New Platform Technologies for Viral and Therapeutic Evolution Assays

TECHNOLOGY AREA(S): Bio Medical OBJECTIVE: Design and develop new bioreactor technology suitable for long-term assays of cellular and viral evolutionary dynamics, emulating human-like continuous conditions. Demonstrate and validate the technology in a relevant application. PROPOSALS ACCEPTED: Phase I and DP2. Please see the 17.1 DoD Program Solicitation and the DARPA 17.1 Direct to Phase II Instructions for DP2 requirements and proposal instructions. DESCRIPTION: There is a critical DoD need to capture long-term evolutionary dynamics of viral mutations in the laboratory under human-like conditions to aid in the design of evolving therapies (See DARPA INTERCEPT program [1]) and to assess efficacy of these and traditional static therapies in an evolutionary environment. The state-of-the-art approach to tracking long-term viral dynamics is flask-based serial passaging, which continually transfers a small fraction of the viral output from a given flask of infected cells onto a new flask of uninfected cells. Although a powerful technique, the serial passaging method has significant limitations: low abundance viral mutants often are discarded, dilution affects cell and virus characteristics, the rate of viral evolution is prolonged, and the long-term in vivo evolutionary dynamics are not recapitulated. Maintaining appropriate cell influx and viral efflux rates in the face of viral amplification is key to recapitulating virus-host dynamics; this dynamic state is required to determine the instantaneous and steady state in virus stocks and host cell infection status (e.g., infected, uninfected, dying), which in turn is used to monitor virus mutation. One of the central challenges to overcoming these limitations is to design filters or other methods to controllably and quantitatively remove desired cells and virions. DARPA seeks to promote the design and development of new bioreactor technology to support long-term evolutionary studies for chronic infectious or non-infectious diseases. The reactor must have continuous operation and support controllable rates on influx of fresh cells and nutrient media and the separation and removal of viral particles and different types of cells without clogging or fouling. Removed particles and cells must be sorted by their type. The reactor must support operation for sustained periods of time (minimum of 45-90 days). The system must support controlled sampling of the reactor contents one or more times per day. The sample should be unbiased and support additional assays of cells and supernatant such as high-throughput sequencing, molecular imaging, single cell analysis, etc. The filtering technology for removal and sorting should be characterized and validated. Designs should leverage advances in microfluidics, continuous flow, novel filtration mechanisms, or other relevant technologies. The implementation of real-time monitoring methods (i.e. optical methods) are strongly encouraged. Bioreactor systems that effectively combine more than one phase (gas, liquid, solid) or include 2- or 3-dimensional tissue-like structures will be considered favorably. PHASE I: Develop key requirements (including ranges of influx and efflux rates gathered from published literature) and establish performance metrics for evaluation of the bioreactor. Define the components and methods to be used for filters, sorting, and other parts of system. Investigate and define risks and risk mitigation strategies. Implement a basic prototype system or a simulated system that demonstrates operating principles and fundamental performance capabilities. Establish use cases. Required Phase I deliverables will include a final report detailing the design of the bioreactor system, requirements, fabrication process, and preliminary performance results. PHASE II: Finalize the design of Phase I and complete implementation. Evaluate the performance of the system against requirements of rates, sorting, sampling, and constraints. Demonstrate and validate the technology in at least one of the following applications: mutation dynamics in candidate viral evolution, the co-evolution of a virus and an associated therapeutic interfering particle, viral, bacterial or cellular evolution under selection pressure from antiviral drugs, immune agents, antibiotics, or anti-cancer drugs. Demonstrate continuous operation for 45-90 days with one sampling every week. Through appropriate statistical analysis of the samples, demonstrate the similarity of bioreactor dynamics to human-like conditions. Phase II deliverables will include final bioreactor design and working prototype and a final report detailing system performance for the selected application. PHASE III: The end goal of this effort is to provide the community with a new type of continuous bioreactor to recapitulate human-like conditions for the study of long-term evolutionary dynamics of fast mutating pathogens, diseases and emerging pandemics of interest to DOD. The new platform technologies developed under this SBIR are expected to support investigation and design of evolving therapies such as therapeutic interfering particles. This bioreactor will also be useful to identify pathogen mutations that can escape therapies, understand the evolution of cancer cells, and dynamically track effects of therapy. REFERENCES: 1: DARPA INTERCEPT Program. http://www.darpa.mil/news-events/2016-04-07a2: Nowak MA, RM May (2000). Virus Dynamics. Oxford University Press. ISBN: 9780198504177 KEYWORDS: Bioreactor, Evolutionary Dynamics, Continuous Flow, Filters, Mutations, Viral Escape, Long Term Assay