HR001120S0016.pdf

Broad Agency Announcement

Detect It with Gene Editing Technologies (DIGET)

BIOLOGICAL TECHNOLOGIES OFFICE

HR001120S0016

November 27, 2019

HR001120S0016, DIGET

TABLE OF CONTENTS

PART I: OVERVIEW INFORMATION

PART II: FULL TEXT OF ANNOUNCEMENT

1. Funding Opportunity Description

1.1. Program Overview

1.2. Technical Objectives and Program Structure

1.3. Program Metrics

1.4. General Requirements

2. Award Information

2.1. General Award Information

2.2. Fundamental Research

3. Eligibility Information

3.1. Eligible Applicants

3.2. Organizational Conflicts of Interest

3.3. Cost Sharing/Matching

4. Application and Submission Information

4.1. Address to Request Application Package

4.2. Content and Form of Application Submission

Disclosure of Information and Compliance with Safeguarding Covered Defense Information Controls

4.3. Funding Restrictions

4.4. Other Submission Information

5. Application Review Information

5.1. Evaluation Criteria

5.2. Review of Proposals

6. Award Administration Information

6.1. Selection Notices

6.2. Administrative and National Policy Requirements

6.3. Reporting

6.4. Electronic Systems

7. Agency Contacts

8. Other Information

9. APPENDIX 1 – Volume II checklist

PART I: OVERVIEW INFORMATION

Federal Agency Name – Defense Advanced Research Projects Agency (DARPA), Biological Technologies Office (BTO)

Funding Opportunity Title – Detect It with Gene Editing Technologies (DIGET) Announcement Type – Initial Announcement Funding Opportunity Number – HR001120S0016 North American Industry Classification System (NAICS) – 541714 Catalog of Federal Domestic Assistance Numbers (CFDA) – 12.910 Research and

Technology Development Dates o Posting Date: November 27, 2019 o Proposal Abstract Due Date and Time: January 7, 2020, 4:00 PM ET o Full Proposal Due Date and Time: February 25, 2020, 4:00 PM ET o BAA Closing Date: February 25, 2020 o Proposers Day: December 11, 2019 DARPA-SN-20-12 on beta.SAM.gov

Concise description of the funding opportunity – The goal of the DIGET program is to leverage advances in gene editing technologies to develop low-cost, high-trust, sensitive, multiplexed, rapidly reconfigurable, and fieldable diagnostics and biosurveillance technologies to address the need for timely and comprehensive threat detection surveillance to support Department of Defense (DoD) stabilization missions and outpace infectious disease.

Anticipated individual awards – Multiple awards are anticipated.

Types of instruments that may be awarded – Procurement contract, cooperative agreement, or other transaction.

Agency contact

The BAA Coordinator for this effort may be reached at:

DIGET@darpa.mil

DARPA/BTO

ATTN: HR001120S0016

675 North Randolph Street Arlington, VA 22203-2114 https://beta.sam.gov/opp/77b597563adb4dcb9e624c3a3bff78d4/view?keywords=DARPA-SN-20-12 mailto:DIGET@darpa.mil

PART II: FULL TEXT OF ANNOUNCEMENT

1. Funding Opportunity Description

This publication constitutes a Broad Agency Announcement (BAA) as contemplated in Federal Acquisition Regulation (FAR) 6.102(d)(2) and 35.016 and 2 CFR § 200.203. Any resultant award negotiations will follow all pertinent law and regulation, and any negotiations and/or awards for procurement contracts will use procedures under FAR 15.4, Contract Pricing, as specified in the BAA.

The Defense Advanced Research Projects Agency (DARPA) often selects its research efforts through the Broad Agency Announcement (BAA) process. The BAA will appear first on the beta.SAM.gov website, https://beta.sam.gov/, and the Grants.gov website http://www.grants.gov/. The following information is for those wishing to respond to the BAA.

DARPA is soliciting innovative proposals to develop distributed point-of-need and massively multiplexed gene editing-based nucleic acid detection capabilities for diagnostics and biosurveillance that address the following areas for technical innovation: (1) development of in silico tools to aid in design of gene editing guides, tools, and assays for detection of pathogen and host biomarker targets, (2) development of foundational enzymes and reporters to enable sensitive and specific detection of nucleic acid targets, (3) incorporation of detection reagents into assays that yield high sensitivity and specificity results in 15 minutes or less and can be rapidly reconfigured for new targets, (4) integration of detection assays into point-of-need diagnostics and massively multiplexed detection (MMD) devices, and (5) algorithms and analytics tools to assist interpretation of complex assays with clearly presented and easily understood results for decision making. Proposed research should develop innovative technologies to enable usage in field-forward locations and rapid reconfiguration of assays in response to new and emergent threats. Specifically excluded is research that primarily results in incremental improvements to the existing state of practice.

1.1. PROGRAM OVERVIEW

Exposure to regional endemic diseases, global pandemic outbreaks, or other emergent threats can degrade the health and preparedness of U.S. military forces and civilians alike, contributing to the spread of disease and further destabilization of already fraught regions. Therefore, it is critical that field-forward personnel have access to tools that yield unequivocally trusted diagnostic and biosurveillance results to enable rapid and informed decision-making regarding medical support and interventions to prevent or mitigate illnesses and epidemics.

