Chimera specifically targeting degradation of rna virus genome and application thereof

Abstract

The application discloses a chimera specifically targeting and degrading an RNA virus genome and an application thereof, and the chimera is composed of a chimera N and a chimera C; the chimera N is sequentially connected with a MAVS CARD domain, a connecting peptide and an N terminal of RNase A; the chimera C is sequentially connected with a MAVS CARD domain, a connecting peptide and a C terminal of RNase A; and the N terminal of RNase A and the C terminal of RNase A are obtained by segmenting RNase A at a segmentation site. The chimera in the application assembles in situ to form a CARTAC complex with RNA cleavage activity, and then degrades a viral RNA genome, thereby providing a safe, broad-spectrum and effective prevention and treatment strategy for resisting RNA virus infection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a chimera that specifically targets and degrades the genome of an RNA virus and its applications. Background Technology

[0002] RNA virus infections, including emerging and re-emerging epidemics and pandemics, pose a threat to global health. Despite continuous progress in vaccines and treatments, the high mutation rate of RNA viruses leads to significant genetic diversity and the rapid emergence of drug-resistant strains, presenting a severe challenge to prevention and control efforts. Therefore, the development of broad-spectrum antiviral drugs is of great importance in responding to unknown and emerging RNA virus outbreaks.

[0003] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0004] The technical objective of this application is to address the above-mentioned shortcomings by providing a chimera that specifically targets and degrades the genome of RNA viruses and its applications. This application fuses the CARD domain of MAVS with the N-terminal and C-terminal fragments of split-RNase via linker peptides, thereby constructing an inactive CARTAC. N and CARTAC C When the virus infects host cells, it activates the RIG-I-like receptor, thereby recruiting CARTA cells to fuse with CARTAC. N and CARTAC C The in-situ assembly of the CARTAC complex, which has RNA cleavage activity, degrades the viral RNA genome, providing a new, safe, broad-spectrum, and effective prevention and control strategy against RNA virus infection.

[0005] To achieve the above objectives, this application provides the following technical solution:

[0006] According to one aspect of this application, a chimera for specifically targeting and degrading RNA virus genomes is provided, characterized in that the chimera is composed of chimera N and chimera C; chimera N is sequentially linked by a MAVS CARD domain, a linker peptide, and the N-terminus of RNase A; chimera C is sequentially linked by a MAVS CARD domain, a linker peptide, and the C-terminus of RNase A; the N-terminus and C-terminus of RNase A are obtained by splitting RNase A at a cleavage site.

[0007] In some embodiments, the cleavage site is one of 39 / 40, 57 / 58, or 68 / 69.

[0008] In some embodiments, the cleavage site is 68 / 69, the N-terminal amino acid sequence of RNase A is shown in SEQ ID NO:5, and the C-terminal amino acid sequence of RNase A is shown in SEQ ID NO:7.

[0009] In some embodiments, the MAVS CARD domain is a domain that targets and combines with the RLRs CARD domain.

[0010] In some embodiments, the amino acid sequence of the MAVS CARD domain is shown in SEQ ID NO:4.

[0011] In some embodiments, the linker peptide is one of a short linker peptide, a flexible linker peptide, or a semi-flexible linker peptide.

[0012] In some embodiments, the amino acid sequence of the short linker peptide is shown in SEQ ID NO:1, the amino acid sequence of the flexible linker peptide is shown in SEQ ID NO:2, and the amino acid sequence of the semi-flexible linker peptide is shown in SEQ ID NO:3.

[0013] In some embodiments, the linker peptide is a semi-flexible linker peptide.

[0014] In some embodiments, the amino acid sequence of chimera N is shown in SEQ ID NO:6, and the amino acid sequence of chimera C is shown in SEQ ID NO:8.

[0015] According to another aspect of this application, a chimera that specifically targets and degrades the genome of an RNA virus is provided for use in the preparation of a medicament for inhibiting RNA viruses.

[0016] In some embodiments, the RNA virus is a positive-sense RNA virus and / or a negative-sense RNA virus.

