Targeting herpes simplex virus with CRISPR–Cas9 cures herpetic stromal keratitis in mice

Yin, Di; Ling, Sikai; Wang, Dawei; Dai, Yue; Jiang, Hao; Zhou, Xujiao; Paludan, Søren R; Hong, Jiaxu; Cai, Yujia

doi:10.1038/s41587-020-00781-8

Cited by 108 publications

(104 citation statements)

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“…CRISPR/Cas9, besides being a versatile tool for research, also holds promising therapeutic potential for the treatment of persistent virus infections, as illustrated by the application to excise the integrated SIV genome in vivo [46,47]. Additionally, several studies showed successful targeting of herpesvirus genomes with either CRISPR/Cas9 or other programmable endonucleases, such as reducing latent EBV [14,48], latent KSHV [49], quiescent and latent HSV-1 genomes in vitro and in vivo [13,50] and, importantly, reducing herpes keratitis occurrence in vivo [23]. In this study, we showed that in vitro editing of VZV genomes during lytic infection is feasible.…”

Section: Discussionmentioning

confidence: 99%

“…2.12. Reconstitution of pOka-VLTM1I and pOka-VLTM1IR Viruses from BAC Genomes VZV strain pOka-based VLTM1I and its repaired VLTM1IR recombinant viruses were reconstituted in MeWo cells by transfection of pOka-BACVLTM1I and pOka-BACVLTM1IR genomes using Lipofectamine2000 (Thermo Fisher Scientific) and the BAC cassette within the reconstituted viruses was subsequently removed in MeWo-Cre cells [23,24].…”

Section: Generation Of Bacterial Artificial Chromosome (Bac) Mutant and Repaired Genomes By Redα/β-mediated Linear Recombinationmentioning

confidence: 99%

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Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection

Braspenning

Lebbink

Depledge

et al. 2021

Viruses

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Primary varicella-zoster virus (VZV) infection leads to varicella and the establishment of lifelong latency in sensory ganglion neurons. Reactivation of latent VZV causes herpes zoster, which is frequently associated with chronic pain. Latent viral gene expression is restricted to the VZV latency-associated transcript (VLT) and VLT-ORF63 (VLT63) fusion transcripts. Since VLT and VLT63 encode proteins that are expressed during lytic infection, we investigated whether pVLT and pVLT-ORF63 are essential for VZV replication by performing VZV genome mutagenesis using CRISPR/Cas9 and BAC technologies. We first established that CRISPR/Cas9 can efficiently mutate VZV genomes in lytically VZV-infected cells through targeting non-essential genes ORF8 and ORF11 and subsequently show recovery of viable mutant viruses. By contrast, the VLT region was markedly resistant to CRISPR/Cas9 editing. Whereas most mutants expressed wild-type or N-terminally altered versions of pVLT and pVLT-ORF63, only a minority of the resulting mutant viruses lacked pVLT and pVLT-ORF63 coding potential. Growth curve analysis showed that pVLT/pVLT-ORF63 negative viruses were viable, but impaired in growth in epithelial cells. We confirmed this phenotype independently using BAC-derived pVLT/pVLT-ORF63 negative and repaired viruses. Collectively, these data demonstrate that pVLT and/or pVLT-ORF63 are dispensable for lytic VZV replication but promote efficient VZV infection in epithelial cells.

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Section: Discussionmentioning

confidence: 99%

Section: Generation Of Bacterial Artificial Chromosome (Bac) Mutant and Repaired Genomes By Redα/β-mediated Linear Recombinationmentioning

confidence: 99%

Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection

Braspenning

Lebbink

Depledge

et al. 2021

Viruses

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“…1a) 26 . We have previously shown delivery of Cas9 mRNA with lentivirus-derived VLP mediated efficient genome editing in vivo in the different disease models 27,28 , however, it is unclear if VLP can serve as a vaccine platform. To package the full-length spike mRNA into VLPs, we inserted MS2 stem-loop repeats in its 3' terminus between the stop codon and the polyA signal.…”

Section: Design and Characterization Of Vlp-based Sars-cov-2 Mrna Vaccinementioning

confidence: 99%

Dendritic cell targeting virus-like particle delivers mRNA for in vivo immunization

