May 8, 2013

Update on HSV Research

Almost all of our research on HSV-1 is now done in collaboration with Prof. David Bloom at the University of Florida. Dr. Bloom is a world leader in studying HSV pathogenesis and latency in animal models and this skill set makes his laboratory a natural partner for my research group, which focuses on molecular virology and analysis of gene expression.

We continue to work on different aspects of HSV infection, with a view to developing treatments for HSV that have the potential to cure, not just ameliorate, this infection, as currently available drugs do. My group is currently focused on two main areas of research relevant to HSV.

1) The role of virally encoded microRNAs in maintaining latent HSV infections

Some time ago, my laboratory showed that HSV-1 and HSV-2 encode viral microRNAs, small regulatory RNAs that, among other things, repress the expression of HSV regulatory proteins that function to induce viral productive replication, i.e., cold sores. We therefore suggested that these HSV microRNAs function to stabilize viral latency and that inhibition of microRNA function might induce viruses to leave the latent state. Simultaneous treatment with antiviral drugs might then prove able to purge the viral reservoir. Working with the Bloom laboratory, we have now generated viral mutants lacking several viral microRNAs and we are in the process of examining how this affects viral replication. In a paper that just appeared in the Journal of Virology (Flores et al., 2013), we demonstrate that viral microRNAs indeed repress viral protein expression in infected cells and that viruses lacking these microRNAs are indeed more able to actively proliferate in cultured neuronal cells. These experiments are currently being extended to animal models in the Bloom laboratory. The good news is that our hypothesis, that is, that viral microRNAs facilitate viral latency, appears to be correct. The bad news is that loss of viral microRNAs does not seem to be sufficient to fully block viral entry into latency. We therefore are currently somewhat less optimistic that targeting viral microRNAs would be likely to be a good treatment approach, with the potential to cure HSV latency. However, we are continuing to work on this question with the Bloom laboratory.

2) Direct destruction of the HSV DNA genome

In latently infected cells, the HSV DNA genome forms a small extrachromosomal circle that is highly stable. This chromosomal circle does not express any viral proteins, only the microRNAs described above and a large viral non-coding RNA, called the latency associated transcript (LAT), which is of uncertain function. Because the latent HSV genome expresses very few RNAs, most of this genome is densely covered with proteins—so-called heterochromatin—and is essentially inaccessible. Only a small portion, near where the microRNAs and LAT are transcribed, is in an open conformation, so-called euchromatin. Recently, the Bloom laboratory has carefully mapped the open, accessible areas of the HSV genome in latently infected cells.

This information is now especially valuable because research focusing on bacteria in plants (basic research is important!) has identified proteins that act as programmable DNA cleavage enzymes. Essentially these are proteins that can be designed to specifically destroy a particular DNA in a cell while leaving all other DNA, e.g., the human genome, entirely untouched. We have focused on developing several types of DNA-specific “smart bombs”, and we have now developed smart bombs that efficiently cleave and destroy the HSV genome in cultured live cells without causing any cellular toxicity.

While the Cullen lab was developing these DNA sequence-specific smart bombs, the Bloom lab has been developing gene delivery vehicles, essentially acting like “guided missiles.” Their approach is based on using a type of innocuous virus called Adeno Associated Virus (AAV), as a delivery vehicle or “viral vector.” AAV has been widely used in clinical trials in humans for the delivery of genes able to treat a range of genetic diseases, and large scale facilities for the production of high-titer, clinical trial-grade AAV vectors exist both in North Carolina, near Duke, and in Florida, near UFL. The Bloom lab has now figured out a way to infect up to ~99% of all neurons in the trigeminal ganglion in mice with an AAV which has been gutted and the contents replaced with a protein that can be easily traced because it glows green. Now that the guided missile is ready, we are replacing this “green gene” with the various smart bombs and we hope to soon test whether one or more of these will destroy the HSV-1 genome in latently infected neurons in mice. We are very excited about this research, as it offers the potential to directly destroy HSV DNA genomes and clear latent viral infections. Please note that these studies are at an early stage in mice. We expect that pre-clinical development will take at least two years before we can even envision starting clinical trials, assuming that we continue to make good progress. Moreover, we would require substantial sums to generate the AAV vectors required for a clinical trial, which likely will require a partnership with a biotech or pharmaceutical company. However, this approach really does offer a potential way forward and we are cautiously optimistic.

