CRISPR antiviral inhibits neurotrophic JC polyomavirus in 2D and 3D culture models through dual-gRNA excision by SaCas9
Without an effective antiviral, JC virus (JCV) has persisted throughout multiple epochs of immunosuppression, causing the opportunistic demyelinating disease, progressive multifocal leukoencephalopathy (PML). This study proposes a novel therapy using a dual-gRNA, SaCas9, CRISPR antiviral targeting J...
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Elsevier
2025-06-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253125001106 |
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| author | Angela Rocchi Shuren Liao Hong Liu Chen Chen Senem Çakır Anna Bellizzi Hassen S. Wollebo Ilker K. Sariyer Kamel Khalili |
| author_facet | Angela Rocchi Shuren Liao Hong Liu Chen Chen Senem Çakır Anna Bellizzi Hassen S. Wollebo Ilker K. Sariyer Kamel Khalili |
| author_sort | Angela Rocchi |
| collection | DOAJ |
| description | Without an effective antiviral, JC virus (JCV) has persisted throughout multiple epochs of immunosuppression, causing the opportunistic demyelinating disease, progressive multifocal leukoencephalopathy (PML). This study proposes a novel therapy using a dual-gRNA, SaCas9, CRISPR antiviral targeting JCV transcription factor, large tumor antigen (LT-Ag), and capsid protein, viral protein 1 (VP1). This treatment was validated using traditional two-dimensional cell culture. A recombinant cell line was produced from SVG astrocytes (SVGA) via lentiviral inoculation and puromycin selection. Following infection, sanger sequencing identified uniform excision of the circular dsDNA genome of JCV, significantly reducing viral load per genomic copy number on qPCR, viral proteins on western blot, and infectivity of viral progeny on adoptive transfer. Following this proof-of-concept using cell lines, translatability of results was advanced using three-dimensional, heterogeneous cerebral organoids (COs). COs were infected and treated with the lentivirus-packaged CRISPR antiviral. As observed in monolayer culture, a truncated genome was confirmed with sequencing, reducing viral load per genomic copy number on qPCR, protein levels on immunofluorescent imaging, and infectivity on adoptive transfer. The high efficacy of this JCV-targeting CRISPR antiviral in the context of cerebral organoids expounds on its value for the currently untreatable JCV and PML. |
| format | Article |
| id | doaj-art-37fbaf33d4a64f51b89a8249e0e9cf54 |
| institution | DOAJ |
| issn | 2162-2531 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj-art-37fbaf33d4a64f51b89a8249e0e9cf542025-08-20T03:12:08ZengElsevierMolecular Therapy: Nucleic Acids2162-25312025-06-0136210255610.1016/j.omtn.2025.102556CRISPR antiviral inhibits neurotrophic JC polyomavirus in 2D and 3D culture models through dual-gRNA excision by SaCas9Angela Rocchi0Shuren Liao1Hong Liu2Chen Chen3Senem Çakır4Anna Bellizzi5Hassen S. Wollebo6Ilker K. Sariyer7Kamel Khalili8Department of Microbiology, Immunology and Inflammation, Center for Neurovirology and Gene Editing, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USADepartment of Microbiology, Immunology and Inflammation, Center for Neurovirology and Gene Editing, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USADepartment of Microbiology, Immunology and Inflammation, Center for Neurovirology and Gene Editing, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USADepartment of Microbiology, Immunology and Inflammation, Center for Neurovirology and Gene Editing, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USADepartment of Microbiology, Immunology and Inflammation, Center for Neurovirology and Gene Editing, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USADepartment of Microbiology, Immunology and Inflammation, Center for Neurovirology and Gene Editing, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USADepartment of Microbiology, Immunology and Inflammation, Center for Neurovirology and Gene Editing, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USADepartment of Microbiology, Immunology and Inflammation, Center for Neurovirology and Gene Editing, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USA; Corresponding author: Ilker K Sariyer, Department of Microbiology, Immunology and Inflammation, Center for Neurovirology and Gene Editing, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USA.Department of Microbiology, Immunology and Inflammation, Center for Neurovirology and Gene Editing, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USA; Corresponding author: Kamel Khalili, Department of Microbiology, Immunology and Inflammation, Center for Neurovirology and Gene Editing, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USA.Without an effective antiviral, JC virus (JCV) has persisted throughout multiple epochs of immunosuppression, causing the opportunistic demyelinating disease, progressive multifocal leukoencephalopathy (PML). This study proposes a novel therapy using a dual-gRNA, SaCas9, CRISPR antiviral targeting JCV transcription factor, large tumor antigen (LT-Ag), and capsid protein, viral protein 1 (VP1). This treatment was validated using traditional two-dimensional cell culture. A recombinant cell line was produced from SVG astrocytes (SVGA) via lentiviral inoculation and puromycin selection. Following infection, sanger sequencing identified uniform excision of the circular dsDNA genome of JCV, significantly reducing viral load per genomic copy number on qPCR, viral proteins on western blot, and infectivity of viral progeny on adoptive transfer. Following this proof-of-concept using cell lines, translatability of results was advanced using three-dimensional, heterogeneous cerebral organoids (COs). COs were infected and treated with the lentivirus-packaged CRISPR antiviral. As observed in monolayer culture, a truncated genome was confirmed with sequencing, reducing viral load per genomic copy number on qPCR, protein levels on immunofluorescent imaging, and infectivity on adoptive transfer. The high efficacy of this JCV-targeting CRISPR antiviral in the context of cerebral organoids expounds on its value for the currently untreatable JCV and PML.http://www.sciencedirect.com/science/article/pii/S2162253125001106MT: RNA/DNA EditingCRISPRantiviralgene editingJC virusprogressive multifocal leukoencephalopathy |
| spellingShingle | Angela Rocchi Shuren Liao Hong Liu Chen Chen Senem Çakır Anna Bellizzi Hassen S. Wollebo Ilker K. Sariyer Kamel Khalili CRISPR antiviral inhibits neurotrophic JC polyomavirus in 2D and 3D culture models through dual-gRNA excision by SaCas9 Molecular Therapy: Nucleic Acids MT: RNA/DNA Editing CRISPR antiviral gene editing JC virus progressive multifocal leukoencephalopathy |
| title | CRISPR antiviral inhibits neurotrophic JC polyomavirus in 2D and 3D culture models through dual-gRNA excision by SaCas9 |
| title_full | CRISPR antiviral inhibits neurotrophic JC polyomavirus in 2D and 3D culture models through dual-gRNA excision by SaCas9 |
| title_fullStr | CRISPR antiviral inhibits neurotrophic JC polyomavirus in 2D and 3D culture models through dual-gRNA excision by SaCas9 |
| title_full_unstemmed | CRISPR antiviral inhibits neurotrophic JC polyomavirus in 2D and 3D culture models through dual-gRNA excision by SaCas9 |
| title_short | CRISPR antiviral inhibits neurotrophic JC polyomavirus in 2D and 3D culture models through dual-gRNA excision by SaCas9 |
| title_sort | crispr antiviral inhibits neurotrophic jc polyomavirus in 2d and 3d culture models through dual grna excision by sacas9 |
| topic | MT: RNA/DNA Editing CRISPR antiviral gene editing JC virus progressive multifocal leukoencephalopathy |
| url | http://www.sciencedirect.com/science/article/pii/S2162253125001106 |
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