JSTOin theNews

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August 2016 | Vol. 6 No. 8

Approved for public release, distribution is unlimited

Tiny Technologies Combat Nerve Agents

Paper Wins as New Detection Tool for Zika

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DTRA Mentors Next Generation

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On the front cover: Airman 1st Class Brendan Rapp, 673d Aerospace Medical Squadron public health technician, counts mosquitoes at Joint Base Elmendorf-Richardson, Alaska. According to the World Health Organization, mosquitoes are the greatest menace when it comes to disease-transmitting insects. They are responsible for several million deaths and hundreds of millions of cases every year involving malaria, dengue and yellow fever. (U.S. Air Force photo by Airman 1st Class Valerie Monroy)

On the back cover:A UH-60 Black Hawk helicopter from the 1st Air Cavalry Brigade, 1st Cavalry Division, enters the landing pattern near Robert Gray Army Airfield. Although the 1st ACB returned from Iraq three months ago, they are already training for future missions. (U.S. Army photo/Sgt. Travis Zielinski)

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Lead DoD science and

technology to anticipate,

defend and safeguard

against chemical and

biological threats for the

warfighter and the nation.

DEFENSE THREAT REDUCTION AGENCY & USSTRATCOM Center for Combating WMD & Standing Joint Force Headquarters-Elimination

J9 Research and Development Directorate Joint Science and Technology Office

8725 John J Kingman Road, Stop 6201, Fort Belvoir, VA 22060

www.dtra.mil

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Endocytosis

Receptor

Endosome

Nucleus

Segregation & recycling of

receptor

Release of payload

Movement to nucleus

Targeted nanoparticle with payload bound to receptor

Schematic of the intranasal delivery of nanostructured liposomal carriers of nerve agent countermeasures. Figure courtesy of Esther Chang, SynerGene Therapeutics, Inc.

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RECEPTOR-MEDIATED UPTAKE OF PAYLOAD

Exactly how small is a nanometer? The average human hair is 50 micrometers wide and one nanometer is 1/50,000th the width of a single strand of hair. Using nano-sized structures to combat nerve agents could lead to enormous advancements to protect warfighters.

Funded by the Defense Threat Reduction Agency’s Joint Science and Technology Office, researchers are leveraging nano science to combat organophosphonate nerve agents (OPNA) for improved warfighter safety. The efforts, managed by Dr. Brian Pate, are part of JSTO’s Nanostructured Countermeasure Platforms for Chemical Warfare Agents (NCP-CWA) program that explores enhanced delivery methods of prophylactics and therapeutics.

Currently there are no approved broad-spectrum prophylactic countermeasures available to protect the warfighter against multiple chemical threats. The JSTO portfolio aims to fill this gap with its bioscavenger program, yet the current technology has some disadvantages including high costs and dosing requirements, potential immunogenicity, and short-term stability in circulation.

In addition, the JSTO portfolio for nerve agent therapeutics currently lacks the ability to treat effects on the brain. However, the NCP-CWA program addresses the gaps in the prophylactic and therapeutic medical countermeasures program by exploring alternative platform approaches, readily tailorable to specific threat agents.

Circulatory stability is an important factor in drug design. Protein-based bioscavenger therapeutics are only

available in the circulatory system for a few days while the desired target is between 10 and 60 days. Prolonged circulatory stability of a medical countermeasure would afford the warfighter advanced protection against chemical threats.

Projects in the NCP-CWA program aim to improve the circulatory stability of protein prophylactics and therapeutics via novel mechanisms that leverage nanoscale phenomena.

One such project exploits the individual’s own red blood cells (RBC). The strategy is to encapsulate the protein therapeutic in a mixture of polymers that target RBCs. Upon attachment to RBCs, the protein becomes available throughout the circulatory system to scavenge chemical threats. In addition, attachment to RBCs improves the circulatory stability of the protein.

A second key project within the program aims to promote protein stability, enhance circulatory stability and eliminate immune response using a two-pronged approach. This strategy requires attachment of the protein therapeutic to a polymer, which is then encapsulated in a gel. This conjugation/encapsulation approach was applied to two different proteins and showed up to a three-fold improvement in circulatory stability for one protein and an improvement of 25-fold for the second protein.

The encapsulated particles showed little or no immunogenicity in a study conducted in rats. The results were published in the Journal of Controlled Release, “Butyrylcholinesterase Nanocapsule as a Long Circulating Bioscavenger with Reduced Immune Response.”

A third project in JSTO’s NCP-CWA program focuses

Combat Nerve AgentsTINY TECHNOLOGIES

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SME @ email address4

Paper Wins as New Detection ToolThe recent Zika virus outbreak, and its emergence as a concern at the forefront of global healthcare, demonstrates the need for a diagnostics platform capability that can be rapidly adapted and responsive to emerging viral threats, whether natural or manmade, to protect our warfighters and civilian populations.

Researchers from the Defense Threat Reduction Agency’s Joint Science and Technology Office are at the forefront of developing a paper-based diagnostic device to detect viral threats, such as Zika. Managed by JSTO’s Dr. Ilya Elashvili,

the project is led by Dr. James J. Collins and his team from the Massachusetts Institute of Technology.

Published in the journal Cell, “Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components,” explains how the team created a portable diagnostic platform that allows rapid, specific and low-cost detection of the Zika virus at clinically relevant concentrations. For this effort, Dr. Collins’ group merged two biotechnologies, which they previously developed.