State of the art diagnostics (Dx) and biosurveillance (BSV) systems are unable to keep pace with disease outbreaks, fail to provide high trust information to support decision making at the time and place of need, and do not address disease severity. These shortcomings stem from a lack of the right balance of cost, reagent and equipment needs, inability to easily update for new targets, slow time to answer, low number of targets per test, and, most importantly, underperformance on two key metrics important for trustworthy detection – sensitivity (correctly identify a true positive sample) and specificity (correctly identify a true negative sample). Most point-of-need detection is performed with lateral flow immunoassays – a low complexity test in which https://beta.sam.gov/ http://www.grants.gov/ antibodies patterned on nitrocellulose or other media bind protein targets in samples such as clinical specimens (e.g., blood, serum, urine) or other environmental samples (e.g., mosquito lysate, livestock biofluids, etc.). While these low-cost (<$1) tests are easy to operate and tolerate a wide variety of storage conditions, they suffer from poor sensitivity and specificity and are slow to reconfigure for new targets. More complex molecular assays and genetic testing – including PCR, microarrays, and whole genome sequencing – can provide higher sensitivity and specificity and greater multiplex capacity, but are largely incompatible with field-forward stabilization missions due to reagent and equipment needs and/or the time needed to yield an answer (hours to days). In moderate resource conditions, such testing is limited to a few systems in centralized laboratories akin to traditional clinical Dx. BSV workflows similarly require shipment of samples to a centralized laboratory for these more complex molecular assays, slowing sample to answer turnaround time and adding significant cost. Additionally, current assays for detecting host biomarkers that can assess disease severity are primarily lab-based and are not widely available outside of research settings, leaving a significant unmet need for both the military and public health institutions.

In recent years, gene editing technologies have proven to be a promising molecular tool for the detection and modification of genetic material in a manner that is precise, sensitive, rapid, cost-effective, and broadly accessible. Fundamental advances in gene editing technologies have been extended to include detection of disease-relevant nucleic acid targets in samples with high sensitivity and specificity, highlighting their potential utility for a range of Dx and BSV applications. For example, the Cas13-based SHERLOCK1 (Specific High-sensitivity Enzymatic Reporter unLOCKing) and Cas12-based DETECTR2 (DNA Endonuclease Targeted CRISPR Trans Reporter) platforms can differentiate between nucleic acid sequences that differ by a single nucleotide and rely on the collateral activity of the gene editing enzymes to boost signal and assay sensitivity.

Pre-amplification of the target nucleic acids is still typically required to reach single-molecule detection sensitivity, and gene-editing-based diagnostic platforms employ a variety of isothermal target amplification techniques, such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP). However, progress has been made toward rendering pre-amplification unnecessary. For example, the CRISPR-Chip system, which uses CRISPR-Cas9 immobilized on a graphene field-effect transistor to enable electrical detection of nucleic acids, is able to yield results within 15 minutes with femtomolar sensitivity and without target amplification or bulky instrumentation3, and the SHERLOCK platform utilizes effector enzymes in tandem to boost sensitivity by 3.5-fold4. Additional advances directly impact the potential of gene editing-based diagnostics in the field, including INSPECTR5 (Internal Splint- Pairing Expression Cassette Translation Reaction), which employs synthetic biology-based gene

1 Gootenberg et al., Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017 April; 356: 438-442.

2 Chen et al., CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018 April; 360: 436-439.

3 Hajian et al., Detection of unamplified target genes via CRISPR-Cas9 immobilized on a graphene field-effect transistor. Nature Biomedical Engineering. 2019 June; 3: 427-437.

4 Gootenberg et al., Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6.

Science. April 2018; 360: 439-444.

5 Pardee et al., Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell.

2016 May: 165: 1255-1266.

networks to create a paper-based, shelf-stable, diagnostic that works at ambient temperatures, and HUDSON6 (Heating Unextracted Diagnostic Samples to Obliterate Nucleases), a rapid pre-treatment method that enables nucleic acid detection directly from clinical samples, such as bodily fluids, without additional sample preparation. The availability of orthogonal reporter molecules and orthogonal Cas enzymes can also facilitate multiplexing4. Taken together, these recent advances indicate that gene editor-mediated nucleic acid detection technologies have the potential to provide a powerful, flexible new capability for rapid Dx and BSV in the field.

While initial proofs of concept in support of future fieldable gene-editing based detection are encouraging, there remains significant technology innovation and development that is required to deliver the intended radically improved Dx and BSV capabilities. To achieve this goal, DIGET will design, develop, prototype, and deploy two novel nucleic acid detection devices for the simultaneous detection of multiple targets: 1) a disposable point-of-need diagnostic for up to 10 targets, and 2) a massively multiplexed detection (MMD) device for 1,000 or more targets. Both devices must be simple to operate, low-cost, and rapidly reconfigurable to provide high impact, high quality, and trusted information that enhances decision-making. The devices will share a common upstream assay development pipeline and will have different but complementary uses in the field where they could be used individually or together depending on need. The disposable point-of-need device will improve the speed and efficacy of triage and treatment and enhance the standard of care for the military and public health domains; and the MMD device will enable a more comprehensive analysis for early threat detection, high throughput low-volume analysis of samples, assessment of patient status, and improved situational awareness to improve standard of care and inform countermeasure deployment. Successful DIGET technologies will be versatile enough for use in active combat zones, disaster response, and Role/Echelon 1 (point-of-need) and Role/Echelon 2 (MMD) medical settings worldwide.

