Spleen-targeted self-replicating RNA-LNP and use thereof

Abstract

Provided are a spleen-targeted self-replicating RNA-LNP and the use thereof. Also provided is a method for expressing a peptide or protein of interest, or a non-coding RNA in the spleen or lymph node, the method comprising administering to the spleen or lymph node a self-replicating RNA molecule, wherein the self-replicating RNA molecule comprises, from the 5' end to the 3' end, a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a sequence of interest, a 3' UTR, and a poly(A) tail, and the RNA replicase is a non-structural protein derived from the Everglades virus (EVEV), Rio Negro virus (RNV) or Mosso das pedras virus (MDPV).

Description

Spleen-targeted self-replicating RNA-LNPs and their applications

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application No. 202510121971.X, filed on January 24, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to a method for expressing a target peptide or protein, or non-coding RNA, in the spleen or lymph node, comprising administering a self-replicating RNA molecule to the spleen or lymph node, wherein the self-replicating RNA molecule comprises, from the 5' end to the 3' end, a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a target sequence, a 3' UTR, and a poly(A) tail, wherein the RNA replicase is capable of amplifying the self-replicating RNA molecule and a subgenomic RNA molecule containing the target sequence, wherein the target sequence comprises an open reading frame encoding a target peptide or protein, or a non-coding RNA sequence, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), or Mostas Pedras virus (MDPV). Background Technology

[0004] In recent years, mRNA drug technology has experienced rapid development, significantly altering the landscape of modern pharmaceutical technology. This progress is primarily driven by technological breakthroughs and global health needs. Firstly, innovations and breakthroughs in mRNA synthesis, modification, and delivery technologies have resolved its instability and low delivery efficiency, making the research and production of mRNA drugs possible. During the COVID-19 pandemic, Pfizer-BioNTech and Moderna's mRNA vaccines demonstrated the potential of this technology through rapid development and effective protection, increasing global acceptance of mRNA technology. The flexibility and versatility of mRNA platforms enable them to quickly adapt to different disease needs, finding wide application in vaccine development, cancer immunotherapy, protein replacement therapy for rare diseases, and gene editing, among other fields. Therefore, from proof-of-concept to market application, mRNA drug technology has become an important component of modern medicine.

[0005] In the application of mRNA drug technology, the delivery method is a crucial factor in achieving its efficacy and safety. Currently, lipid nanoparticles (LNPs) are the most commonly used mRNA delivery carriers, as they can protect mRNA from degradation and promote its entry into cells. However, the characteristics of LNP delivery systems also present significant challenges, one of which is that mRNA delivered via LNPs mainly accumulates in the liver for expression, a tendency that limits the application of mRNA drugs in other target organs. Achieving efficient targeted delivery remains one of the major challenges facing mRNA drugs. To overcome this limitation, researchers are exploring various strategies to optimize mRNA delivery.

[0006] The most direct strategy is to inject mRNA drugs directly into the target site or tissue using local injection methods, increasing local mRNA concentration and reducing systemic distribution and side effects. This strategy effectively improves treatment specificity, but its application is limited by the feasibility of the operation and the accessibility of the target site. For some disease scenarios requiring systemic treatment, local delivery may not be suitable. The second strategy is to use active targeting, which involves modifying the surface of LNPs with specific ligands to achieve targeted delivery to specific cell types or tissues. This strategy helps increase the concentration of mRNA in the target tissue, thereby enhancing the therapeutic effect. However, this requires a deep understanding of the specific markers on the target cell surface, and the preparation methods of ligand-conjugated LNPs are relatively complex. Furthermore, the conjugated ligands usually do not play a dominant role in the targeting of LNPs and cannot fundamentally change the targeting behavior of LNPs. The third common targeting strategy is to develop novel ionizable lipids. LNPs prepared using these new lipids have different physicochemical properties, thus affecting their in vivo distribution and cellular uptake behavior. These carriers achieve different targeting functions through variations in material type and surface properties. However, the biocompatibility and toxicity of each new material need to be extensively tested, and the complexity and scalability of production also pose challenges in the application process.

[0007] These representative targeted delivery strategies reveal that most research in exploring targeted pathways for mRNA drugs focuses on the design and optimization of delivery vectors. However, current research on whether the loaded mRNA itself affects drug targeting is relatively limited, leaving a knowledge gap.

[0008] Self-replicating RNA (saRNA, also known as self-amplifying RNA) has attracted widespread attention because it can leverage the viral replicase system to continuously amplify in vitro synthesized RNA within cells, achieving higher and more persistent expression of target proteins. saRNA sequences are derived from modifications of the bicistronic genomes of positive-sense RNA viruses (e.g., alphaviruses, flaviviruses, lentiviruses, measles viruses, and rhabdoviruses). In addition to conventional mRNA elements such as the cap, 5'UTR, 3'UTR, and poly(A) tail, saRNA contains a very large open reading frame at its 5' end, encoding four non-structural proteins (nsPs) of the positive-sense RNA virus. The viral structural protein genes originally located after the subgenomic promoter (SGP) are replaced with genes encoding the target proteins. Once saRNA enters the host cytoplasm, it first translates into the four non-structural proteins (nsP1, nsP2, nsP3, and nsP4), which then polymerize to form an RNA-dependent RNA polymerase complex, also known as RNA replicase. The RNA polymerase complex first synthesizes a complementary antisense RNA from the sense RNA. Then, using this antisense RNA as a template, it synthesizes a copy of the original full-length RNA sense RNA and multiple subgenomic sense RNAs encoding the target protein downstream of the SGP. The former further enters the amplification cycle, while the latter translates the target protein. This is why saRNA can achieve efficient and sustained expression of the target protein at low doses.

[0009] Bathula et al. encapsulated Venezuelan equine encephalitis virus (VEEV)-saRNA expressing luciferase in lipid nanoparticles (LNPs) and administered it to mice intramuscularly, intradermally, intraperitoneally, intranasally, intravenously, or subcutaneously, and then imaged the luciferase luminescence in the mice. The results showed that the in vivo distribution of saRNA was completely different depending on the administration method. For example, after intranasal administration, luciferase luminescence was mainly observed in the lungs; after intraperitoneal administration, luminescence was mainly observed in the spleen, kidneys, and lungs; while intravenous administration resulted in luciferase luminescence being mainly concentrated in the spleen and lungs (Bathula NV, et al., (2024) Delivery vehicle and route of administration influences self-amplifying RNA biodistribution, expression kinetics, and reactogenicity. J Control Release. 374:28-38). Summary of the Invention

[0010] The inventors of this application constructed self-replicating RNA (saRNA) expressing firefly luciferase (FLuc) based on Venezuelan equine encephalitis virus (VEEV), Mosso das Pedras virus (MDPV), Rio Negro virus (RNV), and Everglades virus (EVEV) from the alphavirus family. These saRNAs and FLuc-expressing mRNAs were then encapsulated in fluorescently labeled LNPs and injected into mice via the tail vein. Twenty-four hours after injection, the distribution of LNPs and RNA in seven organs of the mice was tracked.

[0011] The results showed that the delivery patterns of saRNA-LNPs and mRNA-LNPs were consistent, with over 80% of RNA-LNPs concentrated in the liver, and a small number distributed in lymph nodes, brain, lungs, spleen, and heart. Surprisingly, only the FLuc expression distribution of mRNAs was consistent with the LNP distribution, while the FLuc expression of each saRNA was mainly distributed in extrahepatic areas such as the spleen, lymph nodes, heart, and kidneys, with very low FLuc expression in the liver. For example, as shown in Figure 6, VEEV-saRNA FLuc expression was higher in the spleen, heart, and kidneys; MDPV-saRNA expression was mainly in lymph nodes and spleen; RNV-saRNA FLuc expression in the spleen accounted for about half of the total expression in the tested organs; and EVEV-saRNA FLuc expression in the spleen even more dramatically accounted for 72.68% of the total expression in the tested organs.

[0012] Therefore, it can be seen that although different administration methods can affect the delivery of saRNA-LNPs, the organ preference of saRNAs in vivo is the key factor affecting their amplification and expression of the target sequence. Taking EVEV-based saRNAs as an example, only about 6.38% of saRNA-LNPs were delivered to the spleen, but their FLuc expression accounted for 72.68% of the total expression in the tested organ. For example, only 2.04% of MDPV saRNA-LNPs were delivered to lymph nodes, but their FLuc expression in lymph nodes accounted for 47.14% of the total expression in the tested organ; only 0.68% of RNV saRNA-LNPs were delivered to lymph nodes, but their FLuc expression in lymph nodes accounted for 18.04% of the total expression in the tested organ. As another example, only 0.96% of VEEV saRNA-LNPs were delivered to the heart, while FLuc expression in the heart accounted for 22.45% of the total expression in the tested organ.

[0013] The organ-specific expression preferences of these saRNAs can be leveraged to select suitable saRNAs for efficient amplification and expression of target sequences in specific organs, especially in in vitro expression scenarios. In in vivo applications, these saRNAs can be combined with organ-targeting vectors such as LNPs to further reduce impact on other organs. Because EVEV saRNA exhibits a particularly strong preference for the spleen, low-dose naked sequence intravenous injection can be attempted to minimize amplification and expression in other organs without affecting the target sequence amplification and expression in the spleen. Similarly, because MDPV saRNA and RNV saRNA exhibit a particularly strong preference for lymph nodes, low-dose naked sequence intravenous injection can be attempted to minimize amplification and expression in other organs without affecting the target sequence amplification and expression in lymph nodes.

[0014] Therefore, in a first aspect, this application provides a self-replicating RNA molecule encapsulated by lipid nanoparticles (LNPs).

[0015] Self-replicating RNA molecules may contain a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a target sequence, a 3' UTR, and a poly(A) tail from the 5' end to the 3' end. This RNA replicase can amplify the self-replicating RNA molecule and its subgenomic RNA molecules containing the target sequence (such as the target sequence and the 3' UTR).

[0016] This self-replicating RNA molecule can be a single-stranded RNA molecule.

[0017] The RNA complex enzyme can be a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), Mosta Spedras virus (MDPV), or Venezuelan equine encephalitis virus (VEEV). The non-structural protein may include nsP1, nsP2, nsP3, and / or nsP4. In some embodiments, the non-structural protein may be nsP123 and nsP4. In some embodiments, the non-structural protein may be nsP1234.

