HR001118S0037-Amendment-01.pdf

Broad Agency Announcement PReemptive Expression of Protective Alleles and Response

Elements (PREPARE)

BIOLOGICAL TECHNOLOGIES OFFICE

HR001118S0037

June 6, 2018

HR001118S0037, PREPARE

TABLE OF CONTENTS

PART I: OVERVIEW INFORMATION

PART II: FULL TEXT OF ANNOUNCEMENT

1. Funding Opportunity Description

1.1. Background and Program Overview

1.2. Program Structure

1.3. Technical Areas, Platform Integration, and Capability Demonstrations

1.4. Program Metrics

1.5. 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

3.4. Other Eligibility Criteria - Collaborative Efforts

4. Application and Submission Information

4.1. Address to Request Application Package

4.2. Content and Form of Application Submission

4.3. Funding Restrictions

4.4. Other Submission Requirements

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

8.1. Frequently Asked Questions (FAQ)

8.2. Agency Level Protests

8.3. Proposers Day

9. APPENDIX 1 – Volume II Checklist

List of Abbreviations

ADME Absorption, Distribution, Metabolism, and Excretion ARS Acute Radiation Syndrome BuChE Butyrylcholinesterase CD Capability Demonstration CRISPR Clustered Regularly Interspaced Short Palindromic Repeats CRISPRa/i CRISPR activator/inhibitor CTI Controlled Technical Information CUI Controlled Unclassified Information DARPA/PRC DARPA’s Public Release Center DEARE Delayed Effects of Acute Radiation Exposure DoD Department of Defense ELSI Ethical, Legal, and Societal Implications EUA Emergency Use Authorization FDA Food and Drug Administration G-CSF Granulocyte-Colony Stimulating Factor GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor gRNA guide ribonucleic acid Gy Gray (unit of ionizing radiation dose) IND Investigational New Drug MCM Medical Countermeasure MoPs Mechanisms of Protection OP Organophosphate PGM Programmable Gene Modulator PGM-MCM Programmable Gene Modulator – Medical Countermeasure PI Principal Investigator PM Program Manager POC Proof of Concept SoA State of the Art ssRNA Single-stranded ribonucleic acid TA Technical Area TPP Target Product Profile

PART I: OVERVIEW INFORMATION

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

Funding Opportunity Title – PReemptive Expression of Protective Alleles and Response Elements (PREPARE)

Announcement Type – Initial announcement Funding Opportunity Number – HR001118S0037 Catalog of Federal Domestic Assistance Numbers (CFDA) – 12.910 Research and

Technology Development Dates o Posting Date: June 6, 2018 o Proposal Abstract Due Date: July 12, 2018 o Proposal Due Date: August 27, 2018 o Any other relevant date(s) - Proposers Day, June 13, 2018 https://www.fbo.gov/spg/ODA/DARPA/CMO/DARPA-SN-18-45/listing.html

Concise description of the funding opportunity: The PREPARE program aims to develop programmable gene modulators for humans that can provide specific, effective, safe, and transient medical countermeasures and prophylaxes to combat biological, chemical, and/or radiological threats to public health and national security.

Anticipated individual awards - Multiple awards are anticipated.

Types of instruments that may be awarded - Procurement contract, cooperative agreement or Other Transactions.

Agency contact o Points of Contact The BAA Coordinator for this effort may be reached at:

PREPARE@darpa.mil

DARPA/BTO

ATTN: HR001118S0037

675 North Randolph Street Arlington, VA 22203-2114 https://www.fbo.gov/spg/ODA/DARPA/CMO/DARPA-SN-18-45/listing.html mailto:PREPARE@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) is soliciting innovative proposals to develop a platform capability for the production of Medical Countermeasures (MCMs) that bolster natural genetically-encoded defenses against biological, chemical, and/or radiological threats to public health and national security. Proposed research should investigate disruptive approaches for the identification of gene targets that, when modulated, confer protection or resistance against these threats; the development of novel tools to modulate the activity of target gene(s); and the generation of clinical formulations that enable delivery to the appropriate cells and tissues in vivo. The integration of these approaches into a platform that can produce novel MCMs should enable a specific, potent, safe, and transient approach for directly modulating the activity of target genes to limit the damaging and potentially fatal consequences of a broad range of threats to warfighters, first responders, and civilian populations.