1.2. TECHNICAL OBJECTIVES AND PROGRAM STRUCTURE

DIGET will encompass a four-year effort organized as two sequential two-year phases of increasing technical complexity. During Phase I, performer teams will establish and integrate the fundamental chemistry and hardware tools necessary to address each of the Technical Areas and will build benchtop (point-of-need) and breadboard (MMD) device prototypes to provide laboratory proof-of-concept for the point-of-need and MMD platforms. Phase II will focus on further development of prototypes, testing with a broad range of clinical and environmental samples, manufacturing, and ultimately field testing to generate data in support of submissions for Food and Drug Administration (FDA) approval. Intermediate and end-of-phase milestones, as outlined in this BAA, will be required in each phase to evaluate progress throughout the program.

The program consists of two Technical Areas (TAs) to be addressed concurrently:

1. Technical Area 1 (TA1): Detection assay design and development;

2. Technical Area 2 (TA2): Device development and deployment.

6 Myhrvold et al., Field-deployable viral diagnostics using CRISPR-Cas13. Science. 2018 April; 360: 444-448.

The objective of TA1, detection assay design and development, is to establish the foundational chemistries to support gene editing-based detection tools and assays for pathogen and host nucleic acid targets. Proposers must target nucleic acids for detection, broadly defined to include DNA and RNA from pathogens for diagnostic purposes and host biomarkers (e.g., cell-free circulating DNA, mRNA transcripts) for prognostic purposes. The objective of TA2, device development and deployment, is to establish the requisite hardware and engineering advances to implement the detection assay chemistries in a low-cost, disposable point-of-need device and a simple, rugged MMD device. Proposers must also develop the front- and back-end bioinformatics pipelines required to design sensitive and specific nucleic acid detection assays and generate actionable data from massively multiplexed (≥1,000 targets) assays. Proposers must also plan to verify performance of both the point-of-need and MMD devices in Capability Demonstrations and a field test and submit a regulatory approval package to the FDA for a proposer-defined, DoD-relevant indication prior to completion of the program.

Both TAs must be developed concurrently over the duration of the effort. Proposers are responsible for ensuring their team has the requisite technical expertise, capabilities, and facilities to address both TAs. Proposals that fail to address both TAs and DoD-relevant indications will be considered non-responsive and not considered for review.

1.2.1. Technical Areas

TA1. DETECTION ASSAY DESIGN AND DEVELOPMENT

In TA1, proposers should focus on the foundational chemistries and tools required for the development of sensitive and specific detection assays to be used in point-of-need and MMD devices. This development will include generation of the enzymes, reporters, probes, and the associated upstream computational pipeline to support reagent and assay design. Proposed approaches must be compatible with multiplexed assays (i.e., ≥10 targets for point-of-need devices and ≥1000 targets for massively multiplexed approaches) and yield assays that are interoperable (i.e., adaptable for use on both point-of-need and MMD devices) and rapidly reconfigurable (i.e., components can be updated to respond to new targets in less than a day).

Proposed reagents must be able to be generated in outbreak relevant timescales (<24 hours) and quantities to meet manufacturing goals of increasing speed and scale throughout the program.

Proposers must design their reagents and assays to address DoD relevant panels of targets, and it is anticipated that multiple panels will be proposed. There may be panels of the proposer’s choosing, but the panels developed must also include panels of DoD relevant targets. These targets must specifically include pathogens and disease biomarkers associated with at least two of the following:

respiratory illnesses (e.g., severe acute respiratory syndrome, influenza), febrile illnesses (e.g., viral hemorrhagic fevers), vector-borne illnesses (e.g., malaria, viral encephalitis), gastrointestinal illnesses (e.g., cholera, giardiasis), and/or sepsis (e.g., bacterial, viral, fungal).

Of the two or more panels of DoD relevant targets selected by the proposer, at least one of these panels must also address host biomarkers to report severity of disease. Proposers may also choose to explore panels of targets of commercial interest. Proposers must clearly define the make-up of their target panels, provide technical rationale for their selections, and include targets that will report comprehensive information about a given threat type (e.g., pathogen subtype, host biomarkers for disease severity, antimicrobial or anti-viral resistance).

It is anticipated that the core nucleic acid detection and signal amplification enzymes will be gene editing-based systems (e.g., Cas122, Cas131, or other novel editors); however, any technology that can achieve the desired assay sensitivity and specificity and other DIGET performance metrics in the form factors required will be considered. For proposers planning to use gene editing enzymes, proposals must describe current enzyme performance (e.g., off-target frequency, turnover, amplification, multiplex compatibility, etc.) and identify strategies for iteration/optimization and troubleshooting/risk mitigation. Because of the desired multiplexing capability, enzymes must operate in an orthogonal manner and not interfere with each other.