[0018] In some embodiments, the RNA replicase may be a non-structural protein derived from Everglades virus (EVEV) or a functional variant thereof, and the open reading frame encoding the RNA replicase may contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:9.

[0019] In some embodiments, the RNA replicase may be a non-structural protein derived from Rio Negro virus (RNV) or a functional variant thereof, and the open reading frame encoding the RNA replicase may contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8.

[0020] In some embodiments, the RNA replicase may be a non-structural protein or a functional variant thereof derived from the Moscow das Pedras virus (MDPV), and the open reading frame encoding the RNA replicase may contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:7.

[0021] In some embodiments, the RNA replicase may be a non-structural protein or a functional variant thereof derived from Venezuelan equine encephalitis virus (VEEV), and the open reading frame encoding the RNA replicase may contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:6.

[0022] RNA replicase can amplify self-replicating RNA molecules, including the ability to transcribe a complementary RNA strand from the self-replicating RNA molecule, and the ability to transcribe a self-replicating RNA molecule from the transcribed RNA strand. RNA replicase can also amplify subgenomic RNA molecules, including the ability to transcribe a complementary RNA strand from the self-replicating RNA molecule, and the ability to transcribe a subgenomic RNA molecule from the transcribed RNA strand. In some embodiments, the amount of subgenomic RNA molecules amplified by the RNA replicase is greater than the amount of self-replicating RNA molecules amplified. Subgenomic RNA molecules may contain a target sequence. Subgenomic RNA molecules may contain a target sequence and a 3' UTR.

[0023] RNA replicase can have the activities of RNA-dependent RNA polymerase, protease, detransferase, terminal adenylate transferase, methyltransferase and / or guanylate transferase.

[0024] The 5'UTR, promoter, and / or 3'UTR of a self-replicating RNA molecule can cooperate with RNA replicase to amplify the self-replicating RNA molecule and / or subgenomic RNA molecule. In some embodiments, the 5'UTR, promoter, and 3'UTR of a replicable RNA molecule can cooperate with RNA replicase to amplify the self-replicating RNA molecule and subgenomic RNA molecule.

[0025] In some implementations, the 5'UTR, promoter, and 3'UTR may be derived from the same viral genome as the RNA replicase.

[0026] The promoter can be a subgenomic promoter of a virus.

[0027] In some embodiments, the 5'UTR, promoter, and 3'UTR may be derived from Everglades virus (EVEV) and contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:5, 13, and 20, respectively.

[0028] In some embodiments, the 5'UTR, promoter, and 3'UTR may be derived from Rio Negro virus (RNV) and contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:4, 12, and 19, respectively.

[0029] In some embodiments, the 5'UTR, promoter, and 3'UTR may be derived from Mostas Pedras virus (MDPV) and contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:3, 11, and 18, respectively.

[0030] In some embodiments, the 5'UTR, promoter, and 3'UTR may be derived from Venezuelan equine encephalitis virus (VEEV) and contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2, 10, and 17, respectively.

[0031] The 5' cap can be a natural 5' cap or a 5' cap analogue. A 5' cap analogue can be Cap-AU or Cap-AG. The poly(A) tail can contain or consist of consecutive adenosine nucleotides, for example, it can contain the nucleotide sequence shown in SEQ ID NO:21. Alternatively, the poly(A) tail can contain 2-5 consecutive adenosine nucleotide fragments separated by spacer sequences, wherein the spacer sequences can contain 1-20 nucleotides, and each consecutive adenosine nucleotide fragment can contain 10-100 consecutive adenosine nucleotides.

[0032] The target sequence can be any sequence. In some embodiments, the target sequence may contain an open reading frame encoding a target peptide or protein. The target peptide or protein may be a disease-related antigen or a therapeutic agent. In some embodiments, the target sequence may contain a non-coding RNA sequence. The non-coding RNA may be, for example, miRNA, tracrRNA, etc.

[0033] Self-replicating RNA molecules may also include an internal ribosome entry site (IRES) on the 3' side of the target sequence. The IRES can be any suitable IRES, such as an IRES derived from Coxsackie B3 virus (CVB3). An IRES derived from Coxsackie B3 virus (CVB3) may contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:15.

[0034] Self-replicating RNA molecules may also contain an open reading frame encoding an immunosuppressive protein on the 3' side of the target sequence, such as the 3' side of the internal ribosome entry site (IRES). The immunosuppressive protein may be an interferon-inhibiting protein (IIP), such as the vaccinia virus E3L protein. E3L may contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:16.

[0035] Self-replicating RNA molecules may also contain a second 5'UTR on the 5' side of the target sequence, such as between the promoter domain and the target sequence.

[0036] In some embodiments, the self-replicating RNA molecule may include a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a second 5' UTR, a target sequence, a 3' UTR, and a poly(A) tail from the 5' end to the 3' end, wherein the RNA replicase is capable of amplifying the replicable RNA molecule and its subgenomic RNA molecules containing the second 5' UTR, the target sequence, and the 3' UTR.

[0037] In some embodiments, the self-replicating RNA may include a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a second 5' UTR, a target sequence, an internal ribosome entry site (IRES), an open reading frame encoding an immunosuppressive protein, a 3' UTR, and a poly(A) tail from the 5' end to the 3' end, wherein the RNA replicase is capable of amplifying the self-replicating RNA molecule and its subgenomic RNA molecule comprising the second 5' UTR, the target sequence, the internal ribosome entry site (IRES), the open reading frame encoding the immunosuppressive protein, and the 3' UTR.

[0038] Lipid nanoparticles (LNPs) can be organ-targeted LNPs, such as spleen-targeted LNPs, lymph node-targeted LNPs, or heart-targeted LNPs.

[0039] Organ-targeting LNPs can be LNPs containing antibodies (especially scFv or nanobodies) or ligands that target the organ.

[0040] In some embodiments, the LNP may comprise permanently cationic lipids (e.g., 1,2-dioleoyl-propyl-3-trimethylammonium chloride (DOTAP), DDAB, or EPC), for example, 10-15% (e.g., 10-15 wt% or mol%) of permanently cationic lipids. In addition to permanently cationic lipids, the LNP may comprise ionizable cationic lipids, amphiphilic phospholipids, cholesterol, and PEGylated lipids, for example, comprising SM102, cholesterol, DSPC, and DMG-PEG-2000 in a molar ratio of 50:38.5:10:1.5; or 5A2-SC8, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and DMG-PEG in a molar ratio of 15:15:30:3.

[0041] In some embodiments, the LNP may comprise negatively charged lipids (e.g., 1,2-dioleoyl-sn-glycerol-3-phosphate (18PA)), for example, 10-40% (e.g., 10-40 wt% or mol%) of negatively charged lipids. In addition to negatively charged lipids, the LNP may comprise ionizable cationic lipids, amphiphilic phospholipids, cholesterol, and PEGylated lipids, for example, comprising SM102, cholesterol, DSPC, and DMG-PEG-2000 in a molar ratio of 50:38.5:10:1.5; or 5A2-SC8, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and DMG-PEG in a molar ratio of 15:15:30:3.

[0042] In some embodiments, the LNP may comprise negatively charged lipids, such as anionic lipids, like 14PA and 18BMP. In addition to anionic lipids, the LNP may comprise ionizable cationic lipids, amphiphilic phospholipids, cholesterol, and PEGylated lipids, such as SM102, cholesterol, DSPC, and DMG-PEG-2000 in a molar ratio of 50:38.5:10:1.5; or 5A2-SC8, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and DMG-PEG in a molar ratio of 15:15:30:3.

[0043] Accordingly, this application provides a composition comprising the self-replicating RNA molecule of this application. In some embodiments, the composition may be a pharmaceutical composition and may also comprise a pharmaceutically acceptable carrier.

[0044] In a second aspect, this application also provides a method for expressing a target peptide or protein or non-coding RNA in an organ selected from lymph nodes, kidneys, lungs, spleens, and hearts, comprising administering a self-replicating RNA molecule to the organ, wherein the self-replicating RNA molecule comprises, from the 5' end to the 3' end, a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a target sequence, a 3' UTR, and a poly(A) tail, wherein the RNA replicase is capable of amplifying the self-replicating RNA molecule and a subgenomic RNA molecule containing a target sequence (such as the target sequence and the 3' UTR), wherein the target sequence contains an open reading frame encoding a target peptide or protein, or a non-coding RNA sequence, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), Venezuelan equine encephalitis virus (VEEV), or Mosta Spedras virus (MDPV). In some embodiments, this application provides a method for expressing a target peptide or protein, or non-coding RNA, in the spleen or lymph node, comprising administering a self-replicating RNA molecule to the spleen or lymph node, wherein the self-replicating RNA molecule comprises, from the 5' end to the 3' end, a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a target sequence, a 3' UTR, and a poly(A) tail, wherein the RNA replicase is capable of amplifying the self-replicating RNA molecule and a subgenomic RNA molecule containing the target sequence (such as the target sequence and the 3' UTR), wherein the target sequence contains an open reading frame encoding a target peptide or protein, or a non-coding RNA sequence, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), Venezuelan equine encephalitis virus (VEEV), or Mosta Spedras virus (MDPV).

[0045] RNA replicase can amplify self-replicating RNA molecules, including the ability to transcribe a complementary RNA strand from the self-replicating RNA molecule, and the ability to transcribe a self-replicating RNA molecule from the transcribed RNA strand. RNA replicase can also amplify subgenomic RNA molecules, including the ability to transcribe a complementary RNA strand from the self-replicating RNA molecule, and the ability to transcribe a subgenomic RNA molecule from the transcribed RNA strand. In some embodiments, the amount of subgenomic RNA molecules amplified by the RNA replicase is greater than the amount of self-replicating RNA molecules amplified. Subgenomic RNA molecules may contain a target sequence. Subgenomic RNA molecules may contain a target sequence and a 3' UTR.

[0046] RNA replicase may have RNA-dependent RNA polymerase, protease, detransferase, terminal adenylate transferase, methyltransferase, and / or guanylate transferase activities. In some embodiments, the organ is the spleen, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), or Mosta Spedras virus (MDPV).

[0047] In some embodiments, the organ is a lymph node, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), Venezuelan equine encephalitis virus (VEEV), or Mostas Pedras virus (MDPV).

