Application of VZV latency-associated transcript (VLT / circVLT) in treatment of varicella zoster

Abstract

The invention discloses an application of a VZV latent related transcript (VLT / circVLT) in treatment of varicella zoster. The herpes zoster is a public health problem urgently needing to be solved in China at present, and how to remove VZV latent in neurons is an unsolved problem. The invention discloses a method for activating latent VZV by using VLT / circVTL, and removing the VZV in combination with a VZV vaccine or an antiviral drug. A method for reactivating and removing VZV latent in neurons is realized, and an effective scheme can be provided for prevention and treatment of herpes zoster.

Description

[0001] The present invention belongs to the field of pharmaceutical technology, and in particular relates to the application of VZV latency-related transcripts (VLT / circVLT) in the treatment of varicella-zoster. Background Art

[0002] Varicella-zoster virus (VZV) is the pathogen that causes varicella (Varicella) and herpes zoster (HZ). VZV can be transmitted through aerosolized droplets or direct contact and infect respiratory mucosal epithelial cells. Infected T cells can transport VZV to the skin through the blood circulation, resulting in a systemic densely distributed vesicular rash commonly known as chickenpox (Gershon et al., 2015). Varicella is common in children under 10 years old, with an incidence of about 140 million (Chinese Medical Association Dermatologist Branch Herpes Zoster Expert Consensus Working Group, 2018; Kawai et al., 2014). For people with weakened immunity, such as the elderly, VZV can cause herpes zoster (HZ) accompanied by neuralgia, called herpes zoster-related pain (ZP), with an annual incidence of 3-5% (Herpes Zoster Expert Consensus Working Group of the Dermatology Branch of the Chinese Medical Doctor Association, 2018).

[0003] The VZV genome is about 125 kb, encoding 71 classic open reading frames (ORFs) (Zhang et al., 2010). During the VZV activation phase (i.e., lytic infection), all 71 ORFs can transcribe a large amount of viral RNA. However, during the latent period, the VZV genomic DNA is a circular structure; and almost all genes are in a dormant state, with only VZV latency-associated transcripts (VLT) expressed near ORF61 (Depledge et al., 2018). VZV can infect the dorsal root ganglia (DRG) or trigeminal ganglia (TG) and establish lifelong latent infection (latent infection) (Thiele et al., 2011). The cell bodies of primary sensory neurons are located in DRG and TG, which are important tissues for pain transmission (Bertae et al., 2017). When the body's immunity is low, latent VZV can be reactivated and replicate progeny viruses in large quantities, traveling along sensory nerve fibers to the innervated skin ( Figure 1)(Gershon et al., 2015). In this process, VZV destroys the infected DRG or TG neurons, leading to neuronal dysfunction, ectopic discharges, and peripheral and central sensitization, causing severe neuralgia (Chinese Medical Association Dermatology Branch Herpes Zoster Expert Consensus Working Group, 2018). How to eliminate VZV lurking in neurons has always been an unresolved problem. Circular RNA (circRNA) is a class of single-stranded RNA molecules with a covalently closed circular structure at the head and tail through back-spliced ​​junction. In the early stage of the present invention, it was discovered that exon 5 of VLT was reversely spliced ​​with exon 2 to form a circular RNA, namely circVLT (Yang et al., 2024). Summary of the invention

[0004] The present invention provides the use of VZV latency-related transcripts (VLT / circVLT) in the treatment and prevention of varicella zoster. The present invention can be used to prevent or treat herpes zoster.

[0005] The present invention comprises three parts:

[0006] The first part is to improve the body's immunity through VZV vaccine and use the body's immunity to eliminate VZV. The VZV vaccine includes varicella vaccine or shingles vaccine in the form of live attenuated vaccine, mRNA vaccine, viral protein vaccine, etc.

[0007] The second part uses VLT to regulate VZV latency-reactivation, wherein the VLT includes VZV latency-related transcripts including linear RNA or homologous circular RNA transcribed between ORF60 and ORF64 during VZV latency or reactivation and the proteins encoded by them.

[0008] The third part is to further eliminate VZV using antiviral drugs, which include acyclovir and its analogs.

