Abstract
We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.
Highlights
We evaluate gene editing of Herpes simplex virus (HSV) in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection
We took advantage of a relatively simple mouse model of HSV infection to perform a set of iterative studies to increase the efficiency of AAV-delivered gene editing enzymes targeting HSV
While HSV does not reactivate spontaneously from ganglia of living mice, the virus does reactivate from mouse neurons after explantation, and our results demonstrate 95% (SCG) to 55% (TG) reduction in viral genomes produced de novo in ganglionic explants after meganuclease treatment of latently infected mice
Summary
We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. We show that AAV-delivered meganucleases, but not CRISPR/Cas[9], mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Gene editing using CRISPR/Cas[9], meganucleases, or similar enzymes offers the possibility of directly targeting latent genomes for disruption or elimination while preserving neurons, eliminating the possibility of viral reactivation and pathogenesis[13,14,15,16,17,18,19,20]. In vivo gene editing of latent HSV genomes within TG sensory neurons of mice has been previously demonstrated using HSV-specific meganucleases delivered via adenoassociated virus (AAV) vectors, but the levels of gene editing were modest (
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