[0017] In some embodiments, the positive-sense RNA virus is one or more of Japanese encephalitis virus, dengue virus, novel coronavirus, enterovirus 71, or chikungunya virus; the negative-sense RNA virus is one or more of Ebola virus, fever with thrombocytopenia syndrome virus, or Crimean-Congo hemorrhagic fever virus.

[0018] Compared with the prior art, the advantages and positive effects of this application are as follows: This application fuses the CARD domain of MAVS with the N-terminal and C-terminal fragments of split-RNase respectively via linker peptides, thereby constructing an inactive CARTAC N and CARTAC CWhen the virus infects host cells, it activates the RIG-I-like receptor, thereby recruiting CARTA cells to fuse with CARTAC. N and CARTAC C The in-situ assembly of the CARTAC complex, which has RNA cleavage activity, degrades the viral RNA genome, providing a new, safe, broad-spectrum, and effective prevention and control strategy against RNA virus infection. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This diagram illustrates how the present application exerts its antiviral effect.

[0021] Figure 2 The analysis shows the activity recovery of split-RNase A in Example 1 of this application.

[0022] Figure 3 This paper illustrates the analysis of the degradation effect of CARTAC on 5'-ppp RNA in Example 3 of this application.

[0023] Figure 4 The analysis of the degradation effect of CARTAC on dsRNA in Example 4 of this application is shown.

[0024] Figure 5 This demonstrates the effectiveness of CATRAC in inhibiting different RNA viruses in Example 5 of this application.

[0025] Figure 6 This demonstrates the in vivo therapeutic effect of CATRAC on a VSV mouse infection model as shown in Example 6 of this application.

[0026] Figure 7 This demonstrates the in vivo therapeutic effect of CATRAC on a SeV mouse infection model as shown in Example 7 of this application. Detailed Implementation

[0027] To better understand the above-mentioned objectives, features, and advantages of this application, the application will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

[0028] RNA virus infections, including emerging and re-emerging epidemics and pandemics, pose a serious threat to global health. Despite continuous progress in vaccines and treatments, the high mutation rate of RNA viruses leads to significant genetic diversity and the rapid emergence of drug-resistant strains, presenting a severe challenge to prevention and control efforts. Therefore, the development of broad-spectrum antiviral drugs is of great importance in responding to unknown and emerging RNA virus outbreaks.

[0029] Despite the diversity of RNA viruses, they all share a common molecular characteristic: the 5' end of the genome of negative-strand RNA viruses often contains a triphosphate structure (5'-ppp). Furthermore, RNA viruses form double-stranded RNA (dsRNA) intermediates during replication. These viral 5'-ppp-RNAs and dsRNAs can be specifically recognized by RIG-I-like receptors (RLRs) in host cells, such as RIG-I and MDA5. RIG-I primarily recognizes 5'-ppp RNA and short-chain dsRNAs, while MDA5 recognizes long-chain dsRNAs. Although RIG-I and MDA5 differ in the types of RNA they recognize, their structures are similar, both containing two N-terminal CARD domains, a DExD / H-box RNA helicase domain, and a C-terminal domain (CTD). In the resting state, the CARD signaling domain is masked by the helicase domain. Upon binding to a RIG-I-like receptor, the viral RNA releases the self-inhibitory state of the CARD domain, allowing it to polymerize through CARD-CARD interactions. This multimer further interacts with the CARD domain of the mitochondrial antiviral signaling protein (MAVS) to form prion-like MAVS oligomers on the mitochondrial membrane, thereby activating the TBK1 / IRF3 / NF-κB signaling pathway and promoting the production of type I interferon and pro-inflammatory cytokines.