1.1. BACKGROUND AND PROGRAM OVERVIEW

Influenza pandemics, organophosphate poisoning, opioid overdose, and exposure to ionizing radiation are representative of the broad spectrum of biological, chemical, and radiological threats that can significantly impact public health and national security. These seemingly disparate threats share a common challenge: despite decades of laboratory experimentation and significant research investment, broad-acting, longer-lived, and effective MCMs remain elusive.

While current pharmaceutical and biologic MCMs confer some protection for these categories of threats, strategies to rapidly and flexibly discover, design, manufacture, scale, and administer these solutions remain inadequate for public health emergencies, pandemic preparedness, and force protection. The current one threat/one MCM paradigm is not sufficient to address the diverse and rapidly evolving threat space.

The human genome encodes a number of genes whose products can confer some protection against these threats, however, the time between threat exposure and expression of protective genes is often too long, leading to severe symptoms or even death. DARPA’s PReemptive Expression of Protective Alleles and Response Elements (PREPARE) program expands upon recent advances in genome engineering, insights into host/threat response, and in vivo delivery to implement a novel paradigm for the development of a common molecular architecture for MCMs that bolster innate host defenses to combat an array of national security and public health threats. PREPARE is anticipated to deliver a capability to generate programmable gene modulator MCMs (PGM-MCMs) to enable safe, transient, and preemptive activation of intrinsic host defenses against a variety of threats to health, as well provide novel solutions for the direct neutralization of pathogenic viral genomes.

Importantly, transient and clinically relevant PREPARE PGM-MCM interventions may also provide an alternative to permanent somatic cell genome editing approaches that permanently alter host primary DNA sequences (e.g., CRISPR-Cas9). PGM-MCMs may act at the level of the transcriptome, the epigenome, and the epitranscriptome. PGM-MCMs should consist of noncanonical and non-permanent genome editors, defined as transient and programmable modulators of endogenous gene targets, transcripts, and pathways that do not alter primary DNA sequence, cause life-long gene expression changes in the host, or create DNA lesions in a host genome in any way1.

PGM-MCMs should be designed in a manner that enables somatic interventions at common pathway nodes to enhance protective host responses across multiple threats, promote universal neutralization of viral threats, and be easily re-targeted and re-configured for rapid adaptation and response to new and emergent threats. This architecture should be compatible with the potential for stockpiling common MCM reagents (e.g., an RNA editor) and time-of-need manufacture of PGM-MCM reagents (e.g., guide RNAs) to enable the most agile, timely, and robust response to known and emergent public health and national security threats.

Specific proofs of concept demonstrated under the PREPARE program will focus on measurable progress towards the development and testing of PGM-MCMs to address four primary threats that represent a range of threats to public health and national security that may be encountered.

The four primary threats that PREPARE will address are:

Influenza viral infection Opioid overdose Organophosphate (OP) poisoning Exposure to gamma radiation

MCM development for influenza poses unique challenges due to strain variation and the ability to rapidly mutate and resist existing MCMs or evade the immune system. Highly conserved components of the influenza virus have provided insights into possible universal treatment or prophylaxis options, but these have proved elusive thus far. In addition, the demonstrated ability to synthesize and engineer biological threats de novo (e.g., 1918 influenza) further emphasizes the need for agnostic MCM platforms that are easily re-configurable and compatible with simple and rapid manufacture. Direct neutralization of viral genomes in highly conserved regions may afford near-universal protection for the host and reduce the likelihood of developing viral resistance. Approaches that modulate host defenses against viruses, or bolster immune responses, may further amplify protection. For example, recent studies have identified host gene targets that, when modulated, confer protection against influenza infection in experimental systems2,3.

1 Wegrzyn et al., Genome Editing: Insights from Chemical Biology to Support Safe and Transformative Therapeutic Applications. ACS Chem Biol. 2018 Feb 16;13(2):333-342.

2 Han et al., Genome-wide CRISPR/Cas9 Screen Identifies Host Factors Essential for Influenza Virus Replication.

Cell Rep. 2018 Apr 10;23(2):596-607.

