Nadia Amrani, Kevin Luk, Pankaj Singh, Mason Shipley, Meltem Isik, Martina Donadoni, Anna Bellizzi, Kamel Khalili, Ilker K. Sariyer, Donna Neumann, Jennifer Gordon, Guo-Xiang Ruan
{"title":"由单一 AAV9 载体传递的 CRISPR-Cas9 介导的基因组编辑可抑制潜伏兔角膜炎模型中的 HSV-1 再激活","authors":"Nadia Amrani, Kevin Luk, Pankaj Singh, Mason Shipley, Meltem Isik, Martina Donadoni, Anna Bellizzi, Kamel Khalili, Ilker K. Sariyer, Donna Neumann, Jennifer Gordon, Guo-Xiang Ruan","doi":"10.1016/j.omtm.2024.101303","DOIUrl":null,"url":null,"abstract":"Herpes simples virus 1 (HSV-1) keratitis is a major cause of blindness globally. During primary infection, HSV-1 travels to the trigeminal ganglia and establishes lifelong latency. Although some treatments can reduce symptom severity and recurrence, there is no cure for HSV-1 keratitis. We used CRISPR-Cas9 to co-target gene sequences encoding two essential HSV-1 proteins, ICP0 and ICP27, as a potential therapy for HSV-1 keratitis. In HSV-1-infected Vero cells, the HSV-1 viral load and titer were significantly reduced by plasmid transfection or AAV2 vector transduction expressing Cas9 nuclease from (SaCas9) and paired guide RNAs (gRNAs). Off-target assessment showed minimal off-target editing activity from the selected gRNAs. We then tested our CRISPR-Cas9 gene editing approach in a latent rabbit model of HSV-1 keratitis. Corneal scarification with all-in-one AAV8(Y733F)-SaCas9 or AAV9-SaCas9 vector reduced viral shedding by over 50%. Interestingly, intravenous administration of the same AAV9-SaCas9 vector eliminated viral shedding in 92% of treated eyes. In addition, treated trigeminal ganglia showed a reduction in HSV-1 DNA and RNA expression. Our results support the utility of single-dose AAV9 all-in-one CRISPR-Cas9 gene editing as a safe and effective strategy for treating HSV-1 keratitis.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.6000,"publicationDate":"2024-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"CRISPR-Cas9-mediated genome editing delivered by a single AAV9 vector inhibits HSV-1 reactivation in a latent rabbit keratitis model\",\"authors\":\"Nadia Amrani, Kevin Luk, Pankaj Singh, Mason Shipley, Meltem Isik, Martina Donadoni, Anna Bellizzi, Kamel Khalili, Ilker K. Sariyer, Donna Neumann, Jennifer Gordon, Guo-Xiang Ruan\",\"doi\":\"10.1016/j.omtm.2024.101303\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Herpes simples virus 1 (HSV-1) keratitis is a major cause of blindness globally. During primary infection, HSV-1 travels to the trigeminal ganglia and establishes lifelong latency. Although some treatments can reduce symptom severity and recurrence, there is no cure for HSV-1 keratitis. We used CRISPR-Cas9 to co-target gene sequences encoding two essential HSV-1 proteins, ICP0 and ICP27, as a potential therapy for HSV-1 keratitis. In HSV-1-infected Vero cells, the HSV-1 viral load and titer were significantly reduced by plasmid transfection or AAV2 vector transduction expressing Cas9 nuclease from (SaCas9) and paired guide RNAs (gRNAs). Off-target assessment showed minimal off-target editing activity from the selected gRNAs. We then tested our CRISPR-Cas9 gene editing approach in a latent rabbit model of HSV-1 keratitis. Corneal scarification with all-in-one AAV8(Y733F)-SaCas9 or AAV9-SaCas9 vector reduced viral shedding by over 50%. Interestingly, intravenous administration of the same AAV9-SaCas9 vector eliminated viral shedding in 92% of treated eyes. In addition, treated trigeminal ganglia showed a reduction in HSV-1 DNA and RNA expression. Our results support the utility of single-dose AAV9 all-in-one CRISPR-Cas9 gene editing as a safe and effective strategy for treating HSV-1 keratitis.\",\"PeriodicalId\":54333,\"journal\":{\"name\":\"Molecular Therapy-Methods & Clinical Development\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.6000,\"publicationDate\":\"2024-08-14\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular Therapy-Methods & Clinical Development\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1016/j.omtm.2024.101303\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"MEDICINE, RESEARCH & EXPERIMENTAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Therapy-Methods & Clinical Development","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1016/j.omtm.2024.101303","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MEDICINE, RESEARCH & EXPERIMENTAL","Score":null,"Total":0}
CRISPR-Cas9-mediated genome editing delivered by a single AAV9 vector inhibits HSV-1 reactivation in a latent rabbit keratitis model
Herpes simples virus 1 (HSV-1) keratitis is a major cause of blindness globally. During primary infection, HSV-1 travels to the trigeminal ganglia and establishes lifelong latency. Although some treatments can reduce symptom severity and recurrence, there is no cure for HSV-1 keratitis. We used CRISPR-Cas9 to co-target gene sequences encoding two essential HSV-1 proteins, ICP0 and ICP27, as a potential therapy for HSV-1 keratitis. In HSV-1-infected Vero cells, the HSV-1 viral load and titer were significantly reduced by plasmid transfection or AAV2 vector transduction expressing Cas9 nuclease from (SaCas9) and paired guide RNAs (gRNAs). Off-target assessment showed minimal off-target editing activity from the selected gRNAs. We then tested our CRISPR-Cas9 gene editing approach in a latent rabbit model of HSV-1 keratitis. Corneal scarification with all-in-one AAV8(Y733F)-SaCas9 or AAV9-SaCas9 vector reduced viral shedding by over 50%. Interestingly, intravenous administration of the same AAV9-SaCas9 vector eliminated viral shedding in 92% of treated eyes. In addition, treated trigeminal ganglia showed a reduction in HSV-1 DNA and RNA expression. Our results support the utility of single-dose AAV9 all-in-one CRISPR-Cas9 gene editing as a safe and effective strategy for treating HSV-1 keratitis.
期刊介绍:
The aim of Molecular Therapy—Methods & Clinical Development is to build upon the success of Molecular Therapy in publishing important peer-reviewed methods and procedures, as well as translational advances in the broad array of fields under the molecular therapy umbrella.
Topics of particular interest within the journal''s scope include:
Gene vector engineering and production,
Methods for targeted genome editing and engineering,
Methods and technology development for cell reprogramming and directed differentiation of pluripotent cells,
Methods for gene and cell vector delivery,
Development of biomaterials and nanoparticles for applications in gene and cell therapy and regenerative medicine,
Analysis of gene and cell vector biodistribution and tracking,
Pharmacology/toxicology studies of new and next-generation vectors,
Methods for cell isolation, engineering, culture, expansion, and transplantation,
Cell processing, storage, and banking for therapeutic application,
Preclinical and QC/QA assay development,
Translational and clinical scale-up and Good Manufacturing procedures and process development,
Clinical protocol development,
Computational and bioinformatic methods for analysis, modeling, or visualization of biological data,
Negotiating the regulatory approval process and obtaining such approval for clinical trials.