)-LNP) and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant was collected (eluted), and the magnetic beads were resuspended in PBS (bound). The eluted and bound supernatants were diluted 1:10 with 0.05% Triton-X 100 and incubated at 95°C for 10 minutes. The supernatant was cooled to room temperature and the RNA concentration was measured using the Ribogreen RNA assay kit.

[0315] As shown in Figure 7, mRNA on the outer surface (mRNA-LNP) or inner surface ((mRNA)-LNP) of LNPs was immunoprecipitated in the presence of recombinant NPs, and was also partially immunoprecipitated in the absence of recombinant NPs.

[0316] Immunoprecipitation of NP-binding mRNA present on the outer or inner surface of LNPs in the presence of recombinant NP proteins. Magnetic beads (Dynabeads) washed with PBS were coated with 10 μg of anti-NP mAb and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound anti-NP mAb was removed. Next, the magnetic beads were washed with PBS to remove any unbound residual anti-NP mAb, coated with 20 μg of recombinant NP protein, and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound recombinant NP was removed. The magnetic beads were washed with PBS to remove any further unbound recombinant NP protein. Next, the magnetic beads were coated with LNPs containing mRNA on either the outer surface (NP-mRNA-LNP) or the inner surface ((NP-mRNA)-LNP) and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant was collected (eluted), and the magnetic beads were resuspended in PBS (bound). The eluted and bound supernatant was diluted 1:10 with 0.05% Triton-X 100 and incubated at 95°C for 10 minutes. The supernatant was cooled to room temperature, and the RNA concentration was measured using the Ribogreen RNA assay kit.

[0317] Immunoprecipitation of NP-binding mRNA present on the outer or inner surface of LNPs in the absence of recombinant NP protein. Magnetic beads (Dynabeads) washed with PBS were coated with 10 μg of anti-NP mAb and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound anti-NP mAb was removed. Next, the magnetic beads were washed with PBS to remove any unbound residual anti-NP mAb. Next, the magnetic beads were coated with LNPs containing mRNA on either the outer surface (NP+mRNA-LNP) or the inner surface ((NP+mRNA)-LNP) and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant was collected (eluted), and the magnetic beads were resuspended in PBS (bound). The eluted and bound supernatants were diluted 1:10 with 0.05% Triton-X 100 and incubated at 95°C for 10 minutes. The supernatant was cooled to room temperature and the RNA concentration was measured using the Ribogreen RNA assay kit.

[0318] As shown in Figure 8, NP-binding mRNA on the outer surface (NP+mRNA-LNP) or inner surface ((NP+mRNA)-LNP) of LNPs was immunoprecipitated in the presence of recombinant NPs, and was also partially immunoprecipitated in the absence of recombinant NPs.

[0319] LNP preparation that reduces the amount of ionized lipids LNPs were prepared using Dlin-MC3, DSPC, cholesterol, and DMG-PEG. Lipids were prepared to standard molar concentrations in ethanol and then mixed with ionized lipids (Dlin-MC3) and DMG-PEG in different ratios. Luciferase mRNA was prepared with or without RNA bound to the NPs. The ratio of NPs to mRNA was 2:1. The mRNA was then encapsulated in the lipid mixture using a nanoassembler.

[0320] Immunoprecipitation of mRNA encapsulated within LNPs, where the amount of ionized lipids decreases in the presence of recombinant NP proteins. Magnetic beads (Dynabeads) washed with PBS were coated with 10 μg of anti-NP mAb and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound anti-NP mAb was removed. Next, the magnetic beads were washed with PBS to remove any unbound residual anti-NP mAb, coated with 20 μg of recombinant NP protein, and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound recombinant NP was removed. The magnetic beads were washed with PBS to further remove any unbound recombinant NP protein. Next, the magnetic beads were coated with mRNA encapsulated in LNPs with a reduced amount of ionized lipids and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant was collected (eluted), and the magnetic beads were resuspended in PBS (bound). The eluted and bound supernatant was diluted 1:10 with 0.05% Triton-X 100 and incubated at 95°C for 10 minutes. The supernatant was cooled to room temperature, and the RNA concentration was measured using the Ribogreen RNA assay kit.