3 Heaton et al., A CRISPR activation screen identifies a pan-avian influenza virus inhibitory host factor. Cell Rep.

2017 Aug 15; 20(7): 1503–1512.

MCMs against opioid overdose or organophosphate pesticides require rapid intervention to improve patient prognosis. Currently, the state of the art (SoA) for mitigation of opioid (e.g., fentanyl) or organophosphate (e.g., pesticides) poisoning are small molecules such as naloxone or atropine/pralidoxime, respectively. While effective, these small molecule MCMs require immediate administration to reverse effects of poisoning and require repeat dosing due to short half-lives. SoA biologics such as human-derived butyrylcholinesterase (BuChE) for the stoichiometric neutralization of OPs need to be recombinantly produced or purified from human plasma, then intravenously administered as a prophylaxis against organophosphate exposure, which is costly, not scalable, and requires frequent re-administration.

For treatment of acute radiation syndrome (ARS) that may result from radiation therapy or occupational exposures, FDA-approved treatments are limited to recombinant human proteins G- CSF (Neupogen® and its PEGylated form Neulasta®) and GM-CSF (Leukine®), administered to stimulate hematopoietic response. These MCMs do not address the full range of ARS symptoms and require frequent re-administration and close monitoring of patient health.

In order to bridge the gap between experimental proof of concept and clinical translation of PGM-MCMs to solve these public health and national security challenges, development of the PREPARE capability will require innovation across several key component areas to generate a platform that can be applied in an agnostic manner for any threat category. This platform should be designed to determine: which genes exhibit intrinsic protections against the threat; which programmable gene modulation tools are most appropriate to modulate the activity of those genes for a given threat; and how and where the PGM-MCMs should be delivered in vivo for greatest protective effect against a threat (Figure 1). The integrated PREPARE platform components and resulting PGM-MCMs should be iteratively tested and optimized in vivo against various threat challenges with increasing complexity and performance requirements on a path towards clinical translation and regulatory application submission; for example, Food and Drug Administration (FDA) Investigational New Drug (IND) or Emergency Use Authorization

(EUA).

Figure 1 Schematic of PREPARE program objectives.

1.2. PROGRAM STRUCTURE

PREPARE is structured into a four (4) year effort consisting of two (2) phases for two (2) years each (Figure 2).

Figure 2 PREPARE Program Structure & Timeline.

Phase I

Technical Area Component Development Proposers must indicate their primary selection of the relevant threats that they intend to pursue under the PREPARE program from the following: influenza, opioids, organophosphates, or gamma radiation. In Phase I, proposers should concurrently develop and integrate component innovations spanning three Technical Areas (TAs):

TA1: Gene Target Identification TA2: Programmable Gene Modulator Development TA3: Clinical Formulations

During Months 1 to 12 of Phase I, proposer teams should generate foundational capabilities to identify targets within genomes that can counter the health risks posed by influenza, opioids, organophosphates, or gamma radiation (TA1); develop libraries of programmable gene modulators to control protective gene activity (TA2); and develop formulations to deliver the programmable gene modulators to the cells and tissues where their activity is required (TA3), and should establish proof-of-concept that these capabilities can be applied to reduce the effects of the selected threats in in vitro systems.

Platform Integration and Capability Demonstration 1 Beginning in Month 13, proposers are required to functionally integrate TA1, TA2, and TA3 components into a platform that enables the production of PGM-MCMs for testing against the selected threat(s) in vivo. Phase 1 should culminate with a proof-of-concept Capability Demonstration 1 (CD1) to test the performance of PGM-MCMs against SoA MCMs in a relevant in vivo animal model against the proposer-selected threat. By the end of Phase I, proposers must also demonstrate multiplex capability (i.e., >1 target modulated, such as multiple protective genes modulated or multiple sites within a viral genome neutralized) and/or generalizability (i.e., >1 threat protected against, such as multiple influenza strains) of PGM-MCMs for a variety of genomic targets and threats in vitro and/or in vivo. CD1 must be completed by Month 22. During Phase I, proposers must also draft a Target Product Profile (TPP) and engage with the appropriate regulatory bodies (e.g., FDA pre-pre-IND and/or EUA discussions) to inform PGM- MCM development and strategy for eventual clinical translation.