The first of the two biotechnologies uses a riboregulator concept to construct programmable ribonucleic acid (RNA) sensors called toehold switches, which can be rationally designed to bind and sense virtually

The rapid design, assembly and validation of paper-based biomolecular sensors for the portable detection of Zika virus is shown here. Using sequence information from online databases, primers for isothermal RNA amplification and toehold switch-based RNA sensors are designed in silico using algorithms. Sensors are embedded into paper discs and freeze-dried along with a cell-free transcription and translation system, to be deployed in the field as colorimetric diagnostics. In the diagnostic test, viral RNA is extracted, isothermally amplified, and used to rehydrate the freeze-dried sensors, resulting in a color change from yellow to purple in the presence of Zika RNA. Strain differentiation at single-base resolution is provided by a CRISPR-based module which connects to a secondary paper-based test that also provides colorimetric output.

ROCK, PAPER, ZIKA:

SME @ email address 5POC: Dr. Ilya Elashvili, ilya.elashvili.civ@mail.mil

any RNA sequence. This sensing portion of the toehold is placed upstream of a ribosome binding site (RBS) to create a RBS-locking hairpin structure, which block RNA binding to ribosome and thereby prevents the translation of a downstream reporter protein.

When targeted, “trigger” RNA binds the toehold, peeling the hairpin and opening the blockade, allowing the RNA to bind to the ribosome and subsequent translation of the reporter protein (LacZ enzyme). This converts a yellow substrate to a purple product, indicating the presence of the virus.

The second technology involves a paper-based, freeze-dried, cell-free protein expression platform that allows for the deployment of these toehold switch sensors outside of a research laboratory by providing a sterile method for storage and distribution of genetic circuits at room temperature.

In order to achieve the sensitivity needed in the diagnostic tests, the researchers employed widely used nucleic acid sequence-based amplification (NASBA) to isothermally

amplify extracted RNA. As a result, the researchers were able to develop a low-cost diagnostic platform for the detection of clinically relevant (low femtomolar) concentrations of Zika virus from infected monkey plasma. It is noteworthy that there was no significant detection of the closely related Dengue virus.

Furthermore, by employing a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based module that can cut preprogrammed sequences in NASBA amplified product and thereby prevent reporting the selected target sequences, the researchers were able to discriminate different genotypes of Zika virus (i.e., American Zika virus from African Zika virus).

This diagnostic tool can be stored for more than a year at room temperature and does not require highly skilled personnel or expensive equipment to operate. This makes it suitable for current or emerging viral threats for deployed warfighters in low-resource areas, where detection and containment are critically needed.

Using sequence information from online databases, primers for isothermal RNA amplification and toehold switch-based RNA sensors were designed in silico using purpose-built algorithms. Once synthesized, the resulting sequence-specific toehold sensors can be assembled and validated in less than seven hours. In under a day, validated sensors can be embedded into paper and freeze-dried along with a cell-free transcription and translation system to be deployed in the field as stable diagnostics. For the diagnostic test, extracted RNA is isothermally amplified via the nucleic acid sequence-base and used to rehydrate the freeze-dried paper sensors. The detection of the appropriate trigger RNA is indicated by a color change in the paper disc from yellow to purple, which can be quantified by a portable electronic reader for even greater diagnostic sensitivity.

Figures courtesy of Dr. James Collins, Massachusetts Institute of Technology.

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SME @ email address6 POC: Dr. Brian Pate; brian.d.pate.civ@mail.mil

TINY TECHNOLOGIES (continued from Page 3)

on mitigating the toxic effects of OPNAs on the brain. Nerve agents enter the brain and bind to their target resulting in seizures. Currently, there are no effective therapeutics to protect the brain prior to the onset of seizures. The NCP-CWA strategy involves formulating a small molecule drug with liposomes for intranasal drug delivery. Early results demonstrate a dramatic improvement in the survivability of mice when exposed to a nerve agent simulant.

Together, the early successes of the NCP-CWA program points the way toward more effective means to protect and treat warfighters from exposure to nerve agents. Reducing or removing the threat posed by these chemical warfare agents, among the deadliest threats faced on today’s battlefields, offers the potential to provide a new means to offset enemy capabilities to equip the U.S. with the needed edge to facilitate victory in current and future military engagements.

Currently, the United States ranks 29th in math and 22nd in science among industrialized nations. Since the children of today are the future of tomorrow, U.S. leaders are placing an emphasis on getting high school-aged children involved in the science, technology, engineering and mathematic (STEM) fields.

Supporting the government’s initiative, members of the Defense Threat Reduction Agency’s Joint Science and Technology Office are mentoring high school students in a variety of projects to garner interest in STEM to develop the next generation of DoD scientists.

For the fourth consecutive year, JSTO members took STEM education to the next level at their annual Joint Science

and Technology Institute held in Aberdeen, Md., from July 25 to August 5. The program combined student education and teacher development into a two-week STEM-intensive camp. The coveted camp drew 84 high school and junior high school students and eight teachers from more than 1,000 applicants.

Students and teachers travelled from DoD schools all over the world, representing seven countries, including American Samoa, Italy, South Korea,

Ukraine and the United States. Twenty states were represented as well, from Arizona to Virginia and the District of Columbia.

Participants spent their two weeks experimenting with the latest technologies; 3D printers, drones, Raspberry Pi, robotics and chemistry. These hands-on activities allowed our next generation of scientists to explore several STEM fields while working with leading DoD scientists and researchers.

Supporting STEM activities is one way DTRA’s JSTO is helping to grow the next generation of scientists, making the world safer and empowering the youth of today to develop the technologies of tomorrow.

DTRA Mentors Next Generation of

DoD Scientists

Within the Defense Threat Reduction Agency’s

Research and Development Directorate resides the

Joint Science and Technology Office for Chemical

and Biological Defense. This publication highlights

the organization’s advancements in protecting

warfighters and citizens through the innovative

application of science and technology research.DTRA

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