In order to detect specific nucleic acid targets in RNA and DNA, proposers must define the probes necessary for identifying the target nucleic acids within a sample, most likely guide RNAs or crRNAs for compatibility with gene editing-based detection strategies. Both broad probes (i.e., targeting sequences that are present across a number of strains/biomarkers) and specific probes (i.e., targeting unique sequences that can identify subtype, rare mutations, and host biomarkers of disease severity) are necessary and must be compatible with both point-of-need and MMD devices. These probes must be designed to maximize orthogonality to other targets and avoid unintended non-specific activity or spontaneous or erratic signal amplification.

Design and discovery of broad and specific target probes for thousands of pathogens and host biomarkers will require a bioinformatics pipeline to support the development of DIGET technologies. Recent advances have demonstrated a wide array of in silico tools to aid in gene editing tool creation and target refinement. Proposers must define their in silico pipeline, including any specific machine learning and artificial intelligence tools required for probe, enzyme, and/or assay design. Proposers must also include strategies to leverage pre-existing data sets to inform their design strategies, indicate how reference genes will be utilized, and how pathogen and host variability will be addressed in their design of universal Dx and BSV tools. In silico models should also support the enzyme design, build, and test process and help optimize overall in vitro assay performance. Modeling and simulation results should complement experimental investigation that quantifies the enzyme system efficacy and characterizes both on-target and off-target system activities. Furthermore, proposers must describe how the in silico pipeline and assay workflows will be agile enough to rapidly (less than a day) design assays for novel or emergent threats, including timelines for designing, building, testing, and validation of new probes from the moment a new target is identified to the time a new test is delivered. This computational pipeline could be adapted from existing, publically available tools or a novel pipeline developed for the DIGET program. If used, reference genomes or other data sets used to build these informatics tools must be specified in the proposal.

To enable sensitive detection of nucleic acid targets, signal amplification will be necessary.

Phase I approaches may be coupled with target amplification (e.g., LAMP, RPA); however, proposers should clearly explain how pre-amplification could be phased out by the Phase I demonstration prior to Phase II. Signal amplification may include approaches that leverage the collateral activity of gene editing enzymes to activate reporter molecules, synthetic biology-based genetic circuit reporters, or other robust approaches. Proposers must define their reporter molecules and strategies to develop assays that have utility for both the point-of-need and MMD devices, which may require different sets of reporters to generate results. Proposers must measure and report the dynamic range of assays, as well as limit of detection (LOD) for each target tested (see Tables 1 and 2: >4-logs dynamic range, <10 copies LOD).

Proposals must clearly define a simple single-step user-performed or automated assay workflow and include strategies that are compatible with collection methods of small sample volume (e.g., finger stick, mouth or nasal swab, sputum specimen, in-stream urine testing), require little-to-no sample preparation (preferred), and approaches to overcome the current state of the art need for pre-amplification of nucleic acid targets in the sample (by the end of Phase I). Assays must be generalizable, that is, compatible with a variety of sample types (e.g., clinical, environmental, and other lab samples) and matrices (e.g., blood, saliva, water, mosquito lysate) that could be encountered in the field. Further, the ability to detect multiple targets simultaneously (i.e., multiplex) is a key feature of both the point-of-need and MMD assays. It is anticipated that the point-of-need assays will measure 10 unique nucleic acid targets per sample tested by the end of the program. For MMD assays, proposers should describe strategies to achieve measurement of 1000 unique targets in a single sample, as well as a single target across 1000 unique samples and variations therein, to provide a maximally versatile tool for field and biosurveillance use.

Proposers should specifically articulate the sample volume requirements for the assays to achieve program goals for sensitivity and specificity, not to exceed 150 ul volume for point-of-need, or

1.5 mL for MMD, consistent with Role/Echelon 1 and Role/Echelon 2 settings, respectively.

Proposers must describe their method of sample collection, and justify sample volume requirements with statistical analysis for probability of detection (e.g., probit curve or confidence interval) of a given sequence at clinically relevant concentrations. Biofluid sample collection should focus on non-invasive or minimally invasive procedures. In order to enable rapid detection using small sample volumes, proposers are encouraged to consider advances in microfluidic technologies that have demonstrated ultrafast kinetics, thousands of reaction compartments at nanoliter scale, and a variety of form factors ranging from media-based, lab-on-a-disk, and lab-on-a-chip at the point-of-need. Proposers must also ensure that compartmentalization of samples into microfluidic droplets will not compromise the sensitivity of the assays, and provide statistical data analysis to support proposed strategies.

By the end of the DIGET program, assays must meet program goals outlined in Table 1.

Proposers must describe how assay performance metrics such as sensitivity, specificity, dynamic range, and time to result will be achieved, and how performance will improve over the course of the program. Proposers must also identify how they will access a variety of benchmark and testing samples that will be used to inform and facilitate detection assay development and to allow demonstration of the reproducibility, generalizability, and performance of the developed assays with real world samples. Samples should be relevant to the chosen concept of operations and of increasing complexity throughout the program. For example, development may begin with contrived and spike-in samples, but must ultimately perform with complex, neat samples.