[0321] Immunoprecipitation of mRNA encapsulated within LNPs, where the amount of ionized lipids decreases in the absence of recombinant NP protein. Magnetic beads (Dynabeads) washed with PBS were coated with 10 μg of anti-NP mAb and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound anti-NP mAb was removed. The magnetic beads were then washed with PBS to remove any unbound residual anti-NP mAb. Next, the magnetic beads were coated with mRNA encapsulated in LNPs with a reduced amount of ionized lipids and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant was collected (eluted), and the magnetic beads were resuspended in PBS (bound). The eluted and bound supernatant was diluted 1:10 with 0.05% Triton-X 100 and incubated at 95°C for 10 minutes. The supernatant was cooled to room temperature and the RNA concentration was measured using the Ribogreen RNA assay kit.

[0322] As shown in Figure 9, a decrease in the amount of ionized lipids increases mRNA immunoprecipitation induced by anti-NP mAbs.

[0323] Immunoprecipitation of NP-binding mRNA encapsulated within LNPs, where the amount of ionized lipids decreases in the presence of recombinant NP proteins. Magnetic beads (Dynabeads) washed with PBS were coated with 10 μg of anti-NP mAb and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound anti-NP mAb was removed. Next, the magnetic beads were washed with PBS to remove any unbound residual anti-NP mAb, coated with 20 μg of recombinant NP protein, and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound recombinant NP was removed. The magnetic beads were washed with PBS to further remove any unbound recombinant NP protein. Next, the magnetic beads were coated with NP-binding mRNA encapsulated in LNPs with a reduced amount of ionized lipids and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant was collected (eluted), and the magnetic beads were resuspended in PBS (bound). The eluted and bound supernatant was diluted 1:10 with 0.05% Triton-X 100 and incubated at 95°C for 10 minutes. The supernatant was cooled to room temperature, and the RNA concentration was measured using the Ribogreen RNA assay kit.

[0324] Immunoprecipitation of NP-binding mRNA encapsulated within LNPs, where the amount of ionized lipids decreases in the absence of recombinant NP protein. Magnetic beads (Dynabeads) washed with PBS were coated with 10 μg of anti-NP mAb and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant containing any unbound anti-NP mAb was removed. The magnetic beads were then washed with PBS to remove any unbound residual anti-NP mAb. Next, the magnetic beads were coated with NP-binding mRNA encapsulated in LNPs with reduced ionized lipid content and incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant was collected (eluted), and the magnetic beads were resuspended in PBS (bound). The eluted and bound supernatant was diluted 1:10 with 0.05% Triton-X 100 and incubated at 95°C for 10 minutes. The supernatant was cooled to room temperature and the RNA concentration was measured using the Ribogreen RNA assay kit.

[0325] As shown in Figure 10, using NP-binding mRNA increases the amount of mRNA that can be immunoprecipitated.

[0326] LNP containing ionized lipids mRNA-LNP or NP+mRNA-LNP was added to magnetic beads (Dynabeads) washed with PBS. The magnetic beads were incubated at room temperature for 20 minutes with shaking. Next, the magnetic beads were placed on a magnet and the supernatant was collected (elution), and the magnetic beads were resuspended in PBS (binding). The eluted and bound supernatants were diluted 1:10 with 0.05% Triton-X 100 and incubated at 95°C for 10 minutes. The supernatant was cooled to room temperature, and the RNA concentration was measured using the Ribogreen RNA assay kit. As shown in Figure 11, LNPs containing ionized lipids do not bind to the magnets used in immunoprecipitation assays.

[0327] Therefore, it was found that the use of NP-binding mRNA leads to an increase in LNP size (Table 5). [Table 6]

Claims

1. An RNA delivery vehicle, wherein the delivery vehicle is (i) Lipid nanoparticles containing the RNA inside, (ii) The RNA delivery vehicle comprising an RNA-binding protein or peptide coated on the outer surface of the lipid nanoparticles.

2. The RNA delivery vehicle according to claim 1, wherein the RNA-binding protein or peptide encapsulates the lipid nanoparticles.