[0030] Based on the conserved mechanism of viral RNA-induced CARD protein aggregation, this study developed a broad-spectrum pattern recognition receptor clustering-activated split-RNase targeting chimera (CARTAC) antiviral strategy. This strategy involves fusing the CARD domain of MAVS with the N-terminal and C-terminal fragments of split-RNase, respectively, to construct inactive CARTAC. N and CARTAC C When the virus infects host cells, it activates the RIG-I-like receptor, thereby recruiting CARTA cells to fuse with CARTAC. N and CARTAC C The CARTAC complex, which cleaves RNA, is assembled in situ, thereby degrading the viral RNA genome. This provides a new, safe, broad-spectrum, and effective prevention and control strategy against RNA virus infection.

[0031] Figure 1 This diagram illustrates the antiviral activity of this application. After an RNA virus infects a host cell, it releases 5'-ppp RNA through uncoating or produces double-stranded RNA during genome replication. These specialized RNAs, distinct from those in the host cell, can be specifically recognized by RIG-I-like receptors (RLRs), such as RIG-I and MDA5, thereby activating RLRs, releasing the self-inhibitory state of the CARD domain, and recruiting M from the CARTAC chimera. CARD This ultimately leads to the activation of split-RNase A, which then targets and degrades viral RNA. For example... Figure 1 As shown, by utilizing the split-RNase strategy, oligomers are assembled through the activation of the MAVS CRAD domain, inducing complementary activation of split-RNase, which then targets and cleaves viral RNA in situ, thus developing a chimera that broadly inhibits RNA virus replication.

[0032] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0033] Example 1: Determination of cleavage sites and selection of linker peptides

[0034] Using the artificial intelligence software Alpha Fold 3 and SPELL server, potential split sites for RNase A were analyzed. These potential split sites were identified as 39 / 40, 57 / 58, and 68 / 69. The N-terminus and C-terminus of RNase A fragments from these different split sites were then incorporated into the C-terminus of the LgBit and Hibit of the split-NanoLuc luciferase system, respectively. These fragments were linked by different linker peptides, including a short linker peptide (L1), a flexible linker peptide (L2), and a semi-flexible linker peptide (L3).

[0035] The amino acid sequence of the short linker peptide (L1) is: GS (SEQ ID NO:1).

[0036] The amino acid sequence of the flexible linker peptide (L2) is: GGGGSGGGGSGGGGR (SEQ ID NO:2);

[0037] The amino acid sequence of the semi-flexible linker peptide (L3) is: LEASPSNPGASNGS (SEQ ID NO:3).

[0038] The gene sequence encoding the above fusion polypeptide sequence was constructed into the PCDNA3.1+ vector.

[0039] The plasmid structure is as follows: CMV promoter → [LgBit or HiBit]-[Linker]-[RNase AN fragment or RNase A-C fragment]-BGH poly(A) signal;

[0040] For example, construct a pair of plasmids with cleavage sites 39 / 40 and linker peptide L1: CMV promoter → LgBit-GS-RNase A (1-39) → BGH poly(A) signal and CMV promoter → HiBit-GS-RNase A (40-134) → BGH poly(A) signal. The same linker peptide is used for the same set of plasmids.

[0041] After transfecting HEK293T cells with the above eukaryotic expression plasmids, the proteins were purified by IP assay. Utilizing the spontaneous binding property of LgBit and Hibit, the RNase A activity of different combinations was measured using an RNase A activity assay kit (Beyotime, P0347M). The recovery of split-RNase A activity was analyzed.

[0042] Figure 2 This paper illustrates the analysis of the activity recovery of split-RNase A in Example 1 of this application, as shown in the figure. Figure 2 As shown, L3 and 68 / 69 sites showed the best effect on restoring RNase A activity. Therefore, 68 / 69 was selected as the cleavage site and L3 was selected as the linker peptide in subsequent experiments.

[0043] Example 2: Constructing CARTAC.

[0044] Based on screened split-RNase A and optimized linker peptides, a clustering-activated split-RNase targeting chimera (CARTAC) chimera was developed to target the broad-spectrum degradation of RNA virus genomes. The N-terminal and C-terminal fragments of the RNase, obtained from the optimal split site selected during screening, were constructed at the C-terminus of a MAVSCARD (1-100 amino acids) membrane and linked together by linker peptide 3 (L3) to construct chimera N (CARTAC). N ) and chimera C (CARTAC C ) plasmid.