[0048] In some embodiments, the organ is the heart, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Rio Negro virus (RNV) or Venezuelan equine encephalitis virus (VEEV).

[0049] In some embodiments, the organ is the spleen or a lymph node, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), or Mostas Pedras virus (MDPV) or Venezuelan equine encephalitis virus (VEEV).

[0050] The 5'UTR, promoter, and / or 3'UTR of a self-replicating RNA molecule can cooperate with RNA replicase to amplify the self-replicating RNA molecule and / or subgenomic RNA molecule. In some embodiments, the 5'UTR, promoter, and 3'UTR of the replicable RNA molecule can cooperate with RNA replicase to amplify the self-replicating RNA molecule and subgenomic RNA molecule. In some embodiments, the 5'UTR, promoter, and 3'UTR can be derived from the same viral genome as the RNA replicase. The promoter can be a viral subgenomic promoter.

[0051] In some embodiments, the open reading frame, 5'UTR, promoter, and 3'UTR encoding the RNA replicase may be derived from Everglades virus (EVEV) and contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9, 5, 13, and 20.

[0052] In some embodiments, the open reading frame, 5'UTR, promoter, and 3'UTR encoding the RNA replicase may be derived from Rio Negro virus (RNV) and contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8, 4, 12, and 19, respectively.

[0053] In some embodiments, the open reading frame, 5'UTR, promoter, and 3'UTR encoding the RNA replicase may be obtained from Mostas Pedras virus (MDPV) and contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:7, 3, 11, and 18.

[0054] In some embodiments, the open reading frame, 5'UTR, promoter, and 3'UTR encoding the RNA replicase may be derived from Venezuelan equine encephalitis virus (VEEV) and contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 6, 2, 10, and 17, respectively.

[0055] The 5' cap can be a natural 5' cap or a 5' cap analogue. A 5' cap analogue can be Cap-AU or Cap-AG. The poly(A) tail can contain or consist of consecutive adenosine nucleotides, for example, it can contain the nucleotide sequence shown in SEQ ID NO:21. Alternatively, the poly(A) tail can contain 2-5 consecutive adenosine nucleotide fragments separated by spacer sequences, wherein the spacer sequences can contain 1-20 nucleotides, and each consecutive adenosine nucleotide fragment can contain 10-100 consecutive adenosine nucleotides.

[0056] The target sequence can be any sequence. In some embodiments, the target sequence may contain an open reading frame encoding a target peptide or protein. The target peptide or protein may be a disease-related antigen or a therapeutic agent. In some embodiments, the target sequence may contain a non-coding RNA sequence. The non-coding RNA may be, for example, miRNA, tracrRNA, etc.

[0057] Self-replicating RNA molecules may also contain an internal ribosome entry site (IRES) on the 3' side of the target sequence. The IRES can be any suitable IRES, such as the IRES derived from Coxsackie B3 virus (CVB3).

[0058] Self-replicating RNA molecules may also contain an open reading frame encoding an immunosuppressive protein on the 3' side of the target sequence, such as the 3' side of the internal ribosome entry site (IRES). The immunosuppressive protein may be an interferon-inhibiting protein (IIP), such as the vaccinia virus E3L protein. E3L may contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:16.

[0059] Self-replicating RNA molecules can also contain a second 5'UTR on the 5' side of the target sequence, such as between the promoter and the target sequence.

[0060] In some embodiments, the self-replicating RNA molecule may include a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a second 5' UTR, a target sequence, a 3' UTR, and a poly(A) tail from the 5' end to the 3' end, wherein the RNA replicase is capable of amplifying the replicable RNA molecule and its subgenomic RNA molecules containing the second 5' UTR, the target sequence, and the 3' UTR.

[0061] In some embodiments, the self-replicating RNA may include a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a second 5' UTR, a target sequence, an internal ribosome entry site (IRES), an open reading frame encoding an immunosuppressive protein, a 3' UTR, and a poly(A) tail from the 5' end to the 3' end, wherein the RNA replicase is capable of amplifying the self-replicating RNA molecule and its subgenomic RNA molecule comprising the second 5' UTR, the target sequence, the internal ribosome entry site (IRES), the open reading frame encoding the immunosuppressive protein, and the 3' UTR.

[0062] The method can be performed in vitro. Administration of the self-replicating RNA molecule to the organ can include, for example, introducing the self-replicating RNA molecule into the cells of the organ via electroporation, encapsulation with liposomes, etc.

[0063] The method can be performed in vivo. Administration of the self-replicating RNA molecule to the organ may include intravenous or peritoneal administration of the self-replicating RNA molecule, wherein the self-replicating RNA molecule is contained in a vector, such as a lipid or polymer vector, including, but not limited to, exosomes, virus-like particles (VLPs), etc.

[0064] The lipid carrier can be lipid nanoparticles (LNPs). LNPs can contain ionizable cationic lipids, amphiphilic phospholipids, cholesterol, and polyethylene glycol (PEG)-modified lipids, such as SM102, cholesterol, DSPC, and DMG-PEG-2000 in a molar ratio of 50:38.5:10:1.5; or 5A2-SC8, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and DMG-PEG in a molar ratio of 15:15:30:3.

[0065] In some implementations, lipid nanoparticles (LNPs) may be organ-targeting LNPs, such as spleen-targeting LNPs, lymph node-targeting LNPs, or heart-targeting LNPs.

[0066] Organ-targeting LNPs can be LNPs containing antibodies (especially scFv or nanobodies) or ligands that target the organ.

[0067] In some embodiments, the LNP may comprise permanently cationic lipids (e.g., 1,2-dioleoyl-propyl-3-trimethylammonium chloride (DOTAP), DDAB, or EPC), for example, 10-15% (e.g., 10-15 wt% or mol%) of permanently cationic lipids. In addition to permanently cationic lipids, the LNP may comprise ionizable cationic lipids, amphiphilic phospholipids, cholesterol, and PEGylated lipids, for example, comprising SM102, cholesterol, DSPC, and DMG-PEG-2000 in a molar ratio of 50:38.5:10:1.5; or 5A2-SC8, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and DMG-PEG in a molar ratio of 15:15:30:3.

[0068] In some embodiments, the LNP may comprise negatively charged lipids (e.g., 1,2-dioleoyl-sn-glycerol-3-phosphate (18PA)), for example, 10-40% (e.g., 10-40 wt% or mol%) of negatively charged lipids. In addition to negatively charged lipids, the LNP may comprise ionizable cationic lipids, amphiphilic phospholipids, cholesterol, and PEGylated lipids, for example, SM102, cholesterol, DSPC, and DMG-PEG-2000 in a molar ratio of 50:38.5:10:1.5; or 5A2-SC8, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and DMG-PEG in a molar ratio of 15:15:30:3. In some embodiments, the LNP may comprise negatively charged lipids, such as anionic lipids, such as 14PA and 18BMP. In addition to anionic lipids, LNPs may contain ionizable cationic lipids, amphiphilic phospholipids, cholesterol, and polyethylene glycol (PEG)-modified lipids, such as SM102, cholesterol, DSPC, and DMG-PEG-2000 in a molar ratio of 50:38.5:10:1.5; or 5A2-SC8, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and DMG-PEG in a molar ratio of 15:15:30:3.

[0069] Accordingly, this application also protects the use of a self-replicating RNA molecule in the preparation of a medicament for treating diseases of organs selected from lymph nodes, kidneys, lungs, spleen, and heart, wherein the self-replicating RNA molecule comprises a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a target sequence, a 3' UTR, and a poly(A) tail from the 5' end to the 3' end, wherein the RNA replicase is capable of amplifying the self-replicating RNA molecule and its subgenomic RNA molecule containing the target sequence (such as the target sequence and the 3' UTR), wherein the target sequence contains an open reading frame encoding a target peptide or protein, or a non-coding RNA sequence, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), Venezuelan equine encephalitis virus (VEEV), or Mosta Spedras virus (MDPV).

[0070] In some embodiments, this application protects the use of a self-replicating RNA molecule for treating diseases of organs selected from lymph nodes, kidneys, lungs, spleen, and heart, wherein the self-replicating RNA molecule comprises, from the 5' end to the 3' end, a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a target sequence, a 3' UTR, and a poly(A) tail, wherein the RNA replicase is capable of amplifying the self-replicating RNA molecule and its subgenomic RNA molecule containing a target sequence (such as the target sequence and the 3' UTR), wherein the target sequence contains an open reading frame encoding a target peptide or protein, or a non-coding RNA sequence, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), Venezuelan equine encephalitis virus (VEEV), or Mosta Spedras virus (MDPV).

[0071] In some embodiments, the organ is the spleen, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), or Mostas Pedras virus (MDPV).

[0072] In some embodiments, the organ is a lymph node, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), Venezuelan equine encephalitis virus (VEEV), or Mostas Pedras virus (MDPV).

[0073] In some embodiments, the organ is the heart, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Rio Negro virus (RNV) or Venezuelan equine encephalitis virus (VEEV).

[0074] In some embodiments, the organ is the spleen or a lymph node, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), Mostas Pedras virus (MDPV), or Venezuelan equine encephalitis virus (VEEV).

[0075] The target sequence can be any sequence. In some embodiments, the target sequence may contain an open reading frame encoding a target peptide or protein. The target peptide or protein may be a disease-related antigen or therapeutic agent. For example, the target peptide or protein may be a normal protein expressed in mammals, such as humans, which can be used to supplement organs lacking this normal protein. In some embodiments, the target sequence may contain a non-coding RNA sequence. For example, the non-coding RNA may be, for instance, miRNA, which can be used to knock down the expression of certain genes in an organ. Alternatively, the non-coding RNA may be a guide RNA used for gene editing, such as CRISPR.

[0076] Self-replicating RNA molecules may also include an internal ribosome entry site (IRES) on the 3' side of the target sequence. The IRES can be any suitable IRES, such as an IRES derived from Coxsackie B3 virus (CVB3). An IRES derived from Coxsackie B3 virus (CVB3) may contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:15.

[0077] Self-replicating RNA molecules may also contain an open reading frame encoding an immunosuppressive protein on the 3' side of the target sequence, such as the 3' side of the internal ribosome entry site (IRES). The immunosuppressive protein may be an interferon-inhibiting protein (IIP), such as the vaccinia virus E3L protein. E3L may contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:16.