[0009] Beneficial effects of the invention: Herpes zoster is a public health problem that needs to be solved urgently in my country, and how to eliminate VZV lurking in neurons is an unresolved problem. This patent discloses for the first time a method of using VLT to activate latent VZV and combining it with varicella vaccine, herpes zoster vaccine or antiviral drugs to reactivate and eliminate VZV lurking in neurons, which can provide an effective solution for the prevention and treatment of herpes zoster. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1Mutation of circRNA at the genome level based on the bacterial artificial chromosome recombination system; (A) Schematic diagram of the sequence organization of the splicing event of VZVcircVLT at ORF61; Based on the rOka strain of VZV (i.e., GFP and firefly luciferase genes were inserted based on the pOka strain), the genomic sequence at the end of exon 5 of circVLT was edited by base substitution using bacterial artificial chromosome recombination technology, and the downstream of the interface site of circVLT exon 5 was marked as M1 (i.e., VZV-△circVLT), purple and upstream M2, orange. (B) The mutant sequences of M1, purple and M2, orange correspond to the positions of mature circVLT; (C) Sanger sequencing detected the corresponding genomic sequence of VZV-△circVLT after mutation;

[0011] Figure 2 circVLT deficiency leads to impaired VZV latent establishment-reactivation; (A) Experimental flow chart for establishing VZV latent-reactivation model in vitro. Glioblastoma cells (SH-SY5Y) were induced to differentiate and treated with high concentrations of acyclovir to establish VZV latent state. Finally, VZV reactivation was induced by removing neurotrophic factors (NGF, BDNF) and adding PI3K inhibitor (wortmannin). (BD) Viral luciferase activity (B), relative content of viral genomic DNA (C) and viral RNA (D) during VZV latent establishment-reactivation.

[0012] Figure 3 Overexpression of circVLT inhibits VLT and promotes ORF61 expression; (A) Overexpression of circVLT isoforms circVLT-419nt, circVLT-542nt and VZV_circRNA3 were used as controls. RT-PCR detection showed that VZVcircVLT and VZV_circRNA3 were successfully overexpressed. (B) qPCR detection of VLT (primers for exon 1 of VLT) and ORF61 expression 48 hours after VZV infection;

[0013] Figure 4circVLT combined with VZV neutralizing antibodies or anti-VZV drugs can eliminate latent VZV; (AB) Experimental flow chart for establishing a VZV in vitro latent-reactivation model and using circVLT combined with VZV neutralizing antibodies or anti-VZV drugs to eliminate latent VZV. Neuroglioma cells (SH-SY5Y) were induced to differentiate and treated with high concentrations of acyclovir to establish VZV latency. Finally, (A) gE / gI / gN antibodies were added to simulate the neutralizing antibodies produced by the body to stimulate the in vivo vaccine and immunize VZV and transfect in vitro synthesized circVLT-419nt and circVLT-542nt; (B) acyclovir was added and in vitro synthesized circVLT-419nt and circVLT-542nt were transfected. (C) The end back-to-back primers (T1|T2) Specific detection of circular VZV genomic DNA; (D) VZV circular virus genomic DNA content before treatment with VZV antibodies gE, gN, and gI, (E) VZV circular virus genomic DNA content after treatment with VZV antibodies gE, gN, and gI combined with circVLT, (F) VZV circular virus genomic DNA content before treatment with acyclovir combined with circVLT, (G) VZV circular virus genomic DNA content after treatment with acyclovir combined with circVLT. DETAILED DESCRIPTION

[0014] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0016] The following is further demonstrated with various embodiments:

[0017] Example 1: circVLT deficiency leads to impaired VZV latent establishment and reactivation

[0018] 1.1 Preparation of VZVcircVLT-deficient virus strain and infection of SH-SY5Y cells

[0019] The genomic sequence at the end of exon 5 of circVLT was edited by base substitution using bacterial artificial chromosome (BAC) to prepare a circVLT-inactivated VZV mutant strain.