[0045] CARTAC N Plasmids, including the MAVS CARD domain (M) that targets and binds to the RLRs CARD domain. CARD ); The N-terminus of RNase A is linked by a linker peptide 3.

[0046] CARTACN Plasmid structure: M CARD -Link peptide 3-RNase N .

[0047] M CARD The amino acid sequence is (SEQ ID NO:4): MPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTSGS.

[0048] RNase N The amino acid sequence is (SEQ ID NO:5): MKPPQFTWAQWFETQHINMTSQQCTNAMQVINNYQRRCKNQNTFLLTTFANVVNVCGNPNMTCPSNKT.

[0049] In other words: CARTAC N The plasmid amino acid sequence is (SEQ ID NO:6):MPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTSGSLEASPSNPGASNGSMKPPQFTWAQWFETQHINMTSQQCTNAMQVINNYQRRCKNQNTFLLTTFANVVNVCGNPNMTCPSNKT.

[0050] CARTAC C The plasmid includes the MAVS CARD domain, which targets and binds to the RLRs CARD domain; the C-terminus of RNase A; and is linked by linker peptide 3.

[0051] CARTAC C Plasmid structure: M CARD -Link peptide 3-RNase C .

[0052] RNase C The amino acid sequence is (SEQ ID NO:7): RKNCHHSGSQVPLIHCNLTTPSPQNISNCRYAQTPANMFYIVACDNRDQRRDPPQYPVVPVHLDRII.

[0053] In other words: CARTAC CThe plasmid amino acid sequence is (SEQ ID NO:8):MPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTSGSLEASPSNPGASNGSRKNCHHSGSQVPLIHCNLTTPSPQNISNCRYAQTPANMFYIVACDNRDQRRDPPQYPVVPVHLDRII.

[0054] The CARTAC in this application consists of two parts, including CARTAC N and CARTAC C Plasmids. The above plasmids were co-transfected into cells. During viral infection, CARTAC... N and CARTAC C They will be recruited into MAVS aggregates and assembled to form active CARTAC.

[0055] Example 3: Analysis of the degradation effect of CARTAC on 5'-ppp RNA.

[0056] Since this chimera exerts its antiviral effect by targeting and degrading viral RNA, this embodiment analyzes its degradation of the specific RNA: 5'-ppp RNA produced by the virus after entering the cell.

[0057] The specific steps are as follows: Construct the T7 RNA polymerase gene into the PCDNA3.1+ vector. Then, combine the above plasmid with CARTAC... N and CARTAC C Plasmids were co-transfected into HEK 293T cells (the Mock group was only transfected with the T7 RNA polymerase eukaryotic expression vector). 24 h post-transfection, cells were transfected again with DNA fragments of the Pepper aptamer containing either the T7 promoter sequence or the CMV promoter sequence. 12 h post-transfection, Hoechst 33342 and 5 μM HBC530 (green fluorescent) staining solution were added to the cell supernatant. After incubation in the dark for 20 min, the cells were washed twice with 1 ml of fresh DMEM medium each time. Because these RNA sequences contain the Pepper aptamer sequence, they can specifically bind to the fluorescent dye HBC. Therefore, confocal microscopy can be used to acquire images, and the intensity of the green fluorescence signal can be analyzed to determine the expression levels of different structural RNAs within the cells.

[0058] Figure 3 This illustrates the analysis of the degradation effect of CARTAC on 5'-ppp RNA in Example 3 of this application, as shown below. Figure 3As shown, 5'-ppp RNA transcribed from the T7 promoter sequence was significantly degraded after CARTAC treatment, while 5'-cap RNA transcribed from the CMV promoter (as a control) was unaffected. These results confirm that CARTAC can specifically degrade 5'-ppp RNA.

[0059] Example 4: Analysis of the degradation effect of CARTAC on dsRNA.

