April 12, 2016
BACKGROUND AND MOTIVATION
Currently there are relatively few prophylactics or therapeutics for viruses, and most that do exist are highly pathogen-specific or have undesirable side effects or other disadvantages. We have developed a radically new, broad-spectrum antiviral therapeutic/prophylactic that has the potential to revolutionize the treatment of viral infections. Our Double-stranded RNA Activated Caspase Oligomerizer (DRACO) approach selectively induces apoptosis (cell suicide) in cells containing viral double-stranded RNA (dsRNA). DRACO should recognize virus-infected cells and rapidly kill those cells without harming uninfected cells, thereby terminating the viral infection while minimizing the impact on the host (PLoS ONE 6:e22572, 2011; Nature Biotechnology 29:885, 2011; U.S. patents 7,125,839, 7,566,694, and others pending). We have previously created an initial version of DRACO and shown that it is nontoxic and effective against 18 different viruses in 13 mammalian cell types. We have also demonstrated that DRACO is nontoxic in mice and rescues mice from lethal challenges with H1N1 influenza, Amapari arenavirus, Tacaribe arenavirus, and Guama bunyavirus in preliminary trials. Our DRACO approach and results have been called “visionary” by the White House (National Bioeconomy Blueprint, April 2012, p. 9), named one of the best inventions of the year by Time magazine (November 28, 2011, pp. 58, 78), and featured on the BBC Horizons TV program (2013).
The herpesvirus family includes several pathogens of great clinical and commercial interest, including herpes simplex 1 and 2 (HSV, which can cause oral, ocular, and genital infections), cytomegalovirus (CMV, a major cause of birth defects and ocular infections, and also a concern for the aging population), Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV-8, which can cause sarcoma and lymphoma), Epstein-Barr virus (EBV, which can cause Burkitt’s lymphoma and other cancers), and varicella zoster virus (VZV, which causes chickenpox and shingles). Members of the herpesvirus family cause lifelong infections, and existing antiviral drugs can help to control their symptoms but cannot cure the infections or ensure that they cannot be transmitted to other people. In principle these viruses should be susceptible to our DRACO approach, although we have not yet tested DRACO against any of these viruses.
Out of the wide range of possible DRACO designs, the one we have now is simply the first we tried. It is almost certainly not the best, even for the viruses we have already tested, and it may or may not be effective against viruses in the herpesvirus family. As drug trials proceed from cells to mice to higher animals to humans, the trials become much more expensive, longer in duration, able to test fewer possibilities per experiment, and higher profile and thus more politically detrimental to further research funding if there is a failure. As just one example, a cell experiment set up by one person might test 2000 wells of cells with different DRACOs and other 2 conditions within a few days, whereas an equally laborious and more expensive mouse experiment might run for several weeks and test only 54 mice, or 6 different conditions (9 mice per condition). Trials with larger animals are even more time- and resource-intensive. Moreover, if a drug design is later found to be inadequate in advanced animal trials, one might have to go back to the very beginning and repeat all the cell and animal trials with alternate and hopefully better drug designs, making the total costs and delays even greater. Thus before proceeding with animal trials for herpesviruses, it is much faster and ultimately much less expensive and less risky to test thoroughly in cells to identify the best DRACO for herpesviruses, and the next-best DRACOs that could be used if the front-runner fails in later trials for any reason. Testing and optimizing the design of new therapeutic molecules in early experiments is a widely accepted practice called structure-activity relationship (SAR) analysis.
Therefore, we would like to use the SAR approach to produce several different DRACOs with various dsRNA-detection domains, apoptosis-induction domains, and cellular delivery tags that should be effective against these viruses, test their efficacy against members of the herpesvirus family in multiple cell types, and iterate and optimize the DRACO designs in order to develop the most effective DRACOs, which could subsequently be tested in animal models of these viruses. We will proceed as quickly as we can with the proposed experiments for herpesviruses, but we need to be thorough or else the risk and cost of a failure further down the road become too high. We should be able to complete the optimization of DRACO against members of the herpesvirus family in cells within four years. The estimated cost of this work is approximately $500K per year, or $2M total over the four years, to cover all necessary costs for lab space, equipment, reagents, personnel, and other expenses.
