Herpes simplex virus 1 (HSV-1) is a ubiquitous viral pathogen that, while rarely lethal, nevertheless has the ability to cause severe morbidity in a subset of patients. Among the more serious potential consequences of HSV-1 infection is the development of HSV-1 keratitis, the most common cause of infectious blindness in the USA, with ~35,000 new cases each year. Currently available drugs do not effectively treat HSV-1 keratitis, and a new treatment modality is therefore clearly needed. In this grant application, we propose to optimize the bacterial CRISPR/Cas mechanism of adaptive antiviral immunity in order to efficiently cleave and inactivate latent HSV-1 genomes in neuronal cells in vivo. Our initial goal is to develop vectors based on adeno-associated virus (AAV), which has been widely used for gene therapy in humans, to express the novel smaller Cas9 protein recently identified in Staphylococcus aureus (Sau), together with small guide RNAs (sgRNAs) specific for HSV-1. After optimization and demonstration of function in cultured cells, these AAV vectors will be introduced into mice containing trigeminal ganglia latently infected by HSV-1 and the potential reduction in viral DNA load and loss of reactivation potential assayed. In this way, we hope to provide proof-of-principle for the idea that CRISPR/Cas systems can be repurposed as a novel tool for the effective elimination of latent HSV-1 DNA genomes in vivo.
Public Health Relevance Statement: Public Health Relevance: Herpes simplex virus 1 (HSV-1) is a ubiquitous pathogen that is responsible for substantial morbidity in a subset of patients, including HSV-1 keratitis, the most prevalent form of infectious disease leading to blindness. Here, we propose using viral vectors to deliver the recently developed CRISPR/Cas system of RNA-guided DNA endonucleases to infected cells in order to cleave and destroy HSV-1 DNA episomes in vivo.
Project Terms: 3' Flanking Region; adeno-associated viral vector; Afferent Neurons; Animal Model; Antiviral Agents; Applications Grants; Axon; Axonal Transport; Bacteriophages; base; Biological Assay; Blindness; Cells; Chromosomes; Cleaved cell; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Communicable Diseases; corneal scar; Cultured Cells; Deoxyribonuclease I; Dependovirus; Development; DNA; DNA Polymerase II; DNA Polymerase III; DNA Sequence; Episome; Epithelium; Eye; Family; Gene Silencing; gene therapy; Gene Transduction Agent; Generations; Genes; Genetic Screening; Genetic Transcription; Genome; Goals; Green Fluorescent Proteins; Guide RNA; Herpes Labialis; Herpesvirus 1, Human; Human; Human body; human disease; Human Genome; Immune; Immune response; Immunity; in vivo; Individual; Infection; Inverted Terminal Repeat; Keratitis; knockout gene; latent infection; Modality; Modeling; Monkeys; Morbidity - disease rate; Mus; Mutate; Neurons; novel; Nuclear; Oral cavity; Pain; pathogen; Patients; Pharmaceutical Preparations; prevent; Promotor (Genetics); protein expression; Proteins; public health relevance; purine; Purines; Refractory; Research; response; retrograde transport; RNA Polymerase III; RNA Sequences; Safety; Site; Skin; Small RNA; Staphylococcus aureus; Streptococcus pyogenes; Stress; Structure of trigeminal ganglion; System; Tissues; tool; transcriptome sequencing; Travel; Trigeminal nerve structure; vector; Viral; viral DNA; Viral Vector; Virion; Virus