[0020] VZV mutant strains were prepared by using the positive and negative screening method of galactokinase (galK) based on the VZVBAC system. Based on the previous work, the wild sequence of the second targeting fragment (5'-AGGTAAGTCCACAAACAAAA-3') was replaced with a 20bp random sequence (5'-CCCACGATATCCCCTTAACT-3'). Targeting fragment 2 was introduced into the SW102-galK strain, and positive strains were screened in a medium containing 2-deoxy-galactose (2-Deoxy-D-galactose, DOG) (clones with galK will use DOG as a carbon source, produce substances that are harmful to themselves, and cannot continue to grow; while recombinant clones do not use DOG to produce toxic effects and can grow). Positive strains were identified by PCR; Sanger sequencing was further confirmed. An in vitro VZV latent-reactivation cell model was established with reference to the method of Baird NL et al. (Baird et al., 2019).

[0021] 1.2 Experimental Results

[0022] Analysis of the circVLT sequence revealed that only exon 5 did not overlap with the VZV gene ( Figure 1 AB). The results of TA cloning and Sanger sequencing experiments showed that the bacterial artificial chromosome recombination system successfully replaced the end of exon 5 of circVLT with a random sequence and prepared a circVLT-inactivated VZV mutant strain ( Figure 1 C).

[0023] Using NGF and BDNF, we successfully induced the differentiation of neuroblastoma cells (SH-SY5Y). After infection with wild-type VZV or circVLT-deficient virus, we treated VZV with high concentrations of acyclovir to establish latency. Finally, we induced VZV reactivation by removing neurotrophic factors (NGF and BDNF) and adding PI3K inhibitor (Wortmannin). The results showed that circVLT deficiency led to impaired VZV latency and reactivation ( Figure 2 ), these results indicate that circVLT is a key factor in VZV latency-reactivation.

[0024] In some embodiments of the present invention, the deoxyribonucleotide or nucleotide sequence comprising the circVLT or VLT is as shown in seq.no.1 to seq.no.19 or has more than 70% similarity, and the circVLT or VLT translated amino acid sequence is as shown in seq.no.20 or has more than 70% similarity.

[0025]

[0026]

[0027]

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[0030]

[0031]

[0032] Note: The nucleotide sequence in the table is represented by ATGC. Those skilled in the art know that when the nucleotide is DNA, it is ATGC; when it is transcribed into mRNA, it is AUGC.

[0033] Example 2: circVLT deficiency leads to impaired VZV latent establishment and reactivation

[0034] 2.1 Overexpression of circVLT in SH-SY5Y cells and infection with VZV-△circVLT.

[0035] 1. The circVLT sequence was synthesized by gene synthesis and constructed into the lentiviral circular RNA expression vector PCDH. The second-generation lentiviral packaging system was used to package the lentivirus.

[0036] 2. SH-SY5Y cells overexpressing circVLT isoforms circVLT-419nt and circVLT-542nt were inoculated in 6-well plates, and infected with VZV-△circVLT virus when the cell density reached 80%. Cell pellets were collected 48 hours after virus infection. The expression of VLT and ORF61 was determined by qPCR.

[0037] 2.2 Experimental Results

[0038] Exons 3, 4, and 5 of VLT are derived from ORF61 ( Figure 1A), which has been reported to inhibit the expression of ORF61 gene in an antisense manner (Depledge et al., 2018). This project found that exons 2, 3, 4 and 5 of circVLT are derived from VLT. The interaction between RNAs, namely competing endogenous RNAs, is an important gene expression regulation pathway (Liu & Chen, 2022). VLT and circVLT are both derived from ORF61 and its surrounding sequences, and the three contain homologous sequences ( Figure 1 A). ORF61 is an immediate early gene of the virus. Its encoded protein pORF61 is a transcription factor of many viral genes and plays an important role in the early stage of VZV activation and replication. CircVLT isoforms 419nt and 542nt were overexpressed in SH-SY5Y cells using circRNA overexpression vectors. Figure 3 A). qPCR results showed that overexpression of circVLT could inhibit VLT and promote the expression of ORF61 ( Figure 3 BC). Therefore, circVLT and VLT are key factors regulating VZV latency-reactivation.

