In vivo disruption of latent HSV by designer endonuclease therapy
Martine Aubert, Emily A. Madden, Michelle Loprieno, Harshana S. DeSilva Feelixge, Laurence Stensland, Meei-Li Huang, Alexander L. Greninger, Pavitra Roychoudhury, Nixon Niyonzima, Thuy Nguyen, Amalia Magaret, Roman Galleto, Daniel Stone, Keith R. Jerome
Martine Aubert, Emily A. Madden, Michelle Loprieno, Harshana S. DeSilva Feelixge, Laurence Stensland, Meei-Li Huang, Alexander L. Greninger, Pavitra Roychoudhury, Nixon Niyonzima, Thuy Nguyen, Amalia Magaret, Roman Galleto, Daniel Stone, Keith R. Jerome
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Research Article Infectious disease

In vivo disruption of latent HSV by designer endonuclease therapy

Abstract

A large portion of the global population carries latent herpes simplex virus (HSV), which can periodically reactivate, resulting in asymptomatic shedding or formation of ulcerative lesions. Current anti-HSV drugs do not eliminate latent virus from sensory neurons where HSV resides, and therefore do not eliminate the risk of transmission or recurrent disease. Here, we report the ability of HSV-specific endonucleases to induce mutations of essential HSV genes both in cultured neurons and in latently infected mice. In neurons, viral genomes are susceptible to endonuclease-mediated mutagenesis, regardless of the time of treatment after HSV infection, suggesting that both HSV lytic and latent forms can be targeted. Mutagenesis frequency after endonuclease exposure can be increased nearly 2-fold by treatment with a histone deacetylase (HDAC) inhibitor. Using a mouse model of latent HSV infection, we demonstrate that a targeted endonuclease can be delivered to viral latency sites via an adeno-associated virus (AAV) vector, where it is able to induce mutation of latent HSV genomes. These data provide the first proof-of-principle to our knowledge for the use of a targeted endonuclease as an antiviral agent to treat an established latent viral infection in vivo.

Authors

Martine Aubert, Emily A. Madden, Michelle Loprieno, Harshana S. DeSilva Feelixge, Laurence Stensland, Meei-Li Huang, Alexander L. Greninger, Pavitra Roychoudhury, Nixon Niyonzima, Thuy Nguyen, Amalia Magaret, Roman Galleto, Daniel Stone, Keith R. Jerome

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Figure 2

In vitro–targeted mutagenesis in neurons isolated from HSV-infected mice.

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In vitro–targeted mutagenesis in neurons isolated from HSV-infected mice...
(A) Schematic of neuronal culture generation. Five mice were infected with 2 × 105 PFU of HSV-1(F) in the right eye following corneal scarification for each time point and left for 7, 14, or 32 days prior to collection of the right TGs. After tissue dissociation by enzymatic digestion, neuronal cultures were established, and cells were cultured in medium supplemented with 100 μM ACV. (B) Experimental timeline. Cells were cotransduced in quadruplicate with either scAAV8-sCMV-HSV1m5 and scAAV8-sCMV-Trex2 or scAAV8-sCMV-HSV1m8 and scAAV8-sCMV-Trex2 at an MOI of 1 × 106 vg/AAV/neuron for 3 days prior to analysis. (C) Timeline for the evaluation of the effect of TSA. Nine mice were infected with 2 × 105 PFU in the right eye following corneal scarification and, 7 days later, the right TGs were collected. After tissue dissociation by enzymatic digestion, neuronal cultures were established and cells were cultured in medium supplemented with 100 μM ACV. Eight wells per condition were cotransduced with scAAV8 vectors expressing HSV1m5 and Trex2, HSV1m8 and Trex2, NV1 and Trex2, or GFP and mCherry at an MOI of 1 × 106 vg/AAV/neuron for 3 days, after which 4 wells were left untreated while the other 4 were treated with 300 nM TSA for 1 day prior to analysis. (D) Mutagenic event detection by T7E1 assay. The HSV regions containing the target site were PCR amplified from total genomic DNA obtained from pooled quadruplicate wells, subjected to T7E1 digest, and separated on a 3% agarose gel. mw, molecular weight size ladder; red asterisks indicate cleavage products. G, GFP ; NV, NV1; mC, mCherry; T, Trex2; M8, HSV1m8, m5, HSV1m5; T7, T7E1. (E) HSV target sequences from enzyme-treated neurons with and without TSA. ACV, Acyclovir.