Yin

Ling

Tian

et al. 2021

Preprint

Self Cite

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mRNA vaccine was approved clinically in 2020. Future development includes delivering mRNA to dendritic cells (DCs) specifically to improve effectiveness and avoid off-target cytotoxicity. Here, we developed virus-like particles (VLPs) as a DC tropic mRNA vaccine vector and showed the prophylactic effects in both SARS-CoV-2 and HSV-1 infection models. The VLP mRNA vaccine elicited strong cytotoxic T cell immunity and durable antibody response with the spike-specific antibodies that lasted for more than 9 months. Importantly, we were able to target mRNA to DCs by pseudotyping VLP with engineered Sindbis virus glycoprotein and found the DC-targeting mRNA vaccine significantly enhanced the titer of antigen-specific IgG, protecting the hACE-2 mice from SARS-CoV-2 infection. Additionally, we showed DC-targeted mRNA vaccine also protected mice from HSV-1 infection when co-delivering the gB and gD mRNA. Thus, the VLP may serve as an in situ DC vaccine and accelerate the further development of mRNA vaccines.

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“…Previous studies focused on the regulatory network in non-CNS macrophages but provided a limited understanding of the regulatory mechanism for the innate antiviral response in CNS. Herpes simplex virus type 1 (HSV-1) is the most common human neurotropic viruses, infection of which causes numerous diseases, including herpes simplex encephalitis (HSE) that has a high mortality rate if left untreated, and herpetic stromal keratitis (HSK) is the leading cause of infectious blindness 14 , 15 . Moreover, the close association between HSV-1 and Alzheimer's disease has been gradually recognized 16 , although the underlying mechanism remains obscure.…”

Section: Introductionmentioning

confidence: 99%

A novel lncRNA linc-AhRA negatively regulates innate antiviral response in murine microglia upon neurotropic herpesvirus infection

et al. 2021

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Microglia are the primary cellular source of type I interferons (I-IFNs) in the brain upon neurotropic virus infection. Although the I-IFN-based antiviral innate immune response is crucial for eliminating viruses, overproduction led to immune disorders. Therefore, the relatively long-lasting I-IFNs must be precisely controlled, but the regulatory mechanism for the innate antiviral response in microglia remains largely unknown. Long non-coding RNAs (lncRNAs) are being recognized as crucial factors in numerous diseases, but their regulatory roles in the innate antiviral response in microglia are undefined. Methods: The high-throughput RNA sequencing was performed to obtain differentially expressed lncRNAs (DELs) in primary microglia infected with or without the neurotropic herpes simplex virus type 1 (HSV-1). We selected four DELs ranked in the top 15 in basic level and their fold change induced by HSV-1, i.e., FPKM HSV-1 /FPKM Cells .We subsequently found a key lncRNA affecting the innate antiviral response of microglia significantly. We next used dual-luciferase reporter assays, bioinformatical tools, and truncation mutants of both lncRNA and targeted proteins to elucidate the downstream and upstream mechanism of action of lncRNA. Further, we established microglia-specific knock-in (KI) mice to investigate the role of lncRNA in vivo . Results: We identified a long intergenic non-coding RNA, linc-AhRA, involved in regulating the innate antiviral response in murine microglia. linc-AhRA is activated by aryl hydrocarbon receptor (AhR) and restricts I-IFN production in microglia upon neurotropic herpesvirus infection and innate immune stimulation. Mechanistically, linc-AhRA binds to both tripartite motif-containing 27 (TRIM27) and TANK-binding kinase 1 (TBK1) through its conserved 117nt fragment as a molecular scaffold to enhance TRIM27-TBK1 interaction. This interaction facilitates the TRIM27-mediated ubiquitination of TBK1 and results in ubiquitin-proteasome-dependent degradation of TBK1. Consequently, linc-AhRA suppresses I-IFN production through facilitating TBK1 degradation and limits the microglial innate immune response against neurotropic herpesvirus infection. Microglia-specific KI of linc-AhRA mice shows a weakened antiviral immune response upon neurotropic herpesvirus challenge due to a reduction of TBK1 in microglia. Conclusion: Our findings indicate that linc-AhRA is a negative regulator of I-IFN production in microglia to avoid excessive autoimmune responses. These findings uncover a previously unappreciated role for lncRNA conserved fragments in the innate antiviral response, providing a strong foundation for developing nucleotide drugs based on conserved functional fragments within lncRNAs.

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Targeting herpes simplex virus with CRISPR–Cas9 cures herpetic stromal keratitis in mice

Cited by 108 publications

References 50 publications

Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection

Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection

Dendritic cell targeting virus-like particle delivers mRNA for in vivo immunization

A novel lncRNA linc-AhRA negatively regulates innate antiviral response in murine microglia upon neurotropic herpesvirus infection

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