Assay performance must be regularly assessed against gold standards (e.g., FDA approved tests or error-free reference standards) for the proposer-chosen targets. If no gold standard exists, performers must detail how they will quantitatively evaluate performance using approaches based on method(s) that will be acceptable for regulatory review. Proposers must justify their choice of approach, based on the degree of deviation from the classical paradigm and/or goal metrics (e.g., acceptable reference standard available versus reference standard is imperfect versus reference standard does not exist).

Table 1. Final DIGET Program Goals.

Feature Goal Limit of Detection <10 copies of nucleic acid target

Diagnostic Sensitivity and Specificity ≥98%

Dynamic Range >4 logs Target Nucleic Acid

Similarity Detect single-nucleotide differences

Time (Sample to Answer) <15 minutes Multiplex ≥10 per point-of-need; ≥1000 per MMD

Sample Volume Point-of-need: <150 L; MMD: <1.5 mL Sample Preparation Online or simple single step

Sample Throughput Point-of-need: one-use disposable; 1000s samples per day

MMD: >1,000 tests/disposable component; up to ~ 100,000 tests/day from a single device

Cost <$1 per point-of-need run; <$10 per MMD run Target Pre-Amplification None

Generalizability Compatible with >1 sample type/matrix (e.g., blood, sputum, environmental lysates)

Reconfigurability <24 h Interoperability Assays compatible with point-of-need and MMD devices

Stability and Ruggedness Shelf-stable, minimal/no power requirements Integration Fully integrated sample prep, TA1 Assays, and TA2 hardware

Phase I (Base, 24 months): Detection Assay Design and Development

Phase I should yield new computational, chemical, and/or biochemical methods to detect target DNA and RNA, develop novel foundational detection reagents (e.g., enzymes, reporters), integrate these reagents into generalizable assays, stabilize the reagents for ambient temperature storage, and demonstrate one-step, rapid, and sensitive detection.

Proposers will develop reagents that will enable assays with low limits of detection, robust dynamic range, high sensitivity and specificity, and same-hour results. These reagents must be designed with both the point-of-need diagnostic and the MMD devices in mind, enabling multiplex detections across a variety of samples. Proposers will identify/develop computational pipelines for probe and enzyme designs. High throughput screening techniques may complement in silico tools to identify, design, and optimize functional enzymes for detection chemistry in an iterative manner.

Proposers will develop strategies to measure and mitigate potential failure modes, which must be identified and accompanied with proposer-defined characterization and risk mitigation plans.

Some examples of potential failure modes may include, but are not limited to: off-target activity, lack of multiplex compatibility, unsuccessful activation or inactivation of signal amplification, or presence of inhibitors/confounders in samples. The entire design and validation approach, including the in silico pipeline and experimental investigations, must also allow for the revision of a reagent set or development of a new one within a single day by the end of the program.

Proposers will test their assays against a variety of sample and matrix types, including matrices/samples spiked with target nucleic acids to ensure their assays are generalizable across likely field samples. In order to maximize impact and assess reconfigurability, proposers must select two panels of targets with relevance to DARPA/DoD, and test the assays developed in this TA with real world samples appropriate to the proposed use case. These tests can include internal testing performed during development and must include the capability demonstration in Month 22, as described in Section 1.2.5, Capability Demonstrations and Fielding. Proposers should clearly articulate the source, type, and numbers of samples that would be tested in this phase, the quality of data associated with the samples (e.g., clinical outcomes, data from gold standard reference tests already completed), and if these samples are banked or would need to be collected. Sample types must be justified based on their relevance to a defined DoD need (e.g., tropical febrile panel, biosurveillance in a given geographic region, etc.). Proposers will also complete necessary activities to support delivery of DIGET tools to the Independent Verification and Validation (IV&V) teams for testing prior to the completion of Phase I.

By the end of Phase I, performers are expected to:

identify and develop the enzymes, probes, and reporters required for a point-of-need and MMD nucleic acid detection capability that is:

o specific – e.g., exhibits activity only for intended targets and correctly identifies true negative samples without ambiguity;

o sensitive – e.g., detects low abundance samples in complex sample matrices and correctly identifies true positive samples without ambiguity;

o multiplexable – e.g., compatible with simultaneous measurement of multiple nucleic acid targets; and o reconfigurable – e.g., probes can be swapped into assays with ease to detect new targets with minimal re-optimization of assays;

establish an in silico pipeline for probe and assay design, in vitro validations, and rapid reconfigurations;

demonstrate generalizability by being able to perform with at least two different sample types/matrices;

identify risks and mitigation plans for assay integration into the devices and Phase II assay optimization; and define DARPA/DoD-relevant application (e.g., respiratory, febrile, vector-borne, gastrointestinal panel, or host disease severity) and associated samples for testing and establish approach for IV&V.

