Finding Zika one paper disc at a time

"The growing global health crisis caused by the Zika virus propelled us to leverage novel technologies we have developed in the lab and use them to create a workflow that could diagnose a patient with Zika, in the field, within 2-3 hours," said Collins, who is a Wyss Core Faculty member, and Termeer Professor of Medical Engineering & Science and Professor of Biological Engineering at the Massachusetts Institute of Technology (MIT)'s Department of Biological Engineering.

Building off previous work done at Harvard's Wyss Institute by Collins and his team, along with collaborators from Massachusetts Institute of Technology (MIT), the Broad Institute of Harvard and MIT, Harvard Medical School (HMS), University of Toronto, Arizona State University (ASU), University of Wisconsin-Madison (UW-Madison), Boston University (BU), Cornell University, and Addgene, joined their efforts to quickly prototype the rapid diagnostic test and describe their methods in a study published online May 6 in the journal Cell, all within a matter of six weeks. Collins is the paper's corresponding author.

Emerging innovation during the Ebola health crisis

In October 2014, Collins' team developed a breakthrough method for embedding synthetic gene networks – which could be used as programmable diagnostics and sensors – on portable, small discs of ordinary paper.

Stirred by the then-ongoing Ebola outbreak in Africa, they demonstrated a proof-of-concept color-changing diagnostic that could screen for Ebola by embedding in paper a novel kind of synthetic biomolecular sensor designed to screen for specific RNA sequences. These RNA sequences can mark not only the genetic signatures of Ebola but also other RNA viruses including Zika, SARS, measles, influenza, hepatitis C, and West Nile fever. The team believed that one day, the method could be applied in the field to identify viruses with blood, urine, or saliva samples.

However, until recently, the team's paper-based technology has been challenged by the extremely low concentration of virus that is normally found in blood, urine, and saliva. Now, using blood samples from monkeys infected with Zika virus as well as virus recovered from cells infected in the laboratory, the team has validated a next generation technique that overcomes this problem.

A leap for paper-based diagnostics, urged by the Zika outbreak

"The vivid images in the news stemming from the ongoing Zika crisis are heartbreaking," said Keith Pardee, Ph.D., one of the study's co-first authors and an Assistant Professor in the Leslie Dan Faculty of Pharmacy at University of Toronto, who was formerly a Postdoctoral Fellow at the Wyss Institute and BU. "We hope a tool like this can help reduce the impact of the outbreak until a vaccine can be developed."

With field use in mind, Collins' team designed a simple modular workflow comprising three steps: amplification, Zika detection, and CRISPR-Cas9-aided strain identification. CRISPR-Cas9, a gene editing mechanism derived from the immune systems of bacteria, can be used to search entire sequences to find exclusive genetic markers. Leveraging CRISPR-Cas9's talent for sequence recognition, the third part of the team's system uses a CRISPR-Cas9-aided paper-based diagnostic to discriminate between strains whose genetic profiles differ by as little as one nucleotide.

Once a sample's RNA has been amplified using a mixture of enzymes and "primers", DNA sequences that trigger replication, a drop is administered to paper discs that are freeze-dried containing a mixture of cellular components and biological proteins. The droplet of amplified RNA activates the freeze-dried components so that the discs will change color to indicate a positive result for Zika virus. While the result can be read with the naked eye similar to a home pregnancy test, a specially designed electronic reader can also be used to get faster results and could, one day, quantify the amount of viral load in a sample.

If Zika is detected, the third step involves mixing a sample with a freeze-dried CRISPR-Cas9 cocktail and then using that mixture to wet another set of color-changing paper discs. Depending on the type of Zika strain contained in the sample, these discs undergo another set of visible color changes.  Although synthetic biologists and genetic engineers usually put CRISPR-Cas9 to work inside living cells, Collins' team discovered that it functions just as well - and even better in some cases - when freeze dried.

"We have tested our diagnostic systems against closely-related strains of the Dengue virus and found that within the first two steps, our system can readily distinguish Zika from Dengue," said Alexander Green, Ph.D., co-first author on the study and an Assistant Professor in the Center for Molecular Design and Biomimetics at ASU's Biodesign Institute and School of Molecular Sciences, who was formerly a Postdoctoral Fellow at the Wyss Institute and BU. "The addition of the third CRISPR-based step – deploying Cas9 on a paper-based platform for the first time - only enhances the accuracy of detection. As we prepare this technology for translation, we plan to validate our system against dozens or even hundreds of clinical samples."

Combatting future pandemics

All components of the diagnostic system can be freeze-dried for storage and transport while retaining their efficacy. The ability to pinpoint a strain-specific diagnosis in the field could prove valuable to national and global health organizations for tracking the spread of a viral outbreak in real time and for preparing containment strategies and treatment plans.

The diagnostic system developed by Collins' team could be tailored to identify a range of pathogens, and is an extremely cost effective diagnostic platform given its paper-based nature. What's more – the method is robust and could be used to quickly respond and develop new diagnostics in the face of emerging outbreaks.