[0039] Example 3: circVLT combined with VZV neutralizing antibodies or anti-VZV drugs can eliminate latent VZV

[0040] 3.1 circVLT combined with VZV neutralizing antibodies or anti-VZV drugs to treat VZV latent model cells

[0041] Humans are the only host of VZV (Mahalinga et al., 2019). Currently, there is still a lack of a complete animal model for VZV latent reactivation. Therefore, this patent uses induced differentiated human neuroblastoma cells SH-SY5Y cells to infect the VZV latent model for experiments, and uses VZV antibodies gE, gN, and gI to simulate the process of neutralizing antibodies immunized against VZV by vaccine stimulation in vivo. VZV genomic DNA is linear in virus particles; but during the period of cell latency, it forms a circular form of genomic DNA by connecting end to end (Kinchington et al., 1985). Therefore, based on this characteristic, the end back-to-back primers (T1|T2) are designed to specifically detect the circular form of VZV genomic DNA ( Figure 4 C) Figure 4 As shown in A, this example refers to Example 1, using differentiated human neuroblastoma cells SH-SY5Y cells to infect VZV latent model, adding VZV antibodies gE, gN, gI respectively when VZV establishes latency to simulate the neutralizing antibody immunization VZV process stimulated by the vaccine in vivo, and transfecting the in vitro synthesized circVLT; Figure 4As shown in B, acyclovir was added and circVLT synthesized in vitro was transfected when VZV established latency; then the VZV circular virus genomic DNA content was detected before and after circVLT combined with VZV neutralizing antibody treatment.

[0042] 3.2 Experimental Results

[0043] like Figure 4 As shown in D, the content of latent VZV circular genomic DNA in the control group (transfected with circular GFP RNA) and the experimental group transfected with circVLT isoforms circVLT-419nt and circVLT-542nt was consistent before VZV antibody gE, gN, gI treatment; after VZV antibody gE, gN, gI combined with circVLT treatment, the content of latent VZV circular genomic DNA in SH-SY5Y cells was significantly reduced compared with the control group ( Figure 4 DE). Similar to the above, acyclovir combined with circVLT treatment can also significantly reduce the content of latent VZV circular genomic DNA in SH-SY5Y cells ( Figure 4 FG).

[0044] Based on the above experimental data, it can be seen that circVLT can induce latent VZV activation, and combined with VZV vaccine or anti-VZV drugs can eliminate viral particles lurking in neurons.

Claims

1. Application of VZV latency-associated transcripts (VLT / circVLT) in the treatment of varicella-zoster.

2. The use of VZV latency-associated transcript (VLT / circVLT) in the treatment of varicella zoster according to claim 1, characterized in that: VZV linear latency-associated transcripts (VLT) or homologous circular RNA (circVLT) are used to activate latent VZV, and VZV vaccines or antiviral drugs are used to eliminate the activated VZV, thereby achieving reactivation and elimination of latent VZV for the prevention or treatment of herpes zoster.

3. Use of the VZV latency-associated transcript (VLT / circVLT) according to claim 2 in the treatment of varicella-zoster, wherein the VZV latency-associated transcript comprises a linear RNA or homologous circular RNA transcribed between ORF60 and ORF64 during VZV latency and its encoded protein, and a linear RNA or homologous circular RNA transcribed between ORF60 and ORF64 during activation and its encoded protein or polypeptide.

4. Use of the VZV latency-associated transcript (VLT / circVLT) in the treatment of varicella-zoster according to claim 2, wherein the VZV vaccine comprises a varicella vaccine and a herpes zoster vaccine.

5. Use of VZV latency-associated transcripts (VLT / circVLT) in the treatment of varicella-zoster according to claim 2, wherein the antiviral drug comprises acyclovir and its analogs.

6. Use of the VZV latency-associated transcript (VLT / circVLT) in the treatment of varicella-zoster according to claim 3, wherein the varicella vaccine and the herpes zoster vaccine include live attenuated vaccines, mRNA vaccines, viral protein vaccines, and the like.

7. Use of the VZV latency-associated transcript (VLT / circVLT) according to any one of claims 1 to 6 in the treatment of varicella zoster, wherein the method reactivates latent VZV and eliminates it.

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