Phase II (24 months): Detection Assay Development

In addition to the development of a second DARPA/DoD-relevant panel and any required optimization and validation of the detection assays, the majority of Phase II efforts will involve integrating the assays into the point-of-need and MMD device prototypes and field testing.

Please see Section 1.2.4, Integration, and Section 1.2.5, Capability Demonstrations and Fielding for additional information.

TA2. DEVICE DEVELOPMENT AND DEPLOYMENT

TA2 activities should focus on the development of the novel hardware and engineering strategies required to package the detection assays from TA1 into field-forward, deployable devices. The proposers must address both the point-of-need and MMD platforms - two separate devices must be developed in parallel by each proposer team. Both the point-of-need device and MMD must address the following features: unambiguous results in ≤15 minutes, ≥98% sensitivity and specificity, integrated sample processing (as necessary), reconfiguration in <1 day, compatibility with a wide range (>2) of samples and sample types (e.g., clinical samples, environmental samples), and a low-cost, disposable component (e.g., consumable assay cartridges/devices).

As described in the TA1 section, there is a preference for both no reliance pre-amplification of nucleic acids and for little-to-no sample preparation. Thus, the proposal must detail an approach to increase the likelihood of reagent-target reaction without further concentration or purification of the target and in a manner that will ultimately enable detection within 15 minutes. The proposal must also include a detailed description of how both devices will be generalizable across a range of sample types. For example, for a respiratory application, the device would be able to handle samples with dynamic, non-Newtonian viscoelastic properties (e.g., mucus), which would make precise metering challenging. Similarly, devices that sample blood would need to contend with variabilities in clotting, hematocrit, and other properties which could confound results.

Proposals should clearly describe sample handling (i.e., how the user will input the sample to the device), sample preparation (i.e., how the nucleic acid content of the sample will be accessed), if required, and sample requirements for successful detection (e.g., volume, sample matrix compatibility, types of samples) to maximize broad compatibility across use cases. Ideally, sample preparation will not be necessary, but, if included in the proposal, sample preparation should be one-step, simple (e.g., neat sample added to lysis buffer and invert prior to application on device) and clearly described. For the point-of-need device, ideally there is only an application of a small volume of saliva, blood, or other raw sample type directly to the device. If a sample preparation strategy is proposed for the MMD device, it should ideally be in-line and automated. Proposers must define this strategy, including whether it employs novel or off-the-shelf technologies, and how sample preparation will be integrated into the end device and workflow.

The point-of-need diagnostic must be compatible with a one-step operation (e.g., application of the sample) and a visual output of results that is non-ambiguous with sharp cut-offs and a wide dynamic range. The device also must be able to detect at least 10 different targets simultaneously while being handheld, low-cost, and disposable. All technologies and form factors meeting these requirements will be considered. Recent studies have demonstrated rapid, instrument-free nucleic acid detection using paper-based and shelf-stable materials5, lateral flow technology compatible with gene editing tools6, , and a graphene-based field-effect transistor3. There has not been a demonstration of a low-cost (i.e., <$1), disposable device that can detect up to 10 different targets, and achieving this degree of multiplexing will likely require novel materials, reagents, and/or surface chemistries.

The MMD device must incorporate a low-cost (i.e., <$10), disposable component (e.g., cartridge) for single-step operation by the user (i.e., it contains all reagents and fluidic components necessary for automated sample preparation and detection). Further, the disposable component must allow for massive multiplexing of greater than 1,000 simultaneous detection assays. The proposers must also define the companion equipment that will rapidly read the assay results, generate a clear, simple but detailed visualization of the results, include geographic metadata as part of the results, identify secondary infections, and upload the raw data to a biosurveillance network (e.g., cloud-based) for additional analysis and trend prediction. The companion equipment must be compatible with field use, and proposers must address how they will achieve power source, weight, size, and ruggedness fit for this purpose. Based on the scale of the disposable component and the design of the companion equipment, the proposal must include estimates of sample throughput (i.e., how many samples can be analyzed per unit time and per device/disposable component). Proposals should also address arrangement of the targets and if the system enables random-access (e.g., load samples and consumables on the fly with operator walkaway capability). Sample throughput is of special concern during outbreaks/emergencies when the number of samples to be tested increases dramatically.

Proposers must consider manufacturing and scale in the device designs. In order to meet DoD needs and timelines, DIGET devices must be able to be produced at scale (e.g., tens to hundreds of thousands of consumables, hundreds to thousands of companion MMD devices) and devices/consumables must be reconfigurable in order to react and respond to emerging threat information on time scales able to impact a disease outbreak (e.g., days). Proposers must have a manufacturing plan and detail the expected cost of the platforms proposed as well as justify those costs (e.g., bill of materials, current cost of goods). Agile, rapid reconfiguration (<1 day), and arbitrary arrangement of targets (e.g., from the chosen panels of DoD relevance) of the platform must also be described in the proposal, as well as time to manufacture (preferably less than a week) and distribute. Proposers must also clearly articulate approaches for reconfiguration (i.e., how the devices are reconfigured) and at what stage of deployment the reconfiguration can take place (i.e., where the devices can be reconfigured).