The requested funding and the proposed work are critical for helping to move DRACO through the “valley of death” that it is currently in. The early results against other viruses look very promising, but preclinical animal trials and clinical human trials for viruses such as herpesviruses are quite some distance away, and other sponsors have not been willing to fund DRACO to cross that valley. If the requested funding can enable us to demonstrate and optimize DRACO performance against clinically and commercially important herpesviruses in cells, pharmaceutical companies and other sponsors should be willing to carry DRACO through preclinical animal and clinical human trials for these viruses.
Although our initial version of DRACO has been effective against a number of viruses, the dsRNA binding domain, apoptosis induction domain, delivery tag, and other structural features of the DRACO design may need to be optimized to maximize efficacy against herpesviruses, for example in order to optimize binding to herpesvirus-related dsRNA, overcome natural resistance strategies incorporated in herpesviruses, and maximize DRACO delivery to herpesvirus-infected cells and the nuclei thereof. Therefore, we propose a SAR experimental campaign to test and optimize DRACOs against herpesviruses in multiple cell types.
Specifically, we propose to:
• Restart our DRACO production and testing capability, and produce sufficient quantities of DRACOs and negative controls for cell trials against herpesviruses. Multiple DRACOs with different domains will be produced and tested in parallel to conduct the work as 3 efficiently as possible, minimize technical risks, and optimize DRACO performance. DRACOs will be expressed from pET vectors transformed into Rosetta BL21(DE3) E. coli and purified using nickel columns to bind His6-tagged proteins following our standard protocols. For quality control, each newly produced batch will be tested in our proven models such as rhinovirus challenge of normal human lung fibroblast (NHLF) cells before being used in new experiments. Negative controls to be produced will include just the dsRNA-binding domains of DRACOs with appropriate delivery tags, just the apoptosis-induction domains of DRACOs with appropriate delivery tags, and DRACO-free extract produced and purified from the same E. coli strain using the same methods as DRACO. These negative controls will help to ensure that any observed antiviral activity is due to the intended function of intact DRACO and not to other factors.
• Test multiple herpesvirus strains in different human and animal fibroblast, macrophage, and neuronal cell lines in order to verify the best cell/virus systems, cell density, virus dose, assay schedule, and other protocol conditions for measuring viral replication and cytopathic effects. 96-well plates of cells will be challenged with virus at varying multiplicities of infection. We will develop and validate quantitative polymerase chain reaction (qPCR) assays for measuring viral titers in supernatants collected from viruschallenged cells on specified days after infection. We have previously developed qPCR assays for a number of other viruses. In addition to viral titers, cytopathic effects and cell survival will be evaluated by daily visual inspection and by adding Promega CellTiter 96 on selected days and measuring absorbance at 490 nm on a 96-well plate reader. Assay schedules, viral doses, and other parameters will be optimized for these cell/virus systems.
• Conduct tests of DRACOs and negative controls against herpesvirus strains in these validated cell models. 96-well plates of cells will be challenged with virus at varying multiplicities of infection. DRACO will be added at fixed times before or after cells are challenged with virus, with groups of 8 wells per condition. Criteria for demonstrating DRACO efficacy will include measurable reductions in viral titers in the supernatant of virus-challenged cells, measurable reductions in virus-induced cytopathic effects, and/or measurable increases in cell survival in virus-challenged wells.
• Use the results against herpesviruses to improve the DRACO dsRNA binding domain, apoptosis induction domain, delivery tag, other structural features, and production and administration protocols to maximize efficacy against herpesviruses. We already have a large number of designs for DRACO domains and methods, and these experiments will help to select the best ones for herpesviruses. We can do multiple rounds of iteration between producing improved DRACOs and testing them against herpesviruses. This ability to rapidly iterate in cells should help to identify and optimize the best lead DRACOs as quickly and inexpensively as possible before proceeding to animal trials.
We should be able to complete the optimization of DRACO against members of the herpesvirus family in cells within four years. The estimated cost of this work is approximately $500K per 4 year, or $2M total over the four years, to cover all necessary costs for lab space, equipment, reagents, personnel, and other expenses.
This work should demonstrate that DRACOs are effective against infections by multiple herpesviruses, and this data should be very important for persuading major sponsors to fund and advance DRACOs through animal trials and hopefully human trials for these viruses. The results of the proposed work will also hopefully lead to follow-on research to test DRACOs against additional herpesvirus strains or members of other virus families.