[0060] To analyze the degradation of double-stranded RNA (dsRNA) generated by the virus after entering cells by CARTAC, self-amplified RNA (saRNA) expressing GFP protein and GFP mRNA were used for verification (both RNAs were purchased from GenScript; saRNA catalog number: RP-A00015, GFP mRNA catalog number: RP-A00008). SaRNA forms double-stranded RNA during GFP protein expression, while GFP mRNA is directly transcribed to GFP protein without producing double-stranded RNA.

[0061] The specific steps are as follows: [Cartach] N and CARTAC C The plasmids were co-transfected into HEK 293T cells (the Mock group was not transfected with any plasmids). 24 h after transfection, the cells were transfected again with self-amplified RNA (Sa RNA) and GFP mRNA, respectively. 12 h after transfection, the cells were fixed with paraformaldehyde, stained with Hoechst 33342, and images were acquired using a confocal microscope.

[0062] Figure 4 This illustrates the analysis of the degradation effect of CARTAC on dsRNA in Example 4 of this application, such as... Figure 4 As shown, after CARTAC treatment, the GFP protein expressed by Sa RNA was significantly reduced, while the GFP protein expressed by GFP Mrna was unaffected. These results confirm that CARTAC can specifically degrade dsRNA.

[0063] Example 5: Validation of the inhibitory effect of CATRAC on different RNA viruses.

[0064] To confirm that CATRAC has a significant inhibitory effect on both positive-sense RNA viruses (JEV, DENV, CHIKV) and negative-sense RNA viruses (EBOV, SFTSV, CCCHFV). After transfecting cells with the eukaryotic expression plasmid of the chimera, cells were inoculated with 0.1 mol of positive-strand RNA virus or negative-strand RNA virus, respectively. 12 h after inoculation, cells were washed twice with PBS for 2-3 minutes each time, fixed with 4% paraformaldehyde at room temperature for 15 minutes, covered with 0.1-0.2% Triton X-100, and incubated at room temperature for 15-20 minutes. After permeabilization, cells were washed three times with PBS for 5 minutes each time, and then completely covered with 5% BSA and blocked for 1 h. The primary antibody was diluted with dilution buffer and covered with the sample. The sample was incubated overnight at 4°C. The next day, the sample was warmed to room temperature for 20 minutes, washed three times with PBST for 5 minutes each time, incubated with FITC-labeled secondary antibody at 37°C in the dark for 1 hour, stained with DAPI for 5 minutes, and then mounted. The results were observed under a fluorescence microscope.

[0065] The inhibitory effect of the polypeptide chimera on the above viruses was analyzed by immunofluorescence assay.

[0066] Positive-sense RNA viruses include: Japanese encephalitis virus (JEV), dengue virus (DENV), SARS-CoV-2, enterovirus 71 (EV71), and chikungunya virus (CHIKV). Negative-sense RNA viruses include: Ebola virus (EBOV), Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), and Crimean-Congo hemorrhagic fever virus (CCHFV). All of these viruses were obtained from the National Virus Preservation Center.

[0067] Figure 5 This illustrates the validation of the inhibitory effect of CATRAC on different RNA viruses in Example 5 of this application. The Mock group received no treatment, while the NC group was only transfected with the MAVS CARD domain. Figure 5 As shown, the polypeptide chimera CATRAC significantly inhibits the replication of the above viruses.

[0068] Example 6: Validation of the in vivo therapeutic effect of CATRAC on a mouse model of VSV infection.

[0069] To confirm that both lentiviral CATRAC and mRNA-expressed CATRAC have good therapeutic effects on mouse infection models.