Proposers must justify their device hardware, algorithms, and workflow choices using design principles, data, and appropriate technical rationale, based on:

simplicity and ease of use, the ability to rapidly reconfigure the devices to target new nucleic acids, the ability to meet manufacturing goals, user-friendly readouts, the ability to report on emergent threats, sample throughput, and ruggedness.

Proposers must address both the point-of-need and MMD platforms. Proposals that focus on the generation of only the point-of-need or the MMD device will be considered non-responsive and not considered for review.

Phase I (Base, 24 months): Device development

In Phase I, proposers will establish, design, build, and test prototype point-of-need and MMD devices that incorporate assays developed in TA1 as described above. Depending on the proposer-defined DARPA-relevant applications, the types of devices, the form factors of the device, and the user interface may vary. Concurrent with assay development activities in TA1, performers will gather requirements for successful execution of those assays in TA2 devices, as available. Workflows from candidate assays developed in TA1 should be mapped to corresponding manipulations on the device side. For example, sample processing, handling, preparation (including nucleic acid extraction and clean up) may be workflows from the bench which need to be translated onto the device platform. Target identification, subsequent signal amplification steps, and detection should similarly follow.

For the point-of-need device, performers may need to iteratively design, build, and test prototypes to optimize performance. The use of computer-aided design tools, multiphysics simulation of fluid dynamics, and other computational packages may be used to accelerate device design and predict performance. Materials science development and leveraging advances in chromatography may be necessary to enable the detection assay features defined in TA1, especially multiplexing for 10 different targets. Proposals including point-of-need devices that require an additional reader will be considered non-responsive, but non-instrument readers that produce visual outputs without the need for external power sources, such as instant film, will be considered. The type of reporter developed under TA1 (e.g., gene circuit, nanoparticle, precipitating dye) must be arranged on the device such that the results are easily read and understood, making an effective decision-making tool. For example, this could involve spacing lines or dots on a lateral flow test strips with different chromogenic readouts.

For the MMD device, performers may first establish the standalone modules (e.g., sample handling, sample processing, and detection/readout) for performance of detection assays. Sample handling and processing modules must be compatible with a wide range of sample types and sample matrices. The modules must also be capable of metering/routing the sample throughout the assay process to achieve one step operation for the user by the end of Phase I. During Phase I, nucleic acid amplification steps are not preferred, but will be permitted to achieve sensitivity/limit of detection requirements, and these steps should be incorporated into the device platform. It is expected that the approach for amplification would leverage recent advances to miniaturize and accelerate reactions beyond benchtop formats to meet requirements for sensitivity and speed. The detector, or detection modality, must enable rapid, sensitive, and specific detection of the target sequences.

The MMD companion equipment will house the hardware necessary for the back-end algorithmic analysis. The equipment should have a user-friendly interface, and results from massively multiplexed assays should be reported in simple readouts that clearly communicate the identity and levels of the pathogens detected, and the identity and levels of host biomarkers responsible for the disease severity assessment. Additionally, the companion equipment should be able to integrate into existing BSV networks (e.g., upload assay results to existing biosurveillance networks in a compatible format), allowing for further analyses and immediate trend prediction. By the end of Phase I, proposers will develop breadboards that will enable sample analysis with minimal user manipulation and input, using the assays developed in TA1.

At this milestone in the program, the various modules that may be required for sample-to-answer must be co-located on the same breadboard, and capable of automatically and directly accepting the input from the preceding module versus existing as standalone modules that require user intervention.

By the end of Phase I, performers are expected to:

manufacture 500 units of a 10-plex, handheld, instrument-free prototype point-of-need device, integrate MMD device modules into a breadboard-level prototype with 500-plex capabilities, achieve ≥85% sensitivity and specificity, achieve detection of 1,000 copies of target nucleic acids or fewer demonstrate data analytics and capability to upload results, and have coordinated technology onboarding with IV&V partners, shipped platform to partner site, and successfully demonstrated performance in the hands of IV&V partners.

Phase II (24 months): Device deployment

In Phase II, the performers will prototype and ultimately test in the field integrated, deployable, field-forward point-of-need and MMD devices. Please see Section 1.2.4, Integration and Section 1.2.5, Capability Demonstrations and Fielding for additional information.

1.2.4. Integration

The component capabilities from the TAs must work as an integrated whole to generate point-of-need and MMD devices that meet final DIGET program goals and metrics (Tables 1 & 2).

Therefore, proposals must address two integration efforts in Phase II of each TA: 1) full integration of the assays into point-of-need and MMD devices for TA1 and 2) the integration of MMD device modules into a field-forward device that will fit into a mobile platform (e.g., smaller than a Pelican case) for TA2. Integration plans must include any optimization steps, expected risks and risk mitigation strategies, and testing plans to demonstrate successful performance once components are integrated into their prototype formats. Testing plans must include evaluations of the integrated prototypes using the guidelines outlined in previous sections and allow for third-party testing performed by the IV&V teams that will be coordinated by DARPA. Given the goal of submission to the FDA (e.g., for an Investigational Device Exemption (IDE)), internal testing of integrated devices should be based on guidance that will be recognized as a trusted source by FDA.