[0078] Self-replicating RNA molecules can also contain a second 5'UTR on the 5' side of the target sequence, such as between the promoter and the target sequence.

[0079] In some embodiments, the self-replicating RNA molecule may include a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a second 5' UTR, a target sequence, a 3' UTR, and a poly(A) tail from the 5' end to the 3' end, wherein the RNA replicase is capable of amplifying the self-replicating RNA molecule and its subgenomic RNA molecules containing the second 5' UTR, the target sequence, and the 3' UTR.

[0080] In some embodiments, the self-replicating RNA may include a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a second 5' UTR, a target sequence, an internal ribosome entry site (IRES), an open reading frame encoding an immunosuppressive protein, a 3' UTR, and a poly(A) tail from the 5' end to the 3' end, wherein the RNA replicase is capable of amplifying the self-replicating RNA molecule and its subgenomic RNA molecule comprising the second 5' UTR, the target sequence, the internal ribosome entry site (IRES), the open reading frame encoding the immunosuppressive protein, and the 3' UTR.

[0081] In this application, the same nucleotide sequence, such as the nucleotide sequence represented by the same SEQ ID NO, can represent both a DNA sequence and an RNA sequence, the only difference being the substitution of T and U.

[0082] This application reveals the potential contribution of self-replicating RNA (SRNA) to achieving drug targeting. Specifically, it discovers that SRNA not only exhibits efficient and sustained expression in target cells but also demonstrates unique specific targeting capabilities, with different viral types of SRNA specifically expressed in different tissues / organs. This may be due to the interaction between the self-amplification properties or intrinsic sequence characteristics of SRNA and specific intracellular signaling pathways, leading to differences in replication capacity in different tissues and organs, thus achieving extrahepatic targeted expression of SRNA. This discovery opens new avenues for mRNA drug development, providing a novel perspective for optimizing drug design. In the future, it can be combined with targeted delivery vector technologies to achieve more precise and efficient targeted therapeutic effects. This innovation will drive the further development of mRNA drug technology, expanding its potential and utility in various clinical applications.

[0083] Specifically, this invention leverages the differences in replication capacity of self-replicating RNA from different alphavirus types in various tissues and organs, combined with efficient in vivo delivery of LNPs, to develop a self-replicating RNA-LNP delivery technology that can efficiently express target proteins in the spleen or lymph nodes, achieving over 70% high-efficiency expression of the target protein in the spleen. This technology, by enhancing spleen- or lymph node-specific expression, can significantly reduce non-targeted expression in other tissues (such as the liver), thus reducing toxicity and adverse effects. The spleen and lymph nodes play a central role in immune regulation, and this technology can be further applied to vaccine development, immunotherapy, and related gene therapy fields. This technology has significant application prospects in gene therapy, especially in scenarios requiring targeting of immune organs or reduction of systemic toxicity, and may become a new breakthrough tool. Attached Figure Description

[0084] The following detailed description is provided by way of example, but it is not intended to limit this application to the specific embodiments described. A better understanding can be obtained by referring to the accompanying drawings.

[0085] Figure 1 is a schematic diagram of the structure of a self-replicating RNA molecule constructed based on a self-replicating virus.

[0086] Figures 2A-2E show capillary electrophoresis diagrams of self-replicating RNA and mRNA (2E) constructed based on Venezuelan equine encephalitis virus (VEEV, 2A), Mosta Spedras virus (MDPV, 2B), Rio Negro virus (RNV, 2C), and Everglades virus (EVEV, 2D).

[0087] Figure 3 shows the expression of FLuc saRNA-LNP and FLuc mRNA-LNP in HEK293T cells and A549 cells.

[0088] Figure 4 shows the total Fluc expression level in mice 24 hours after tail vein injection of each FLuc saRNA-LNP and FLuc mRNA-LNP.

[0089] Figures 5A and 5B show the distribution of each FLuc saRNA-LNP and FLuc mRNA-LNP in various organs of mice 24 hours after injection (5A), and the percentage of LNP distribution in each organ relative to the total LNP distribution in the tested organs (5B). The bars in Figure 5B show the percentages in the heart, liver, spleen, lung, kidney, brain, and lymph nodes from bottom to top.

[0090] Figures 6A and 6B show the FLuc expression levels of each FLuc saRNA-LNP and FLuc mRNA-LNP in various organs of mice 24 hours after injection (6A), and the percentage of FLuc expression in each organ relative to the total FLuc expression in the tested organs (6B). The bars in Figure 6B, from bottom to top, represent the percentages in the heart, liver, spleen, lung, kidney, brain, and lymph nodes. Detailed Implementation

[0091] Unless otherwise stated, the technical and scientific terms used in this invention have the meanings commonly understood by a person skilled in the art to which this invention pertains.

[0092] Unless otherwise specified, the terms used herein have their common meanings as found in dictionaries, textbooks, and technical reference books, or as commonly understood by those skilled in the art. The following descriptions of some terms are for the purpose of understanding this application only and are not intended to impose any particular limitations on these terms, unless otherwise specified.

[0093] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural form of the object referred to, unless the context clearly specifies otherwise.

[0094] The term "or" refers to a single element among the listed selectable elements, unless the context explicitly indicates otherwise.

[0095] The terms "comprising" or "including" mean that the stated elements, integers, or steps are included, but do not exclude the inclusion of any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps. The terms "consisting of" or "comprises of" generally mean that only the stated elements, integers, or steps are included, without the addition of other elements, integers, or steps.

[0096] The 5' end of a nucleic acid molecule can be a terminal with a free phosphate group, and the 3' end can be a terminal with a free hydroxyl group. The 5' side refers to the position relatively closer to the 5' end in the nucleic acid sequence, while the 3' side refers to the position relatively closer to the 3' end in the nucleic acid sequence.

[0097] In this article, "self-replicating RNA" or "saRNA" refers to an RNA molecule that can be amplified by its own encoded RNA replicase. Specifically, "self-replicating RNA" has a modified genome of a self-replicating virus, which can use itself as a template to amplify a complementary strand according to the base "complementarity" principle. Using this complementary strand as a template, it can amplify a full-length copy of the RNA itself, as well as multiple non-full-length copies (i.e., subgenomic fragments). The RNA molecule itself and its full-length copy can enter a new amplification cycle to amplify more full-length and non-full-length copies.

[0098] "Subgenomic RNA" or "subgenomic transcript" refers to an RNA molecule transcribed from an RNA molecule containing a subgenomic promoter that controls transcription of subgenomic transcripts. Subgenomic transcripts can be obtained in the presence of RNA-dependent RNA polymerases, particularly functional alphavirus non-structural proteins. For example, the term "subgenomic transcript" can refer to RNA transcripts prepared in alphavirus-infected cells using the complementary strand of alphavirus genomic RNA as a template. Subgenomic transcripts can also be obtained using the (-) complementary strand of a transcript containing a subgenomic promoter as a template. Therefore, "subgenomic transcript" refers to RNA molecules obtained by transcribing fragments of alphavirus genomic RNA, as well as RNA molecules obtained by transcribing fragments of replicons.

[0099] In this article, a "promoter" refers to a sequence that controls transcriptomic synthesis by providing recognition and binding sites for RNA polymerase. Promoter regions may also include recognition or binding sites for other factors involved in transcriptional regulation. Promoters can be inducible, initiating transcription in response to an induction signal, or constitutive. Inducible promoters, in the absence of an induction signal, elicit very little or no transcription. Promoters in this article can be subgenomic promoters, such as the subgenomic promoters of alphaviruses. Other specific promoters can be genomic (+) or (-) strand promoters, such as the genomic (+) or (-) strand promoters of alphaviruses.

[0100] A "subgenomic promoter" is a nucleic acid sequence located on the 5' side of the target sequence (e.g., an open reading frame encoding a target peptide or protein) in the RNA molecule of this article. It controls the transcription of this target sequence by providing recognition and binding sites to RNA polymerases (usually RNA-dependent RNA polymerases, particularly functional alphavirus non-structural proteins). Subgenomic promoters may also contain recognition or binding sites for other factors. Subgenomic promoters are typically genetic elements of positive-strand RNA viruses. An alphavirus subgenomic promoter is a nucleic acid sequence contained within the viral genomic RNA. A subgenomic promoter is characterized by enabling transcription, i.e., RNA synthesis, to be initiated in the presence of RNA-dependent RNA polymerases (e.g., functional non-structural proteins). The RNA (-) strand, the complementary strand of the alphavirus genomic RNA, serves as a template for the synthesis of (+) strand subgenomic transcripts, which typically begin at or around the subgenomic promoter.

[0101] In this article, "replication" or "amplification" refers to the process of synthesizing RNA molecules based on the nucleotide sequence of a specified RNA molecule. The synthesized RNA molecule may be identical to or complementary to the template RNA molecule. RNA replication may synthesize DNA intermediates. Alphavirus RNA replication does not involve DNA intermediates; instead, it is mediated by RNA-dependent RNA polymerase, using the first RNA strand or a portion thereof as a template to synthesize the second RNA strand, and the second RNA strand or a portion thereof can serve as a template to synthesize the third RNA strand.

[0102] In this article, "RNA replicase" refers to RNA-dependent RNA polymerase, an enzyme that catalyzes the synthesis of RNA using RNA as a template. Under the catalysis of alphavirus RNA-dependent RNA polymerase, the (-) complementary strand and (+) genomic RNA strand are synthesized sequentially, inducing RNA replication. In nature, RNA-dependent RNA polymerases are typically encoded by all RNA viruses except retroviruses, such as alphaviruses. Specifically, in this application, "RNA replicase" may refer to the non-structural protein of self-replicating viruses, such as alphaviruses.

[0103] "Self-replicating viruses" or "self-replicating viruses" include RNA viruses capable of autonomously replicating within host cells. Self-replicating viruses can have a single-stranded RNA genome and include alphaviruses, flaviviruses, measles viruses, and rhabdoviruses. Alphaviruses and flaviviruses have a sense-sense genome, while measles viruses and rhabdoviruses have an antisense ssRNA. Generally, self-replicating viruses are viruses with a (+)-strand RNA genome that can be directly translated after infecting a cell. This translation provides RNA-dependent RNA polymerase, which subsequently produces sense and antisense transcripts. A "transcriptum" generally refers to a gene transcription product, or a transcription unit, which is a nucleotide molecule complementary to the template strand. The (+) or sense strand can be the strand that contains or encodes genetic information.

