Update on HSV Research
We are continuing our work on using CRISPR/Cas technology to cleave viral genomic DNA as a possible approach to the treatment of chronic diseases caused by human viruses, concentrating at present on Human Papilloma Virus 16 (HPV16), which causes malignant cervical, anal and head-and-neck cancers, as well as on HIV-1, the cause of AIDS. We currently have a paper in press that describes the use of CRISPR/Cas to treat HPV-16+ anal cancer explants in mice. In these animal experiments, we used Adeno Associated Virus (AAV) vectors as a tool to deliver Cas9 to the tumor cells in vivo, which resulted in a highly significant inhibitory effect on tumor growth. We are also about to submit a paper showing that we can not only stably protect T-cells in culture from HIV-1 infection using CRISPR/Cas but also cure the culture, that is remove all replication competent virus, and we hope to repeat these experiments in mice bearing a humanized immune system in the near future. We also have recently made an important breakthrough in improving design and efficacy of AAV-based CRISPR/Cas delivery vectors that could have important implication for the use of CRISPR/Cas as an approach to the treatment of not only HIV-1 and HPV16 but also Hepatitis B virus (HBV) and Herpes Simplex Virus (HSV). Assuming this works as we expect, we then hope to reinvigorate our work on using CRISPR/Cas as a potential cure initially in animal models of HBV and HSV.
My laboratory has decided to terminate our research on the development of treatments for HSV infections due to a chronic lack of funds for this work. However, our previous collaborators, Dr. David Bloom at the University of Florida and researchers at Editas Medicine in Cambridge, MA, are continuing this work and should be contacted directly if you are interested. We are, however, continuing to work on perfecting the CRISPR/Cas technology as a potential treatment for chronic viral infections, using Hepatitis B virus, Human Papillomavirus and Human Immunodeficiency Virus as model systems, and we have observed promising responses in relevant disease models in mice.
My laboratory continues to collaborate with the laboratory of David Bloom, at the University of Florida, and the biotech company Editas, located in Cambridge, Massachusetts, to develop a treatment that will destroy latent HSV genomes and fully cure HSV-induced disease. After much effort, we have now focused on the Cas9 gene encoded by Staph. aureus (SA) as the ideal DNA editing enzyme for use in these studies. In particular, the SA Cas9 gene is only ~3,200 base pairs (bp) in size and this has allowed us to design fully infectious but replication-incompetent viral vectors based on adeno-associated virus (AAV), which represents a highly efficient in vivo delivery system but only has a packaging limit of ~4,800 bp. We have now built and tested several AAV-based vectors, each expressing the SA Cas9 protein, as well as two HSV-1-specific guide RNAs, that are being tested for efficacy in latently HSV-1-infected mice. Our initial goal is to focus on the development of AAV-based SA Cas9 expression vectors as a treatment for HSV-1 induced keratitis, which leads to loss of vision in a significant percentage of infected patients, as a proof-of-concept that this approach can indeed target and destroy HSV-1 DNA genomes in vivo. If successful, then this approach could be readily extended to other latent or active infections caused by HSV-1 and, especially, HSV-2. A review article describing this treatment strategy, and explaining how it works, was recently published by my group: Kennedy & Cullen Virology 2015
We have recently extended our efforts to cure HSV infections by developing DNA editing enzymes as potential HSV treatments. I refer to these as “smart bombs” that can cleave the HSV-1 genome, and destroy the latent virus, if delivered to latently infected neurons using viral vectors. The only viral vectors that really make sense at this point are based on adeno-associated virus (AAV), which has been successfully used in gene therapy trials in humans. The big advantage of AAV is that you can get very high levels of virus—up to 10 billion infectious units per milliliter—and the Bloom lab has clearly shown, using an AAV that expresses green fluorescent protein (gfp), that he can infect essentially every single neuron in the trigeminal ganglia where HSV-1 establishes latency. The problem is that the AAV packaging size, that is the amount of DNA that it can fit into its viral capsid, is only ~4,600 bp.
Our initial efforts to use gene editing to destroy HSV-1, while very successful, used either transcription activator-like endonucleases (TALENs) or bacterial editing enzyme of the CRISPR/Cas9 family, derived from Streptococcus pyogenes (SPy), both of which work well but have size issues. The TALENs work as heterodimeric DNA binding and cleavage enzymes and, while AAV can package one TALEN, it cannot package two. The SPy Cas9 gene is so large that even on its own it is too big for AAV.
So, my long-term collaborator David Bloom and I have taken two approaches. On the one hand, we have generated AAVs that express one or the other TALEN and have generated viral stocks of each. We intend to mix these two stocks and then use the mixture to infect the trigeminal ganglia in mice at high levels of virus such that each neuron should be infected by both AAVs, allowing expression of the TALEN heterodimer and HSV-1 cleavage. These experiments are now in progress in latently HSV-1 infected mice in the lab of Dave Bloom at the University of Florida.
The second approach we have taken is to identify Cas9 proteins encoded by other bacteria that are highly active but small enough to fit into AAV. The one we are currently focused on is derived from Neisseria meningitidis (NMe). This Cas9 gene is only 3,200 bp in size, well below the 4,600 bp cutoff, and works well. Nevertheless, we have now cloned this into AAV, where it is well expressed, and sent it off to Dave Bloom to put into mice. While these are being tested, we are also testing Cas9 genes we have isolated from other bacteria and we are in the process of establishing a collaboration with a biotech company that focuses on the use of Cas9-derived DNA editing enzymes in the treatment of human disease. I have visited their headquarters and they have stated that they are enthusiastic about working with us on the goal of using Cas9 to cure HSV-1 and, especially, HSV-2. Hopefully, this will allow us to move this project along more rapidly, using new resources provided by this company.
So, some progress has been made but we haven’t quite achieved full success. I’m very enthusiastic about the approach we are pursuing, because I really think this could be a way to actually destroy latent HSV genomes and lead to the cure we have all sought. I hope the next update will include the statement that we can at least cure mice! Once that is achieved, I think things will really start to move forward.