CRISPR-Cas9-mediated genome editing delivered by a single AAV9 vector inhibits HSV-1 reactivation in a latent rabbit keratitis model. Molecular therapy. Methods & clinical development 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. 10.1016/j.omtm.2024.101303

A W1282X cystic fibrosis mouse allows the study of pharmacological and gene-editing therapeutics to restore CFTR function. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society BACKGROUND:People with cystic fibrosis carrying two nonsense alleles lack CFTR-specific treatment. Growing evidence supports the hypothesis that nonsense mutation identity affects therapeutic response, calling for mutation-specific CF models. We describe a novel W1282X mouse model and compare it to an existing G542X mouse. METHODS:The W1282X mouse was created using CRISPR/Cas9 to edit mouse Cftr. In this model, Cftr transcription was assessed using qRT-PCR and CFTR function was measured in the airway by nasal potential difference and in the intestine by short circuit current. Growth, survival, and intestinal motility were examined as well. Correction of W1282X CFTR was assessed pharmacologically and by gene-editing using a forskolin-induced swelling (FIS) assay in small intestine-derived organoids. RESULTS:Homozygous W1282X mice demonstrate decreased Cftr mRNA, little to no CFTR function, and reduced survival, growth, and intestinal motility. W1282X organoids treated with various combinations of pharmacologic correctors display a significantly different amount of CFTR function than that of organoids from G542X mice. Successful gene editing of W1282X to wildtype sequence in intestinal organoids was achieved leading to restoration of CFTR function. CONCLUSIONS:The W1282X mouse model recapitulates common human manifestations of CF similar to other CFTR null mice. Despite the similarities between the congenic W1282X and G542X models, they differ meaningfully in their response to identical pharmacological treatments. This heterogeneity highlights the importance of studying therapeutics across genotypes. 10.1016/j.jcf.2024.10.008

Precision medicine advances in cystic fibrosis: Exploring genetic pathways for targeted therapies. Life sciences Personalized medicine has transformed the treatment of cystic fibrosis (CF), providing customized therapeutic approaches based on individual genetic profiles. This review explores the genetic foundations of CF, focusing on mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and their implications for the development of the disease. The advent of genetic testing has enabled the association of specific mutations to disease severity, leading to the development of CFTR modulators like Ivacaftor, Lumacaftor, and Tezacaftor. Beyond CFTR mutations, genetic modifiers, including gene replacement therapy, genetic manipulation, lentivirus, and non-viral gene therapy formulations, along with environmental factors, play critical roles in influencing disease expression and outcomes. The identification of these modifiers is essential for optimizing therapeutic strategies. Emerging biomarkers, including inflammatory markers and pulmonary function indicators, aid in early disease detection and monitoring progression. Omics technologies are uncovering novel biomarkers, enabling more precise disease management. Pharmacogenomics has become integral to CF care, allowing for personalized approaches that consider genetic variations influencing drug metabolism, especially in antibiotics and anti-inflammatory therapies. The future of CF treatment lies in precision therapies, including CFTR modulators and cutting-edge techniques like gene therapy and CRISPR-Cas9 for mutation correction. As research evolves, these advances can improve patient outcomes while minimizing adverse effects. Ethical considerations and regulatory challenges remain critical as personalized medicine advances, ensuring equitable access and the long-term effectiveness of these innovative therapies. 10.1016/j.lfs.2024.123186