[0104] The term "alphavirus" should be interpreted broadly to include any viral particle that possesses the characteristics of an alphavirus. The characteristics of an alphavirus include the presence of a (+) strand of RNA encoding genetic information suitable for replication within a host cell, including RNA polymerase activity. The term includes alphaviruses found in nature, and any variants or derivatives thereof.

[0105] "Non-structural protein" refers to a protein encoded by a virus that does not form part of a viral particle. This term typically includes various viral enzymes and transcription factors used to replicate themselves, such as RNA-dependent RNA polymerases. "Anivirus non-structural protein" refers to individual non-structural proteins of anavirus origin, such as nsP1, nsP2, nsP3, and nsP4, or their polyproteins. In some embodiments, "anavirus non-structural protein" refers to nsP123 and / or nsP4. In other embodiments, "anavirus non-structural protein" refers to nsP1234. A "functional variant" of a non-structural protein refers to a variant that has been mutated compared to the native non-structural protein but still retains all, most, or the desired functions of the non-structural protein.

[0106] An "open reading frame" or "ORF" is a sequence of consecutive bases that begins with a start codon and ends with a stop codon, encoding a complete polypeptide chain. In an mRNA sequence, every three consecutive bases (i.e., a triplet "codon") encode a corresponding amino acid. There is one start codon (AUG) and three stop codons (UAA, UAG, and UGA). The ribosomes begin translation from the start codon, synthesizing and elongating the polypeptide chain along the mRNA sequence. The elongation process terminates when a stop codon is encountered.

[0107] "UTR" or "untranslated region" refers to the sequences located at both ends of a nucleic acid that are not translated. Specifically, the UTR located at the 5' end of the nucleic acid is called the 5'UTR, which usually starts from the 5' cap and extends to the start codon AUG, while the 3'UTR usually extends from the stop codon at the end of the coding region to the poly(A) tail. The nucleotide sequences of the 5' and 3' UTRs of the viral genome are highly conserved, usually forming stem-loop or hairpin structures, and contain cis-acting elements, which are mainly responsible for regulating the translation of viral proteins and the replication of the viral genome.

[0108] The "5' cap," also known as the 7-methylguanylic acid cap (m7G), typically plays a role in recognizing RNA as it enters and exits the cell nucleus. During translation, it helps ribosomes recognize and bind to mRNA.

[0109] A "poly(A) tail" is a sequence composed of multiple adenosine nucleotides that helps prevent enzymatic degradation in the cytoplasm and facilitates transcription termination, as well as the export of mRNA from the nucleus and translation. A poly(A) tail can refer to a continuous poly(A) tail or a segmented poly(A) tail. A continuous poly(A) tail can contain consecutive adenosine nucleotides. A segmented poly(A) tail can contain 2-5 consecutive adenosine nucleotide segments separated by a spacer sequence, where the spacer sequence contains 1-20 nucleotides, and each consecutive adenosine nucleotide segment contains 10-100 consecutive adenosine nucleotides. The spacer sequence is terminated at both ends by non-A bases and can be an A base or a non-A base in the middle.

[0110] Internal ribosome entry sites (IRES) are RNA sequences that form secondary structures to attract transcription initiation complex precursors to translation start codons such as AUG. IRES are typically located in the 5' UTR of RNA viruses, but can also appear at other locations on the mRNA. Bicistrivividae viruses have two open reading frames (ORFs) in their mRNA, and translation of each ORF is guided by two distinct IRES. Some mammalian intracellular mRNAs also possess IRES, possibly located in mRNAs encoding genes involved in stress responses or other survival-critical genes. IRES are also present in small RNA viruses and some pathogenic viruses, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hand-foot-and-mouth disease virus (HFMDV). Although these viral IRES contain different sequences, many share similar secondary structures and initiate translation through similar elements.

[0111] Immunosuppressive proteins are proteins that can inhibit or limit the immune response produced by cells or the body, such as interferon-inhibiting proteins (IIPs). Immunosuppressive proteins or interferon-inhibiting proteins can reduce the immunogenicity of self-replicating RNA by decreasing the immune response of cells or the body, such as the production of interferon.

[0112] "Target sequence" or "GOI" generally refers to the sequence that is intended to be amplified and expressed by self-replicating RNA, including coding sequences for proteins or peptides, or non-coding RNA sequences. In this article, "expression" refers to the process of synthesizing functional gene products from genetic information (i.e., the target sequence or GOI in this text). Gene expression products are usually proteins, but also include functional RNAs such as miRNAs.

[0113] The term "identity" or "sequence identity" as used herein refers to the percentage of nucleotides / amino acids in a sequence that are identical to those in a reference sequence after sequence alignment. If necessary, spaces are introduced in the sequence alignment to achieve the maximum percentage of sequence similarity between the two sequences. Those skilled in the art can use various methods, such as computer software, to perform pairwise or multiple sequence alignments to determine the percentage of sequence similarity between two or more nucleic acid or amino acid sequences. Such computer software includes, for example, ClustalOmega, T-coffee, Kalign, and MAFFT.

[0114] In this article, "spleen-related diseases" refers to diseases of the spleen itself or diseases related to the spleen, such as spleen-related infectious diseases, including spleen abscess and spleen tuberculosis; spleen-related hematologic diseases, including infectious mononucleosis, subacute infective endocarditis and other infectious diseases, systemic lupus erythematosus, leukemia, malignant lymphoma, etc.; and spleen-related tumors, including splenic lymphoma and splenic metastatic tumors, etc.

[0115] In this article, "lymph node-related diseases" refers to diseases of the lymph nodes themselves or diseases related to the lymph nodes, such as lymph node-related inflammatory diseases, including acute lymphadenitis and chronic lymphadenitis; lymph node diseases caused by specific infections, including lymph node tuberculosis; and lymph node-related tumors, including lymphoma and metastatic lymph node tumors.

[0116] Alphaviruses are packaged, positive-sense RNA viruses that replicate in the cytoplasm of infected cells. Many alphaviruses have genomes ranging from 11,000 to 12,000 nt in length, and their genomic RNA typically has a 5' cap and a 3' poly(A) tail. The alphavirus genome encodes both non-structural and structural proteins. Non-structural proteins are involved in viral RNA transcription, modification, replication, and protein modification, while structural proteins are used to form viral particles. The genome typically contains two open reading frames (ORFs). Four non-structural proteins (nsP1-nsP4) are usually encoded by the first ORF located near the 5' end of the genome, while the structural proteins are encoded by the second ORF. Generally, the first ORF is larger than the second. Utilizing the self-replicating nature of alphaviruses, self-replicating RNA molecules carrying exogenous target genes have been constructed. This involves modifying the genome of a positive-sense single-stranded virus, replacing the viral structural protein sequences with exogenous target genes. This self-replicating RNA allows for the continuous and substantial amplification of in vitro synthesized RNA within cells, achieving persistent expression of the exogenous target protein. Traditionally, RNA vaccines require 30-100 micrograms of RNA per injection, with two injections spaced several weeks apart. Using this self-replicating RNA, the injection dose can be significantly reduced to just a few micrograms.

[0117] Different types of alphaviruses in nature exhibit different host preferences and organ-specific infection preferences. For example, New World (NW) alphaviruses are more neurotropic, while Old World (OW) alphaviruses typically cause arthritis-like pathologies. The inventors of this application, through a series of hypotheses and verifications, discovered that saRNAs constructed based on different alphaviruses exhibit amplification and expression preferences for different organs, meaning they demonstrate higher amplification and expression efficiency in certain organs.

[0118] Specifically, the inventors of this application constructed self-replicating RNAs (saRNAs) expressing firefly luciferase (FLuc) based on various alphaviruses. These saRNAs and FLuc-expressing mRNAs were encapsulated in fluorescently labeled LNPs and injected into mice via the tail vein. Twenty-four hours after injection, the distribution of LNPs and RNA in seven organs of the mice was tracked. The results showed that the delivery patterns of each saRNA-LNP and mRNA-LNP were consistent, with over 80% of RNA-LNPs concentrated in the liver, and a small amount distributed in lymph nodes, brain, lungs, spleen, and heart. Surprisingly, only the FLuc expression distribution of mRNA was consistent with the LNP distribution; the FLuc expression of each saRNA was mainly distributed outside the liver in the spleen, lymph nodes, heart, and kidneys, with very low FLuc expression in the liver. For example, as shown in Figure 6, the FLuc expression of VEEV-saRNA was higher in the spleen, heart, and kidney, the expression of MDPV-saRNA was mainly in the lymph nodes and spleen, the FLuc expression of RNV-saRNA in the spleen accounted for about half of the total expression in the tested organs, and the FLuc expression of EVEV-saRNA in the spleen unexpectedly accounted for 72.68% of the total expression in the tested organs.

[0119] Previous researchers encapsulated Venezuelan equine encephalitis virus (VEEV)-saRNA expressing luciferase in lipid nanoparticles (LNPs) and administered them to mice intramuscularly, intradermally, intraperitoneally, intranasally, intravenously, or subcutaneously, and then imaged the luciferase luminescence in the mice. The results showed that the in vivo distribution of saRNA changed depending on the route of administration (Bathula NV, et al., (2024) ibid.). This study suggests that the route of administration significantly affects LNP delivery and thus the distribution of saRNA expression, particularly the significant difference in saRNA distribution in the kidneys caused by peritoneal and intravenous administration.

[0120] In the embodiments of this application, it is clearly shown that the delivery and distribution of LNPs in vivo are not the key factors affecting the distribution of saRNAs; rather, the organ expression preference of each saRNA is. For example, as shown in Figure 5, the distribution of VEEV-LNPs in the spleen and heart accounted for 7.75% and 0.96% of the total tested organs, respectively, while in Figure 6, the FLuc expression levels of VEEV saRNAs in the spleen and heart accounted for 29.24% and 22.45% of the total tested organ expression, respectively, indicating that VEEV saRNAs may prefer to amplify and be expressed in the heart.