The integrated, FDA-relevant product will be an end-to-end system including front-end bioinformatics for design and reconfiguration, sample application, sample preparation, target nucleic-acid detection, signal amplification and readout; and back-end computation for assay data analysis and upload. In order to achieve this goal, the integration of TA1 assays and TA2 prototypes into streamlined point-of-need and MMD devices will require transitioning solution-based assays into formats suitable for long-term storage and performance at field forward locations without the benefits of laboratory equipment and personnel. Additionally, for the MMD device, this effort will include integration of its laboratory-based breadboard components into a fieldable platform. Proposals must describe methods for incorporating all necessary reagents into a shelf-stable format compatible with the requirements and performance metrics for the point-of-need and MMD devices. Proposals must describe how they will ensure long-term stability (i.e., 1 year) of their integrated assays when stored at a range of ambient temperatures and humidity conditions, and how device performance may deteriorate over time at these conditions. Format, stabilization approaches, and integration approach must be justified based on their ability to enable one-step assay execution in the point-of-need and MMD devices with no significant loss in assay performance and the longevity of the assay when stored at ambient temperatures.

Proposals should also describe any optimizations to improve assay performance to meet Phase II goals.

Both the integrated point-of-need and MMD devices must meet or exceed ruggedization metrics in Section 1.3, Program Metrics. Proposals must describe the materials and designs that will be used to mitigate the effects of potential stresses (e.g., drops, vibration, particulates, temperature, etc.), explain why certain conditions are not expected to cause adverse effects, and/or identify stresses that would cause significant to total loss of performance. Proposals must identify the standard to which they are designing (e.g., MIL-STD-810) and detail how resistance to relevant stresses will be evaluated. Ruggedization approaches must be justified based on the number of stresses the final form is expected to mitigate while still meeting the necessary size, weight, power and cost requirements. Furthermore, the integrated forms for both the point-of-need and MMD devices must be produced at an increasing scale in a timely (less than one week) and cost-effective manner.

By the end of Phase II performers are expected to:

demonstrate assay and component integration into final, field-forward point-of-need and MMD devices;

demonstrate sensitive and specific detection of target nucleic acids constituting two DARPA/DoD-relevant panels (respiratory, febrile, vector-borne, or gastrointestinal);

demonstrate ruggedization of devices (metrics detailed in Section 1.3, Program Metrics);

demonstrate detection of <10 copies of target nucleic acid;

demonstrate ≥98% sensitivity and specificity;

define the deterioration of device prototype performance after 1-year storage at multiple, proposer-defined temperature and humidity conditions;

demonstrate manufacturing of 50,000 units of the point-of-need device;

demonstrate manufacturing of 10 units of the MMD device and 1,000 disposable components; and demonstrate successful transfer and testing by IV&V partners.

1.2.5. Capability Demonstrations and Fielding

The integration of novel biochemical assays with prototype devices in an interoperable, deployable platform is critical to the success of the DIGET program. During the four-year program, DIGET performers will complete Capability Demonstrations in each program year to detect nucleic acid sequences from samples of increasing complexity and in decreasing timeframes, to manufacture the point-of-need device in increasing scale, and to demonstrate MMD system performance with an increasingly integrated platform in order to achieve the overall goals of the DIGET program. Devices that are manufactured to meet program metrics are anticipated to be used as consumables for Capability Demonstrations and Field Tests.

During Phase I of the program, individual assays and device module performance will be assessed by Capability Demonstrations at 12 and 22 months, although final integration will not yet be required (i.e., air-gapped modules are acceptable during Phase I). An alpha-prototype of the integrated system will be assessed in the Month 30 Capability Demonstration. By the Field Tests in Month 40, performers must demonstrate end-to-end detection on a fully contained and integrated prototype system.

Phase I, Milestones: Month 12 Phase I, Capability Demo 1: Month 22

Phase II, Capability Demo 2: Month 30 Phase II, Field Test: Month 40

Specific metrics associated with each Technical Area in the Capability Demonstrations are discussed below in Section 1.3, Program Metrics, Table 2. Importantly, subsequent to each Capability Demonstration and Field Test, proposers must transfer their technology to IV&V teams that DARPA will organize for third-party performance assessment and testing. Proposers must include a plan to facilitate transfer of reagents, tools and devices either in parallel or immediately following each Capability Demonstration. Field Tests will comprise testing of point-of-need and MMD devices at domestic and international sites. At a minimum, proposers must define a single domestic or international site for the Year 4 Field Test. In parallel, IV&V teams will test point-of-need and MMD devices at 4 additional sites, to be determined with DARPA, for a total of 5 Field Type sites for each proposer team by the end of the DIGET program. For each Field Test site, teams must test a minimum of 1000 point-of-need tests, and 50 disposable components across 2 MMD devices for a given DoD relevant panel(s), to be agreed upon by the DARPA PM.

In Phase I, proposers should leverage pre-existing sample collections or commercially available samples for testing wherever possible. Later Capability Demonstrations may involve samples collected prospectively for DIGET purposes; however, proposers must clearly…