Thinking big

We all know the scientific method (i.e., observe, hypothesize, experiment, conclude, repeat) and even children can follow this process effectively. The most difficult skill to learn as a scientist is to think big. I remember telling my wife during my first two years of graduate school that there is no way I would be able to develop a ground breaking hypothesis, let alone a technology that may one day prevent or treat disease. How can one individual become such an expert that they can see and test the limits of knowledge? This transition is extremely interesting to me, but it is only the beginning stage in the development and maturation of a scientist. The most successful scientists learn how to identify the most important questions in their field, and then develop tools to answer them.

As a postdoctoral fellow at Yale University School of Medicine, I was approached by a friend and colleague who asked me what I thought of a new ground breaking publication in PLoS ONE regarding a broad-spectrum antiviral. At first, I dismissed the research and my friend because PLoS ONE is considered the "money making" PLoS journal. It's impact factor isn't very high and I knew from first experience that their expectations for publication weren't either. They publish most submissions to fund extensive peer review of their higher-tiered journals (e.g., PLoS Medicine, PLoS Pathogens, etc.). However, before I made a negative statement of the work, I read the paper and the hype that was appearing on the internet. The paper outlined details of a synthetic broad-spectrum antiviral called a Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer (DRACO) protein that killed cells that were infected with a host of disease causing RNA viruses. DRACOs also appeared to work in mouse models of disease.

At first glance, DRACO technology seemed to be an impressive advance in our ability to combat disease causing viruses. However, I did caution my friend that new evidence suggests that humans are a walking ecology of viruses, bacteria, fungi, and parasites, in addition to human cells. I was concerned that DRACOs may also kill cells infected with benign viruses that are considered members of our normal flora. This would lead to the death of non-target cells. I looked forward to further development and clinical trials of DRACO proteins.

Years passed and I often turned to the internet to assess the status of DRACO technology. The original manuscript was published in 2011 and there haven't been any peer reviewed updates since. DRACO technology is patent protected and is being developed in a biotech incubator. These facts make it very difficult for the technology to move into clinical trials and beyond.

In starting up my own laboratory, I began to consider some of the most important questions in virology.

(1) How do viruses contribute to evolution?

(2) How do disease causing viruses emerge?

(3) How do you develop an effective antiviral?

It has always interested me that we have been able to treat bacterial infection for decades, yet viruses have evaded our efforts. In it's most simple terms, this is due to their similarity to us, and their ability to rapidly mutate and generate escape mutants. Viruses do have unique proteins that compose their outer membranes, protein shells, and replication machinery, but they also use many components within the host cell to survive. It is difficult to identify a virus-specific target, and when you do, they mutate and you have to start all over again. This is time consuming and very expensive.

How can we surpass this difficulty in generating effective antivirals? To me, all roads pointed to DRACO technology. I wanted to modify DRACO technology so that we could target a specific virus and limit the number of collateral cells killed during treatment. We attempted this by mining the literature for antibodies that recognized our favorite virus - dengue virus. In particular, we wanted to find genetic sequences that corresponded to the antibody domains that recognized dengue virus so that we could engineer the domains into DRACO technology. In a strange twist of fate, we found those sequences in a previous publication from my postdoctoral mentor (thanks Erol!). We then copied and pasted the sequences on a Microsoft Word document and inserted the final piece (used in DRACO proteins) - a cell killing domain called Apaf 1 CARD. The sequence of this "Frankenstein molecule" was synthesized by Genscript and sent back to us cloned in a mammalian expression vector for less than $1,000.

 

We hypothesized that our protein would bind to dengue virus envelope protein with its antibody domains, which would lead to clustering of our protein on the virus surface or on cellular domains that were enriched with the protein. When our protein clusters together, the Apaf 1 CARD domain is converted into a cell killing mechanism called an apoptosome. It cuts caspase proteins like molecular scissors and leads to a regulated cellular death program called apoptosis. We made a stable cell line that overexpressed our protein and then challenged the cells with dengue virus.

It's Alive!!!

*** The next update will include data showing the effectiveness of our protein.