cIn herpes simplex virus 1 (HSV-1), binding clusters enriched in CTCF during latency have been previously identified. We hypothesized that CTCF binding to CTCF clusters in HSV-1 would be disrupted in a reactivation event. To investigate, CTCF occupation of three CTCF binding clusters in HSV-1 was analyzed following sodium butyrate (NaB)-and explant-induced reactivation in the mouse. Our data show that the CTCF domains positioned within the HSV-1 genome, specifically around the latency-associated transcript (LAT) and ICP0 and ICP4 regions of the genome, lose CTCF occupancy following the application of reactivation stimuli in wild-type virus. We also found that CTCF binding clusters upstream of the ICP0 and ICP4 promoters both function as classical insulators capable of acting as enhancer blockers of the LAT enhancer. Finally, our results suggest that CTCF occupation of domains in HSV-1 may be differentially regulated both during latency and at early times following reactivation by the presence of lytic transcripts and further implicate epigenetic regulation of HSV-1 as a critical component of the latency-reactivation transition.T he human alphaherpesvirus herpes simplex virus 1 (HSV-1), infects sensory neurons, where it establishes a lifelong latent infection as a circular episome associated with histones (6,16,38,40,44). During HSV-1 latency, the latency-associated transcript (LAT) is abundantly transcribed, and lytic regions are, in essence, silent (6,38,40). Recent studies have shown that there are epigenetic components involved in HSV-1 latency and in reactivation (1,13,21,31,36,39). For example, during latency, the LAT regions of the LAT reactivation-critical promoter and LAT 5= exon (containing the LAT enhancer) (8) are enriched in euchromatic histone markers compared to the promoters of the immediateearly (IE) ICP0, ICP4, and ICP27 genes (1,13,21,24,31). Further, these permissive marks established on the LAT are independent of LAT transcription (24). Additional reports indicated that the LAT region of HSV-1 also contains facultative heterochromatin (12, 25), a form of repressed chromatin that can convert to euchromatin via posttranslational histone tail modifications. Consequently, the nearby IE regions are also enriched with repressive histone marks that have been characterized predominantly as facultative heterochromatin (12,25,26,45). These key findings provide evidence that HSV-1 latency is established and maintained, at least in part, by complex epigenetic mechanisms that further poise the virus for reactivation. To support this, recent data show that the enrichments of euchromatic histone marks on the HSV-1 LAT and IE promoters change in response to reactivation stimuli in both rabbit and mouse models latently infected with wild-type HSV-1 (1, 13, 26, 31, 36). Specifically, histone marks on ICP0 and ICP4 rapidly and transiently become more euchromatic in nature, while the LAT region loses enrichment of euchromatic histone marks as LAT is degraded at early times in reactivation in the two different in...

There are seven conserved CTCF binding domains in the herpes simplex virus 1 (HSV-1) genome. These binding sites individually flank the latency-associated transcript (LAT) and the immediate early (IE) gene regions, suggesting that CTCF insulators differentially control transcriptional domains in HSV-1 latency. In this work, we show that two CTCF binding motifs in HSV-1 display enhancer blocking in a cell-type-specific manner. We found that CTCF binding to the latent HSV-1 genome was LAT dependent and that the quantity of bound CTCF was site specific. Following reactivation, CTCF eviction was dynamic, suggesting that each CTCF site was independently regulated. We explored whether CTCF sites recruit the polycomb-repressive complex 2 (PRC2) to establish repressive domains through a CTCF-Suz12 interaction and found that Suz12 colocalized to the CTCF insulators flanking the ICP0 and ICP4 regions and, conversely, was removed at early times postreactivation. Collectively, these data support the idea that CTCF sites in HSV-1 are independently regulated and may contribute to lytic-latent HSV-1 control in a site-specific manner. The role of chromatin insulators in DNA viruses is an area of interest. It has been shown in several beta- and gammaherpesviruses that insulators likely control the lytic transcriptional profile through protein recruitment and through the formation of three-dimensional (3D) chromatin loops. The ability of insulators to regulate alphaherpesviruses has been understudied to date. The alphaherpesvirus HSV-1 has seven conserved insulator binding motifs that flank regions of the genome known to contribute to the establishment of latency. Our work presented here contributes to the understanding of how insulators control transcription of HSV-1.

Glaucoma, a leading cause of irreversible blindness, affects more than 64 million people worldwide and is expected to grow in number due to the aging global population and enhanced methods of detection. Although topical therapies are often effective when used as prescribed, the drawbacks of current medical management methods include poor patient adherence, local and systemic side effects, and in some cases, limited therapeutic efficacy. Novel ocular drug delivery platforms promise to deliver differentiated drug formulations with targeted delivery leveraging patient-independent administration. Several platforms are in various stages of development with promising pre-clinical and clinical data. The Bimatoprost Sustained Release (SR) intracameral implant was approved in the United States in March of 2020, making it the first long-term injectable therapy available for the treatment of glaucoma. This review aims to provide an update on novel sustained release drug delivery systems that are available today as well as those that might be commercialized in coming years.

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Following infection of epithelial tissues, herpes simplex virus 1 (HSV-1) virions travel via axonal transport to sensory ganglia and establish a lifelong latent infection within neurons. Recent studies have revealed that, following intraganglionic or intrathecal injection, recombinant adeno-associated virus (rAAV) vectors can also infect sensory neurons and are capable of stable, longterm transgene expression. We sought to determine if application of rAAV to peripheral nerve termini at the epithelial surface would allow rAAV to traffic to sensory ganglia in a manner similar to that seen with HSV. We hypothesized that footpad or ocular inoculation with rAAV8 would result in transduction of dorsal root ganglia (DRG) or trigeminal ganglia (TG), respectively. To test this, we inoculated the footpads of mice with various amounts of rAAV as well as rAAV capsid mutants. We demonstrated that this method of inoculation can achieve a transduction rate of >90% of the sensory neurons in the DRG that innervate the footpad. Similarly, we showed that corneal inoculation with rAAV vectors in the rabbit efficiently transduced >70% of the TG neurons in the optic tract. Finally, we demonstrated that coinfection of mouse footpads or rabbit eyes with rAAV vectors and HSV-1 resulted in colocalization in nearly all of the HSV-1-positive neurons. These results suggest that rAAV is a useful tool for the study of HSV-1 infection and may provide a means to deliver therapeutic cargos for the treatment of HSV infections or of dysfunctions of sensory ganglia. IMPORTANCEAdeno-associated virus (AAV) has been shown to transduce dorsal root ganglion sensory neurons following direct intraganglionic sciatic nerve injection and intraperitoneal and intravenous injection as well as intrathecal injection. We sought to determine if rAAV vectors would be delivered to the same sensory neurons that herpes simplex virus (HSV-1) infects when applied peripherally at an epithelial surface that had been treated to expose the underlying sensory nerve termini. For this study, we chose two well-established HSV-1 infection models: mouse footpad infection and rabbit ocular infection. The results presented here provide the first description of AAV vectors transducing neurons following delivery at the skin/epithelium/eye. The ability of AAV to cotransduce HSV-1-infected neurons in both the mouse and the rabbit provides an opportunity to experimentally explore and disrupt host and viral proteins that are integral to the establishment of HSV-1 latency, to the maintenance of latency, and to reactivation from latency in vivo. Herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection within the neurons of sensory ganglia. These ganglia are highly specialized structures composed of a diverse assemblage of neuronal and nonneuronal cells. Ganglionic neurons detect a wide variety of sensory inputs, including temperature, touch, and pain, and relay this information into the central nervous system (CNS). Immunohistochemical analyses of infected trigeminal g...