Frequently Asked Questions

Thank you for your interest in our research. We continue to receive a considerable number of inquiries on the report we published in 2008 in the scientific journal Nature. This report received extensive press coverage, some of it misleading. We will briefly answer some of the most common questions that we have been asked, and particularly how our work relates to a new treatment for cold sores, caused by Herpes Simplex Virus 1 (HSV-1), or genital ulcers, caused by the related but distinct HSV-2.

1) What have you accomplished?

Our work provides, for the first time, a molecular understanding of how HSV-1 establishes a life-long latent infection in the nerve cells of the face, and how it reactivates from latency to cause cold sores.

2) Have you developed a new treatment for cold sores?

The work we have performed provides a basis for the development of anti-HSV-1 and anti-HSV-2 drugs that might be able to permanently clear these viruses from patients.

3) Are these drugs being used on people?

No, but we hope to initiate trials in mice to study efficacy and toxicity in the near future. Due to limited funding, we are currently focused on providing more evidence in support of our hypothesis that viral reactivation can be regulated.

4) When might this drug reach the clinical trial stage?

We anticipate several years of animal experiments in mice followed by approximately 1-2 years of toxicity studies in other animals, followed by small studies in healthy volunteers. This timeline is dependent on obtaining additional research funding, which we have not yet succeeded in doing. After that, assuming things go well, this drug might proceed to clinical trials in HSV-1 infected individuals. It is very possible that the
drug candidate might fall out at any of these stages, due to lack of effectiveness or some unanticipated side effect.

5) How does this relate to HSV-2, which causes genital herpes?

Most of our work so far has been on HSV-1, the cold sore virus, but HSV-2 is quite closely related. We are endeavoring to see if the lessons we have learned in HSV-1 also apply to HSV-2, and our recent results indicate that this is indeed the case (Umbach et al., Journal of Virology, 84, pp. 1189-1192, 2010). We hope to eventually start animal trials for an anti-HSV-2 drug, depending on the results with HSV-1 and our financial resources. Our initial data indicate that HSV-1 and HSV-2 may respond to distinct, but similar, drug therapies.

6) Would you be willing to accept a financial contribution to be used for your research focusing on HSV-1 and HSV-2?

Funding is a major constraint on our ability to advance the research, so I am therefore pleased to tell you that the National Institutes of Health has recently awarded a five-year research grant to my laboratory and that of our collaborator Prof. David Bloom at the University of Florida, which will commence in December of this year. This is in addition to a research grant to my lab from a major pharmaceutical company to support our work on herpes that will give us $80,000 a year for two years. This is still far short of what we would need to go to clinical trials, that eventually emerges from our work would not be available unless it is marketed by a company.  We are hopeful that additional experimental evidence further validating our model for the regulation of HSV-1 and HSV-2 latency will eventually persuade NIH, or a pharmaceutical or biotech company, to support drug discovery efforts leading to animal tests and finally, if successful, a clinical trial.  Nonetheless, it still looks like it will be several years before any clinical trials will become possible.  Given that pre-clinical trails are very expensive—probably >$1,000,000 would be required, donations are greatly appreciated.

If you are interested in making a contribution, you may do so in one of the
following ways:

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"Professor Bryan Cullen account 3990310"
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If have any questions, please contact:

Kristina Amidon Bowman
Duke Medicine
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Durham, NC 27701
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7) What can I use to treat chronic HSV-1-induced cold sores or
HSV-2-induced genital ulcers now?

All treatment decisions should be made in consultation with your physician, and no general guidelines will apply to everybody. While there are no drugs that attack latent herpes viruses, three closely related prescription drugs (Zovirax/acyclovir; Famvir/famciclovir and Valtrex/valacyclovir) are potent inhibitors of active virus that work quite well and that you might discuss with your physician.

We hope this information answers your questions and is helpful. Please be assured that we are continuing to work on the problem of developing novel HSV-1 and HSV-2 treatment approaches.