3. The RNA delivery vehicle according to claim 1, wherein the RNA-binding protein or peptide directly binds to unencapsulated RNA and / or partially encapsulated RNA on the outer surface of the lipid nanoparticles.

4. The RNA delivery vehicle according to claim 1, wherein the RNA-binding protein or peptide is a lipidized RNA-binding protein or peptide.

5. The RNA-binding protein or peptide is a) Reduce the toxicity of the lipid nanoparticles, and / or b) Stabilize the RNA and / or c) Protect the RNA from degradation, and / or d) Promote nucleation of the lipid nanoparticles, and / or e) The RNA delivery vehicle according to claim 1, which inhibits the induction of signal transduction by one or more Toll-like receptors.

6. The RNA delivery vehicle according to claim 1, wherein the RNA-binding protein or peptide is modified to remove nuclear localization signals and / or introduce nuclear export signals.

7. The RNA delivery vehicle according to claim 1, wherein the RNA-binding protein or peptide is a viral or nonviral RNA-binding protein or peptide.

8. The RNA delivery vehicle according to claim 7, wherein the viral RNA-binding protein is derived from a class III, class IV, class V, and / or class VI virus.

9. The RNA delivery vehicle according to claim 8, 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, metapneumonia virus, rhinovirus, coronavirus, adenovirus and bocavirus.

10. The RNA delivery vehicle according to claim 9, wherein the viral RNA-binding protein or peptide is a nucleoprotein, a non-structural protein, a substrate protein, and / or a nucleocapsid protein.

11. The RNA delivery vehicle according to claim 7, wherein the viral RNA-binding protein or peptide is a non-structural (NS) protein derived from influenza B virus.

12. The RNA delivery vehicle according to claim 7, wherein the viral RNA-binding protein or peptide comprises the sequences described in SEQ ID NOs: 9 to 11.

13. The RNA delivery vehicle according to claim 7, wherein the nonviral RNA-binding protein or peptide is derived from a cellular protein associated with cell proliferation, cell signaling, and / or antiviral pathways.

14. The RNA delivery vehicle according to claim 13, 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.

15. The RNA delivery vehicle according to claim 13, wherein the cell protein comprises the sequences described in SEQ ID NOs: 1 to 8.

16. The RNA delivery vehicle according to claim 1, wherein the lipid nanoparticles include PEG lipids, structural lipids, neutral lipids and / or cationic lipids.

17. The RNA delivery vehicle according to claim 1, wherein the lipid nanoparticles do not contain cationic lipids.

18. The RNA delivery vehicle according to claim 1, wherein the RNA is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), and antisense RNA.

19. The RNA delivery vehicle according to claim 18, wherein the mRNA is self-replicating mRNA (sa-mRNA) or conventional RNA (cRNA).

20. An immunogenic composition comprising the RNA delivery vehicle described in claim 1.

21. A pharmaceutical composition comprising an RNA delivery vehicle according to claim 1 or an immunogenic composition according to claim 20 and a pharmaceutically acceptable carrier.

22. An RNA delivery vehicle according to claim 1, an immunogenic composition according to claim 20, or a pharmaceutical composition according to claim 21, for use in therapeutic purposes.

23. A method for detecting unencapsulated RNA and / or partially encapsulated RNA on the outer surface of lipid nanoparticles, wherein the method is: (i) Contacting a sample containing lipid nanoparticles with an RNA-binding protein or peptide that binds to unencapsulated RNA and / or partially encapsulated RNA on the outer surface of the lipid nanoparticles to form a complex, (ii) Contacting the complex with a detection protein containing an antibody variable region, wherein the detection protein is bound to the RNA-binding protein or peptide, and the detection protein contains a detectable label, (iii) The method comprising detecting the detectable label, wherein the presence of the detectable label indicates the presence of unencapsulated RNA and / or partially encapsulated RNA on the outer surface of the lipid nanoparticles in the sample.

24. The method according to claim 23, wherein the detectable sign is a magnetic sign.

25. The method according to claim 23, wherein the sample comprises the RNA delivery vehicle described in claim 1.

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