[0070] Six-week-old C57BL / 6 mice were randomly divided into three groups of eight each. The chimera described above was linked to 2A peptide P2A (2A peptide P2A amino acid sequence SEQ ID NO:9: ATNFSLLKQAGDVEENPGP) to construct a bicistronic lentiviral packaging plasmid. This plasmid, along with packaging plasmids (psPAX2 and pMD2.G), was then co-transfected into 293T and other packaging cells. 48-72 hours post-transfection, cell supernatant containing lentiviral particles was collected. The lentivirus was concentrated using a lentivirus concentration kit (Yisheng, catalog number: 41101ES50) and then injected intravenously into mice at a dose of 5 × 10⁵ cells / mL. 7 IU lentivirus was injected into mice (NC group injected with only M expression). CARD The mice were injected with lentiviruses (the Mock group received lentiviruses packaged with empty vectors). Four days after injection, mice were injected via the tail vein with vesicular stomatitis virus (VSV) (from the National Virus Preservation Center) (1 × 10⁻⁶ serovars per mouse). 8 PFU was used to measure mouse body weight and mortality daily after VSV inoculation, and survival curves were plotted.

[0071] Figure 6 This illustrates the in vivo therapeutic efficacy verification of CATRAC on a VSV mouse infection model in Example 6 of this application, as shown below. Figure 6 As shown, the designed chimera significantly improved weight loss and reduced mortality in mice induced by virus inoculation. These results confirm that the peptide chimera can still exert a good anti-VSV effect in vivo.

[0072] Example 7: Validation of the in vivo therapeutic effect of CATRAC on a mouse model of Sendai virus (SeV) infection.

[0073] To verify the therapeutic effect of the polypeptide chimera on respiratory viral diseases, a Sendai virus (SeV) infection model was used as the research subject. Six-week-old C57BL / 6 mice were randomly divided into three groups of eight mice each. The chimera was linked by a 2A peptide P2A to construct a bicistronic plasmid, which was then transcribed into the corresponding mRNA in vitro and encapsulated by an LNP. The LNP was further modified with a CD31 antibody to specifically target lung tissue (the LNP was synthesized by Genscript Biotech). The LNP was injected into mice via the tail vein (5 μg per mouse) (the NC group received injections expressing only M... CARD LNPs (in the Mock group, LNPs without any RNA were injected), and Sendai virus (SeV) was administered via nasal drops 12 hours later (1 × 10⁻⁶ per animal). 5PFU was used to measure the weight and number of deaths of mice daily after inoculation with Sendai virus (SeV), and survival curves were plotted.

[0074] Figure 7 This demonstrates the in vivo therapeutic efficacy of CATRAC on a mouse model infected with Sendai virus (SeV) in Example 7 of this application. Figure 7 As shown, the designed chimera significantly improved weight loss and reduced mortality in mice induced by viral inoculation. These results confirm that the peptide chimera can still exert a good anti-Sev effect in vivo, and that this peptide chimera, delivered via the above-mentioned method, can treat respiratory-related viral infectious diseases.

[0075] It should be noted that, due to the common molecular characteristics of RNA viruses, the chimera in this application is a broad-spectrum chimera. After the virus infects host cells, it activates the RIG-I-like receptor, thereby recruiting fused CARTACs. N and CARTAC C The CARTAC complex, which has RNA cleavage activity, is assembled in situ to form a complex that degrades the viral RNA genome, providing a safe, broad-spectrum and effective way to prevent and control RNA virus infection.

[0076] Through the above specific embodiments, those skilled in the art can easily implement this application. However, it should be understood that this application is not limited to the specific embodiments described above. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to achieve different technical solutions.

Claims

1. A chimera that specifically targets and degrades the genome of an RNA virus, characterized in that, The chimera is chimera N and chimera C, the amino acid sequence of chimera N is shown in SEQ ID NO:6, and the amino acid sequence of chimera C is shown in SEQ ID NO:

8.

2. The use of the chimera of claim 1, which specifically targets and degrades the genome of an RNA virus, in the preparation of a drug for inhibiting RNA viruses; wherein the RNA virus is a positive-sense RNA virus and / or a negative-sense RNA virus.

3. The application according to claim 2, characterized in that, The positive-sense RNA virus is one or more of Japanese encephalitis virus, dengue virus, novel coronavirus, enterovirus 71, or chikungunya virus; the negative-sense RNA virus is one or more of Ebola virus, fever with thrombocytopenia syndrome virus, or Crimean-Congo hemorrhagic fever virus.

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