"In response to an emerging outbreak, we envision a custom-tailored diagnostic system could be ready for use within one week's time," said Collins. "We are currently pursuing multiple opportunities to secure private and public funding in order to commercialize this diagnostic system and make it available to the world's health responders."

"The ability to recapitulate the genetic machinery of living cells in ordinary freeze dried paper provides a way to develop revolutionary sensors and diagnostics in a fraction of the time and with higher sensitivity and specificity than more conventional assays. These inexpensive paper-based tests also can be easily transported out of the laboratory and distributed virtually anywhere around the world.  The potential for applications in health and environmental screening, particularly in low resource areas, is huge," said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children's Hospital, and also Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

In addition to Collins, Pardee, and Green, additional authors on the May 6 Cell paper ("Rapid, low-cost detection of Zika virus using programmable biomolecular components") include: co-first author Melissa K. Takahashi, MIT's Institute for Medical Engineering & Science (IMES); co-first author Dana Braff, Wyss Institute, MIT's IMES, and Boston University (BU)'s Department of Biomedical Engineering; co-first author Guillaume Lambert, Wyss Institute and Cornell University's School of Applied Engineering and Physics; co-author Jeong Wook Lee, Wyss Institute; co-author Tom Ferrante, Wyss Institute; co-author Duo Ma, Center for Molecular Design and Biomimetics at Arizona State University (ASU)'s Biodesign Institute and the School of Molecular Sciences; co-author Nina Donghia, Wyss Institute; co-author Melina Fan, Addgene; co-author Nichole Daringer, MIT's IMES; co-author Irene Bosch, MIT's IMES; co-author Dawn Dudley, Wisconsin National Primate Research Center and Department of Pathology and Laboratory Medicine at University of Wisconsin-Madison (UW-Madison); co-author David O'Connor, Wisconsin National Primate Research Center and Department of Pathology and Laboratory Medicine at UW-Madison; and co-author Lee Gehrke, MIT's IMES and Department of Microbiology and Immunobiology at Harvard Medical School, and Harvard-MIT Program in Health Sciences and Technology.

The work was supported by the Wyss Institute for Biologically Inspired Engineering, MIT's Center for Microbiome Informatics and Therapeutics, the Defense Threat Reduction Agency, Arizona State University (A.G.), and the National Institutes of Health AI100190 (L.G.).

Link to paper (embargo lifting at 12PM ET May 6, note there may be slight lag time until article goes live on Cell website): http://www.cell.com/fulltext/S0092-8674(16)30505-0

PRESS CONTACTS
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MULTIMEDIA CONTACT
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The Wyss Institute for Biologically Inspired Engineering at Harvard University (http://wyss.harvard.edu) uses Nature's design principles to develop bioinspired materials and devices that will transform medicine and create a more sustainable world. Wyss researchers are developing innovative new engineering solutions for healthcare, energy, architecture, robotics, and manufacturing that are translated into commercial products and therapies through collaborations with clinical investigators, corporate alliances, and formation of new startups. The Wyss Institute creates transformative technological breakthroughs by engaging in high risk research, and crosses disciplinary and institutional barriers, working as an alliance that includes Harvard's Schools of Medicine, Engineering, Arts & Sciences and Design, and in partnership with Beth Israel Deaconess Medical Center, Brigham and Women's Hospital, Boston Children's Hospital, Dana–Farber Cancer Institute, Massachusetts General Hospital, the University of Massachusetts Medical School, Spaulding Rehabilitation Hospital, Boston University, Tufts University, Charité – Universitätsmedizin Berlin, University of Zurich and Massachusetts Institute of Technology.

Boston University (http://bu.edu), founded in 1839, is an internationally recognized institution of higher education and research.  With more than 33,000 students, it is the fourth-largest independent university in the United States.  BU consists of 17 schools and colleges, along with a number of multi-disciplinary centers and institutes integral to the University's research and teaching mission.  In 2012, BU joined the Association of American Universities (AAU), a consortium of 62 leading research universities in the United States and Canada. 

The Biodesign Institute at Arizona State University (http://biodesign.asu.edu) works to improve human health and quality of life through its translational research mission in health care, energy and the environment, global health and national security. Grounded on the premise that scientists can best solve complex problems by emulating nature, Biodesign serves as an innovation hub that fuses previously separate areas of knowledge to serve as a model for 21st century academic research. By fusing bioscience/biotechnology, nanoscale engineering and advanced computing, Biodesign's research scientists and students take an entrepreneurial team approach to accelerating discoveries to market. They also educate future generations of scientists by providing hands-on laboratory research training in state-of-the-art facilities for ASU. 

Addgene (http://addgene.org) is a 501(c)3 nonprofit biorepository dedicated to facilitating scientific discoveries by operating a plasmid library for researchers. The repository contains over 45,000 plasmids contributed by 2,700 research labs around the world. Over a half million plasmids have been distributed to 85 countries by Addgene - with 11,000 plasmids currently shipping each month. By authenticating, storing, archiving, and distributing plasmids and their associated data, Addgene is creating a lasting resource for research and discovery scientists around the world. 

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SOURCE Wyss Institute for Biologically Inspired Engineering at Harvard University

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