[0121] Organ expression preferences of saRNAs constructed from different alphaviruses can be utilized to select suitable saRNAs for efficient amplification and expression of target sequences in specific organs, especially in in vitro expression scenarios. In in vivo applications, these saRNAs can be combined with organ-targeting vectors such as LNPs to further reduce impact on other organs. Because EVEV saRNA exhibits a particularly strong preference for the spleen, low-dose naked sequence intravenous injection can be attempted to minimize amplification and expression in other organs without affecting the target sequence amplification and expression in the spleen. Similarly, because MDPV saRNA or RNV saRNA exhibits a particularly strong preference for lymph nodes, low-dose naked sequence intravenous injection can be attempted to minimize amplification and expression in other organs without affecting the target sequence amplification and expression in lymph nodes.

[0122] Therefore, this application primarily protects a method for expressing a target peptide or protein or non-coding RNA in organs selected from lymph nodes, kidneys, lungs, spleens, and hearts, comprising administering a self-replicating RNA molecule to the organ, wherein the self-replicating RNA molecule comprises, from the 5' end to the 3' end, a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a target sequence, a 3' UTR, and a poly(A) tail, wherein the RNA replicase is capable of amplifying the self-replicating RNA molecule and its subgenomic RNA molecule containing the target sequence and the 3' UTR, wherein the target sequence contains an open reading frame encoding a target peptide or protein, or a non-coding RNA sequence, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), Venezuelan equine encephalitis virus (VEEV), or Mosta Spedras virus (MDPV).

[0123] Specifically, when the organ is the spleen, the RNA replicase can be a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rionegro virus (RNV), or Mosta Spedras virus (MDPV); when the organ is a lymph node, the RNA replicase can be a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rionegro virus (RNV), Venezuelan equine encephalitis virus (VEEV), or Mosta Spedras virus (MDPV); when the organ is the heart, the RNA replicase can be a non-structural protein or a functional variant thereof derived from Rionegro virus (RNV) or Venezuelan equine encephalitis virus (VEEV); and when the organ is the spleen or a lymph node, the RNA replicase can be a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rionegro virus (RNV), Venezuelan equine encephalitis virus (VEEV), or Mosta Spedras virus (MDPV).

[0124] Self-replicating RNA molecules may also include an internal ribosome entry site (IRES) on the 3' side of the target sequence. The IRES can be any suitable IRES. Additionally, self-replicating RNA molecules may also include an open reading frame encoding an immunosuppressive protein on the 3' side of the target sequence, such as the 3' side of the internal ribosome entry site (IRES). The immunosuppressive protein can be an interferon-inhibiting protein (IIP), such as the poxvirus E3L protein.

[0125] The replication of self-replicating RNA (dsRNA) begins with the RNA polymerase complex synthesizing a complementary negative-strand RNA intermediate from the positive-strand RNA. This dsRNA amplification intermediate is recognized by the cell's innate immune-related signaling pathways, inducing a strong innate immune response. This includes type I interferon responses mediated by TLR3, 7, and 8 endosome sensing and by MDA5, RIG-I, PKR, and OSA cytoplasmic sensing. This is the main reason why self-replicating RNA possesses strong immunogenicity. While this may be beneficial in the recruitment and activation of antigen-presenting cells and adaptive immune system cells, interferon activation can lead to translational repression and degradation of intracellular mRNA, including genomic and subgenomic RNAs of self-replicating RNA. Therefore, controlling the immunogenicity of self-replicating RNA to promote the recruitment and activation of downstream immune responses while minimizing its negative impact on antigen expression remains a pressing issue in this field. In 2017, Ugur Sahin, to alleviate the inhibitory effect of saRNA translation, first co-delivered a combination of non-replicating mRNAs encoding vaccinia virus immune escape proteins E3 / K3 / B18 with saRNA encoding luciferase. This method significantly inhibited the intracellular PKR and IFN pathways, greatly enhancing the translation efficiency of saRNA-encoded luciferase in mice. In 2021, Robin J. Shattock et al. screened for IIPs that could effectively enhance the expression and immunogenicity of saRNA target proteins by cis-expressing innate immunosuppressive proteins (IIPs) via 2A peptide. The parainfluenza virus PIV5 and MERS ORF4a proteins generated through cis-action increased the expression levels of exogenous gene proteins at the cellular level and in mice, and reduced the immunogenicity of saRNA encoding rabies virus G glycoprotein in rabbits. By inserting exogenous target gene and immunosuppressive protein coding sequences into subgenomic open reading frames, and adding IRES between them, the immunogenicity induced by self-replicating RNA can be reduced, thus decreasing the cellular innate immunity it induces and minimizing cytotoxicity. Furthermore, adding IRES between the coding sequences of the exogenous gene and the immunosuppressive protein, compared to the 2A peptide used in other studies, does not leave extra amino acids on the exogenous gene protein and does not result in uncut fusion proteins, thus offering greater safety.

[0126] Self-replicating RNA molecules can also include a second 5'UTR between the promoter and the target sequence to, for example, enhance the amplification and translation of subgenomic RNA molecules.

[0127] When used in vitro, self-replicating RNA molecules can be introduced into the cells of the organ via methods such as electroporation or encapsulation with liposomes.

[0128] When used in vivo, self-replicating RNA can be encapsulated in a vector, especially a nanocarrier such as a polymer nanocarrier or a lipid nanocarrier, and administered intravenously or via the peritoneum.

[0129] Liposomes, especially lipid nanoparticles (LNPs), are currently the most widely used carriers in clinical practice. They have high delivery efficiency, high stability, and low immunogenicity, and their in vivo delivery effect is superior to that of polymer carriers.

[0130] LNPs are generally composed of four lipid components: ionizable lipids, amphiphilic phospholipids, cholesterol, and polyethylene glycol (PEG)-modified lipids. Ionizable lipids can carry a positive charge at low pH, promoting interactions with negatively charged nucleic acid molecules such as saRNA. Under physiological conditions such as in blood, their charge becomes neutral. When engulfed in endosomes, the acidic conditions cause them to become positively charged, thereby disrupting the endosome membrane and releasing nucleic acid molecules such as saRNA into the cytoplasm. Amphiphilic lipids are related to the cohesiveness of LNPs (i.e., the ability of LNPs to promote membrane fusion after protonation in an acidic environment). Amphiphilic lipids and cholesterol are also responsible for maintaining the stability of LNPs. In particular, cholesterol can fill the gaps between other lipid components, reducing the leakage of the loaded nucleic acid molecules. PEG-modified lipids are responsible for the colloidal stability of LNPs, prolonging their blood half-life.

[0131] Although some of the saRNAs in this application exhibit strong organ preference, they may still affect other non-target organs during in vivo application. One strategy is to reduce the dosage of RNA-LNPs, allowing them to reach the target organ at a lower level, followed by efficient amplification and expression of the saRNAs, while non-target organs remain largely unaffected. Another strategy is to use organ-targeting LNPs. For example, LNPs may contain antibodies or ligands targeting the target organ, enabling them to reach the target organ and bind to antigens or receptors thereon. Alternatively, LNPs can be made organ-biased by adding additional components to the standard four components of the LNP, such as permanently cationic lipids (e.g., 1,2-dioleoyl-propyl-3-trimethylammonium chloride (DOTAP), DDAB, or EPC), negatively charged lipids (e.g., 1,2-dioleoyl-sn-propanetriol-3-phosphate (18PA), anionic lipids (e.g., 14PA and 18BMP)). For example, LNPs can target the spleen when 10-15% (e.g., 10-15 wt% or mol%) of permanently cationic lipids, 10-40% (e.g., 10-40 wt% or mol%) of negatively charged lipids, or anionic lipids such as 14PA and 18BMP are added to them. LNPs can also target the lungs and liver when the percentage of these additional components is adjusted. See Saber N, et al., (2024) Lipid Nanoparticles for Nucleic Acid Delivery Beyond the Liver. Hum Gene Ther. 35(17-18):617-627; Cheng Q, et al., (2020) Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat Nanotechnol. 15(4):313-320.

[0132] The preparation and purification of the saRNA of this application are well known to those skilled in the art, and the operations in the following examples can also be referred to. For example, i) a DNA molecule containing a promoter and a sequence encoding the saRNA molecule of this application from the 5' end to the 3' end can be provided; ii) optionally, the DNA molecule can be made linear; and iii) in vitro transcription can be performed under suitable conditions. Suitable conditions in step iii) include providing RNA polymerase, ATP, UTP, CTP, GTP, cap analogs, etc.

[0133] The technical solution of the present invention will be further described in detail below through embodiments and in conjunction with the accompanying drawings. Unless otherwise stated, the methods and materials of the embodiments described below are all conventional products that can be purchased from the market. Those skilled in the art will understand that the methods and materials described below are merely exemplary and should not be considered as limiting the scope of the present invention.

[0134] Example 1. Preparation of self-replicating RNA and mRNA for expressing FLuc

[0135] Prepare self-replicating RNAs (FLuc saRNAs) expressing firefly luciferase (FLuc) based on Venezuelan equine encephalitis virus (VEEV), Mosta Spedras virus (MDPV), Rio Negro virus (RNV), and Everglades virus (EVEV) from the alphavirus family.

[0136] Specifically, in vitro transcription template DNA for these self-replicating RNAs was designed and synthesized, with the structure shown in Figure 1. From the 5' end to the 3' end, it contains the T7 mini promoter sequence (SEQ ID NO:1), the viral 5' UTR sequence (SEQ ID NO:2, 3, 4 or 5, corresponding to each alphavirus), the viral replicon NSP1-4 sequence (SEQ ID NO:6, 7, 8 or 9, corresponding to each alphavirus), the viral subgene promoter sequence (SEQ ID NO:10, 11, 12 or 13, corresponding to each alphavirus), the second 5' UTR sequence (SEQ ID NO:25), the FLuc coding sequence (SEQ ID NO:14), the CVB3 IRES sequence (SEQ ID NO:15), the E3L sequence (SEQ ID NO:16), the viral 3' UTR sequence (SEQ ID NO:17, 18, 19 or 20, corresponding to each alphavirus), the PolyA sequence (SEQ ID NO:21), and the BspQI restriction enzyme site sequence (GGAAGAGCC) for plasmid linearization. The synthesized double-stranded DNA fragment was cloned into the pUC57-mini-Kana-BsmBI terminator-T7 deletion vector (GenScript).

[0137] Simultaneously, an in vitro transcription template DNA for expressing the ordinary mRNA of FLuc (FLuc mRNA) was designed and synthesized, which contains the T7 promoter sequence (SEQ ID NO:22), HBA5'UTR sequence (SEQ ID NO:23), FLuc coding sequence (SEQ ID NO:14), HBA3'UTR sequence (SEQ ID NO:24), PolyA sequence (SEQ ID NO:21), and BspQI restriction enzyme site sequence (GGAAGAGCC) from the 5' to 3' ends. The synthesized double-stranded DNA fragment was constructed into the pUC57-mini-Kana-BsmBI terminator-less-T7 deletion vector (GenScript).

[0138] The obtained pUC57-FLuc saRNA and pUC57-FLuc mRNA plasmids were transformed into competent cells, seeded on Kana-resistant plates for screening, and single clones were picked. Clones with correct sequences were screened by Sanger sequencing. All the above experiments were performed by Nanjing Genscript Biotech.

[0139] The prepared plasmid was linearized by single digestion with BspQI. The linearized plasmid was recovered by two alcohol precipitation methods, and its concentration was determined by Nanodrop to determine the amount of template to be used subsequently. The linearization effect was identified by 1% agarose gel electrophoresis. The results showed that the linearized product had a single band with no obvious impurities.

[0140] Subsequently, FLuc saRNA (modified with 5-Me-CTP(m5C)) and FLuc mRNA (modified with N1-Me-ψ(m1ψ)) were prepared by in vitro transcription (IVT). Specifically, the transcription system was prepared according to Table 1 and incubated at 37°C for 3 h. Afterward, 10 μl of DNase I was added, mixed well, and incubated at 37°C for 30 min to obtain the IVT stock solution.

[0141] Table 1. In vitro transcription system

[0142] Add 110 μl of enzyme-free water and 100 μl of 8M LiCl solution to the IVT stock solution to make the LiCl concentration 2.5M. Mix well and incubate at -20℃ for at least 30 min. Centrifuge at 12000g for 15 min at 4℃ and discard the supernatant. Add 1 ml of 75% ethanol, mix by inversion, centrifuge at 12000g for 5 min at 4℃ and discard the supernatant. Repeat once more. Centrifuge at 12000g for 2 min at 4℃, aspirate the supernatant, add 100 μl of enzyme-free water to dissolve the RNA, and detect the RNA concentration on a Nanodrop instrument. The results showed that the above linear plasmids could all be transcribed into RNA products, including RNA products containing m5C modification and RNA products containing m1ψ modification.

[0143] Subsequently, each RNA product was subjected to capillary electrophoresis using the Agilent 5200 fragment analyzer system. Specifically, RNA length and integrity were detected using an RNA analysis kit (Agilent, DNF-472-1000) according to the Agilent 5200 fragment analyzer and RNA kit instructions.

[0144] The results are shown in Figures 2A-2E. The capillary electrophoresis results show that the prepared FLuc saRNA and FLuc mRNA are of the correct length and have high integrity (both above 80%).

[0145] Example 2. Preparation of FLuc saRNA-LNP and FLuc mRNA-LNP containing DiR label

[0146] Using a microfluidic nanomedicine preparation system (Microflow M), lipid nanoparticles (LNPs) were used to encapsulate FLuc saRNA and FLuc mRNA prepared in Example 1, respectively. Through a microfluidic device, lipid components (SM102, cholesterol, DSPC, and DMG-PEG-2000, molar ratio: 50:38.5:10:1.5), DiR dye (Thermo, D12731), and saRNA or mRNA self-assembled to form saRNA-LNP or mRNA-LNP complexes. The DiR dye, used for fluorescent labeling of LNPs, comprised 0.5 mol% of the lipid components, and the phosphorus-nitrogen ratio of the lipid components to RNA was 4-6. After buffer replacement and concentration, the final LNP product was obtained. Subsequent observation of the DiR fluorescence signal and the FLuc luminescence signal allowed for determination of the distribution of LNPs and the distribution of mRNA translational expression.

[0147] After LNP preparation, the particle size distribution and zeta potential of the RNA-LNP complex were characterized using a particle size analyzer (Malvern Zetasizer Pro) based on the principle of dynamic light scattering. Simultaneously, the Quant-iT RiboGreen RNA kit (Invitrogen) was used. TM The encapsulation efficiency and concentration of encapsulated saRNA or mRNA in LNP products were determined using Ribogreen (R11490). Encapsulation efficiency refers to the percentage of RNA encapsulated in the lipid bilayer relative to the total amount of RNA input. Ribogreen is a highly sensitive fluorescent nucleic acid dye that cannot penetrate LNPs. Therefore, the amount of RNA free outside the LNP particles in the RNA-LNP complex solution can be detected first. Then, Triton X-100 is used to disrupt the LNP structure, releasing the RNA encapsulated within the RNA-LNP complex into the external solution, thus detecting the total RNA content. The encapsulation efficiency can be calculated based on the difference between the two measurements.

[0148] The results are shown in Table 2. The polydispersity index (PDI) of both FLuc saRNA-LNP and FLuc mRNA-LNP was less than 0.2, indicating good dispersibility and uniformity of the LNP particles. The measured Zeta potential values ​​were all within ±15 mV, indicating that the LNP products have good electroneutrality in a neutral buffer environment. In addition, the encapsulation efficiency of each RNA-LNP was similar, all above 92%.

[0149] Because of its smaller volume after concentration, VEEV-LNP encapsulates RNA at a much higher concentration than other saRNAs.

[0150] Table 2. Characterization of FLuc saRNA-LNP and FLuc mRNA-LNP

[0151] Example 3. In vitro cell expression of FLuc saRNA-LNP and Fluc mRNA-LNP

[0152] The in vitro expression activities of FLuc saRNA-LNP and FLuc mRNA-LNP prepared in Example 2 were tested using the human embryonic kidney epithelial cell line HEK293T and the human non-small cell lung cancer cell line A549.

[0153] Specifically, HEK293T cells or A549 cells in the logarithmic growth phase were seeded into 96-well plates, 100 μl (2 × 10⁶ cells / well) per well. 5Cells were cultured overnight until cell confluence reached approximately 60-70%. 10 ng FLuc saRNA-LNP (VEEV-LNP, MDPV-LNP, RNV-LNP, or EVEV-LNP) or 100 ng FLuc mRNA-LNP was added to each well, and the cells were cultured for another 24 h. Afterward, the supernatant was removed, the cells were collected, and analyzed using Fire-LumiNova. TM The luciferase assay kit (GenScript, L00877C) was used to detect the activity of firefly luciferase Fluc in cells to assess the protein expression levels of various saRNAs and mRNAs.

[0154] As shown in Figure 3, different FLuc saRNA-LNPs and FLuc mRNA-LNPs can effectively express firefly luciferase in both HEK293T and A549 cells. However, the expression levels of different self-replicating RNAs differ significantly between these two cell types, which is speculated to be due to cell-specific preferences of different alphavirus RNA replicases.

[0155] Example 4. In vivo expression of FLuc saRNA-LNP and FLuc mRNA-LNP in mice

[0156] Furthermore, the FLuc saRNA-LNP and FLuc mRNA-LNP prepared in Example 2 were injected into mice, and the distribution of DiR signal and FLuc expression was detected using a small animal in vivo imaging system to evaluate the organ-targeted delivery and organ expression preference of different FLuc saRNA-LNPs and FLuc mRNA-LNPs in vivo. All animal experiments were conducted by Cyagen (Suzhou) Biotechnology Co., Ltd.

[0157] Specifically, male C57BL / 6J mice aged 6-8 weeks were selected and acclimatized for 5 days before the experiment. During the experiment, mice were randomly divided into groups of 3. Each group received an FLuc saRNA-LNP or FLuc mRNA-LNP via tail vein injection at a dose of 0.25 mg / kg. Twenty-four hours after RNA-LNP injection, 100 μl of luciferin (30 mg / mL) was injected intraperitoneally. FLuc imaging analysis was performed on the mice 15 minutes later using a small animal in vivo imaging system.

[0158] As shown in Figure 4, 24 hours after RNA-LNP injection, both FLuc saRNA-LNP and FLuc mRNA-LNP effectively expressed luciferase in mice. Among them, the VEEV-LNP group and the mRNA-LNP group had the highest expression levels, while the MDPV-LNP group had the lowest expression levels.

[0159] Subsequently, the mice were euthanized, and the heart, liver, spleen, lungs, kidneys, brain, and cervical lymph nodes were separated. DiR and FLuc imaging analysis of each tissue was performed using a small animal in vivo imaging system.

[0160] DiR signals reflect the distribution of RNA-LNPs in mice. Figure 5A shows the distribution of each RNA-LNP in different organs, and Figure 5B shows the percentage of each RNA-LNP in each organ relative to the total distribution in the tested organs (the bars represent the percentages in the heart, liver, spleen, lung, kidney, brain, and lymph nodes from bottom to top). It can be seen that 24 hours after RNA-LNP injection, for both FLuc saRNA-LNP and FLuc mRNA-LNP, the DiR signal is mainly concentrated in the liver (over 80%), followed by the spleen (5%-9%), indicating that SM102-based LNPs primarily deliver RNA to the liver and spleen.

[0161] FLuc luminescence signals reflect the translation levels of target proteins after different saRNAs and mRNAs are delivered to various organs. Figure 6A shows the Fluc expression levels of each RNA-LNP in each organ, and Figure 6B shows the percentage of Fluc expression of each RNA-LNP in each organ relative to the total Fluc expression in the measured organs (the bars in the bar chart are from bottom to top: heart, liver, spleen, lung, kidney, brain, and lymph nodes).

[0162] It can be seen that the expression distribution of Fluc in the FLuc mRNA-LNP group is similar to that of the DiR signal, mainly concentrated in the liver (93.53%) and the spleen (3.16%), indicating that FLuc mRNA is translated and expressed in a proportional manner after being delivered to each organ.

[0163] In contrast, the expression level of FLuc saRNA-LNP in different organs did not show a positive correlation with its delivery level. The DiR signal above showed that more than 80% of FLuc saRNA-LNP in each saRNA-LNP group was delivered to the liver, but the FLuc expression level in the liver accounted for only 2.07%-8.23% of the total expression level, while more than 90% of the FLuc signal was distributed in extrahepatic tissues.

[0164] Furthermore, the expression preferences of various FLuc saRNAs in extrahepatic tissues showed significant differences. For example, in the VEEV group, FLuc expression accounted for more than 20% in the kidney, spleen, and heart, with the spleen having the highest proportion at 29.24%. In the MDPV group, FLuc expression was mainly concentrated in the lymph nodes and spleen, accounting for 47.14% and 38.72%, respectively. In the RNV and EVEV groups, FLuc expression was mainly concentrated in the spleen, accounting for 52.83% and 72.68%, respectively. It is evident that each saRNA showed high FLuc signal enrichment in the spleen, which may be related to the spleen's role as a secondary RNA-LNP delivery organ, and the high translation efficiency of each saRNA in the spleen. The EVEV group showed the highest spleen-biased expression, with a Fluc signal of 72.68% in the spleen. In contrast, the expression signals of Fluc in lymph nodes, brain, kidney, lung, and heart tissues were relatively low, indicating the greatest advantage of spleen-targeted expression (72.68% Fluc expression ratio compared to a distribution ratio of 6.38%). The MDPV group showed a high lymph node-biased expression, with a Dir signal of only 2.04% in lymph nodes and a Fluc signal of 47.14% in lymph nodes. In contrast, the expression signals of Fluc in brain, kidney, lung, spleen, and heart tissues were relatively low compared to the Dir distribution signal, indicating a greater advantage of lymph node-targeted expression (47.14% Fluc expression ratio compared to a distribution ratio of 2.04%).

[0165] In summary, each FLuc saRNA, after being delivered via LNP, exhibits different tissue-specific expression in vivo, especially showing high expression efficiency in extrahepatic tissues such as the spleen or lymph nodes. The target sequence of EVEV saRNA-LNP is expressed at a rate as high as 72.68% in the spleen.

[0166] The sequences involved in this application are shown below.

[0167] SEQ ID NO:1-T7 mini starter

[0168] SEQ ID NO:2-VEEV 5'UTR

[0169] SEQ ID NO:3-MDPV 5'UTR

[0170] SEQ ID NO:4-RNV 5'UTR

[0171] SEQ ID NO:5-EVEV 5'UTR

[0172] SEQ ID NO:6-VEEV replicon NSP1-4 sequence

[0173] SEQ ID NO:7-MDPV replicon NSP1-4 sequence

[0174] SEQ ID NO:8-RNV replicon NSP1-4 sequence

[0175] SEQ ID NO:9-EVEV replicon NSP1-4 sequence

[0176] SEQ ID NO:10 - VEEV subgene promoter sequence

[0177] SEQ ID NO:11-MDPV subgene promoter sequence

[0178] SEQ ID NO:12 - RNV subgene promoter sequence

[0179] SEQ ID NO:13-EVEV subgene promoter sequence

[0180] SEQ ID NO:14-FLuc sequence

[0181] SEQ ID NO:15-CVB3 IRES sequence

[0182] SEQ ID NO:16-E3L sequence

[0183] SEQ ID NO:17-VEEV 3'UTR sequence

[0184] SEQ ID NO:18-MDPV 3'UTR sequence

[0185] SEQ ID NO:19-RNV 3'UTR sequence

[0186] SEQ ID NO:20-EVEV 3'UTR sequence

[0187] SEQ ID NO:21-PolyA sequence

[0188] BspQI restriction enzyme cleavage site sequence

[0189] SEQ ID NO:22-T7 promoter sequence

[0190] SEQ ID NO:23-HBA 5'UTR sequence

[0191] SEQ ID NO:24-HBA 3'UTR sequence

[0192] SEQ ID NO:25 - Second 5' UTR sequence

[0193] ***

[0194] The embodiments of the present invention are not limited to those described above. Without departing from the spirit and scope of the present invention, those skilled in the art can make various changes and improvements to the present invention in form and detail, and these are all considered to fall within the protection scope of the present invention.

Claims

1. A method for expressing a target peptide or protein, or non-coding RNA, in the spleen or lymph node, comprising administering a self-replicating RNA molecule to the spleen or lymph node. The self-replicating RNA molecule contains a 5' cap, a 5' UTR, an open reading frame encoding RNA replicase, a promoter, a target sequence, a 3' UTR, and a poly(A) tail from the 5' end to the 3' end. This RNA replicase can amplify the self-replicating RNA molecule and its subgenomic RNA molecule containing the target sequence and 3'UTR. The target sequence contains an open reading frame encoding the target peptide or protein, or a non-coding RNA sequence. The RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), or Mostas Pedras virus (MDPV).

2. The method as described in claim 1, wherein, The RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), wherein the open reading frame encoding the RNA replicase contains a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

9. The RNA replicase is a non-structural protein or a functional variant thereof derived from Rio Negro virus (RNV), wherein the open reading frame encoding the RNA replicase contains a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8, or The RNA replicase is a non-structural protein or a functional variant thereof derived from Mosdas Pedras virus (MDPV), wherein the open reading frame encoding the RNA replicase contains a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

7.

3. The method as described in claim 1 or 2, wherein, This RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), wherein the open reading frame, 5'UTR, promoter, and 3'UTR encoding the RNA replicase contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9, 5, 13, and 20. The RNA replicase is a non-structural protein or a functional variant thereof derived from Rio Negro virus (RNV), wherein the open reading frame, 5'UTR, promoter, and 3'UTR encoding the RNA replicase contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8, 4, 12, and 19, or The RNA replicase is a non-structural protein or a functional variant thereof derived from Mostas Pedras virus (MDPV), wherein the open reading frame, 5'UTR, promoter, and 3'UTR encoding the RNA replicase contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:7, 3, 11, and 18.

4. The method of any one of claims 1-3, wherein administering the self-replicating RNA molecule to the spleen or lymph node comprises introducing the self-replicating RNA molecule into cells of the spleen or lymph node.

5. The method of any one of claims 1-4, wherein administering the self-replicating RNA molecule to the spleen or lymph node comprises administering the self-replicating RNA molecule intravenously or transperitoneally, wherein the self-replicating RNA molecule is in a delivery vector.

6. The method of claim 5, wherein the delivery carrier is a lipid carrier, preferably a lipid nanoparticle.

7. The method of claim 6, wherein the lipid nanoparticles are lipid nanoparticles targeting the spleen or lymph nodes.

8. Use of a self-replicating RNA molecule in the preparation of a medicament for treating spleen-related diseases or lymph node-related diseases, wherein the self-replicating RNA molecule comprises, from the 5' end to the 3' end, a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a target sequence, a 3' UTR, and a poly(A) tail, wherein the RNA replicase is capable of amplifying the self-replicating RNA molecule and a subgenomic RNA molecule comprising the target sequence and the 3' UTR, wherein the target sequence comprises an open reading frame encoding a target peptide or protein, or a non-coding RNA sequence, wherein the RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), or Mostas Pedras virus (MDPV).

9. The use as described in claim 8, wherein, The RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), wherein the open reading frame encoding the RNA replicase contains a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

9. The RNA replicase is a non-structural protein or a functional variant thereof derived from Rio Negro virus (RNV), wherein the open reading frame encoding the RNA replicase contains a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8, or The RNA replicase is a non-structural protein or a functional variant thereof derived from Mosdas Pedras virus (MDPV), wherein the open reading frame encoding the RNA replicase contains a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

7.

10. The use as described in claim 8 or 9, wherein, This RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), wherein the open reading frame, 5'UTR, promoter, and 3'UTR encoding the RNA replicase contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9, 5, 13, and 20. The RNA replicase is a non-structural protein or a functional variant thereof derived from Rio Negro virus (RNV), wherein the open reading frame, 5'UTR, promoter, and 3'UTR encoding the RNA replicase contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8, 4, 12, and 19, or The RNA replicase is a non-structural protein or a functional variant thereof derived from Mostas Pedras virus (MDPV), wherein the open reading frame, 5'UTR, promoter, and 3'UTR encoding the RNA replicase contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:7, 3, 11, and 18.

11. The use as described in any one of claims 8-10, wherein the self-replicating RNA molecule is in a delivery vector, wherein the delivery vector is preferably a lipid carrier, more preferably a lipid nanoparticle.

12. The use as described in claim 11, wherein the lipid nanoparticles are lipid nanoparticles targeting the spleen or lymph nodes.

13. A self-replicating RNA molecule encapsulated by lipid nanoparticles. The self-replicating RNA molecule comprises, from the 5' end to the 3' end, a 5' cap, a 5' UTR, an open reading frame encoding an RNA replicase, a promoter, a target sequence, a 3' UTR, and a poly(A) tail. The RNA replicase is capable of amplifying the self-replicating RNA molecule and its subgenomic RNA molecule containing the target sequence and the 3' UTR. The target sequence contains an open reading frame encoding a target peptide or protein, or a non-coding RNA sequence. The RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), Rio Negro virus (RNV), or Mostas Pedras virus (MDPV).

14. The self-replicating RNA molecule of claim 13, wherein the lipid nanoparticles are lipid nanoparticles targeting the spleen or lymph nodes.

15. The self-replicating RNA molecule as described in claim 13 or 14, wherein, The RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), wherein the open reading frame encoding the RNA replicase contains a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

9. The RNA replicase is a non-structural protein or a functional variant thereof derived from Rio Negro virus (RNV), wherein the open reading frame encoding the RNA replicase contains a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8, or The RNA replicase is a non-structural protein or a functional variant thereof derived from Mosdas Pedras virus (MDPV), wherein the open reading frame encoding the RNA replicase contains a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

7.

16. The self-replicating RNA molecule of claim 15, wherein, This RNA replicase is a non-structural protein or a functional variant thereof derived from Everglades virus (EVEV), wherein the open reading frame, 5'UTR, promoter, and 3'UTR encoding the RNA replicase contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9, 5, 13, and 20. The RNA replicase is a non-structural protein or a functional variant thereof derived from Rio Negro virus (RNV), wherein the open reading frame, 5'UTR, promoter, and 3'UTR encoding the RNA replicase contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8, 4, 12, and 19, or The RNA replicase is a non-structural protein or a functional variant thereof derived from Mostas Pedras virus (MDPV), wherein the open reading frame, 5'UTR, promoter, and 3'UTR encoding the RNA replicase contain nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:7, 3, 11, and 18.

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