Phase II

PGM-MCM Development and Capability Demonstrations 2 and 3 Phase II will focus on optimizing and expanding the capabilities of the integrated PGM-MCM discovery and production platform. Proposers should continue to refine the integrated platform so that it may develop into a robust platform that can be readily tested to generate relevant PGM- MCMs to reduce host risk against a broad range of novel or emerging public health and national security threats. In order to demonstrate measurable progress against PREPARE program goals, proposers should conduct increasingly more challenging Capability Demonstrations – CD2 to be completed by Month 32 and CD3 to be completed by Month 40 – to demonstrate greater in vivo protection; increased formulation versatility; greater understanding of the mechanisms of protection (MoPs) and failure; and thorough in vivo characterizations of PGM-MCM activity to include absorption, distribution, metabolism, and excretion (ADME), immunogenicity, and toxicity studies.

Regulatory Submission Phase II should culminate in an IND or EUA submission to the FDA. Pre-IND or equivalent EUA interactions are anticipated during Phase II to inform PGM-MCM development and eventual clinical translation. Prior to the end of the program (Months 41 to 48), proposers should finalize all necessary IND/EUA submission requirements, data analysis, platform optimization, and plans for technology transition. Although pre-EUA activities may be pursued, they should not replace efforts to develop PGM-MCM products for FDA approval through IND pathways and, where possible, IND and EUA activities should proceed in parallel. Successful teams should advance their most promising final PGM-MCM products for a given threat(s) indication for IND and/or EUA submission to the FDA by the end of the program.

1.3. TECHNICAL AREAS, PLATFORM INTEGRATION, AND CAPABILITY

DEMONSTRATIONS

TA1: GENE TARGET IDENTIFICATION

TA1 is focused on the identification and validation of: (a) endogenous host genes or druggable targets that, when modulated (e.g., up- or down-regulated), confer protection against the selected threats; and/or (b) viral genome targets for neutralization. Proposers must develop agnostic, genome-wide approaches (e.g., CRISPRa/i, etc.) to characterize host genomic, transcriptomic, epigenomic, and/or epitranscriptomic drivers of protection against the selected threats using high-throughput molecular and bioinformatic methodologies. Proposers must also provide insight into the dynamic range and minimum gene expression thresholds that must be achieved to confer host protection against threat(s). If relevant, proposers must also determine the gene regulatory elements that will enable gene modulation (e.g., promoters, enhancers). In the case of viral threats like influenza, it is anticipated that direct modulation and neutralization of the viral genome at one or more target sites may serve to further protect the host from this category of threat and reduce the likelihood of development of resistances or evolutionary escape. Similarly, exposure to health threats may result in a polygenic host protective response where ultimately the modulation of multiple gene targets (i.e., multiplex) may provide optimal protective response.

Modulation of gene targets identified in TA1 should significantly improve physiological outcomes, including survival and reduction of pathologies following exposure to a threat. In addition to de novo data collection during TA1, proposers are encouraged to leverage and expand upon pre-existing human datasets and/or archived patient samples relevant to the pursued threat.

TA1 development should yield foundational data and should be staged such that preliminary screening and target validation may involve, but not require, in vitro experimentation prior to in vivo work in an appropriate animal model. To identify protective gene targets and optimal cell and tissue types, screening and validation against selected threat(s) may be performed: (a) in vitro in relevant organoid and/or microtissue/microphysiological culture systems that best approximate human physiology; and/or (b) in vivo in a threat-relevant healthy animal model. Use of cell cultures that are not representative of healthy cellular biology and physiology will require additional justification; proposals exclusively employing cancer cell lines for gene target discovery will be considered out of scope. Proposers must clearly indicate the approach to determine baseline healthy gene expression states and protective gene expression ranges in a given in vitro or in vivo model, and methods to quantify perturbations to those states. It is critical that proposers ensure that protection from threats through modulation of the identified protective genes can be conferred while avoiding any toxic, immunogenic, carcinogenic, or otherwise adverse effects in healthy organisms.

Successful completion of TA1 activities should yield a component capability that can be used to scan genomes to identify genes that, when modulated, should protect a host against a given threat. By the end of month 12, proposers are expected to use the TA1 component capability to generate a prioritized list of protective candidate genes, relevant regulatory regions, target cell and tissue types, and gene target threshold and range of expression that must be reached in order to confer protection against the proposer-selected threat(s) to be tested in subsequent CDs. These key TA1 deliverables define the gene target(s) specifications that inform the draft TPP and integration with innovations from TA2 and TA3 in a synergistic manner and help refine performance metrics for PGM-MCMs in vivo beginning in Month 13.

TA2: PROGRAMMABLE GENE MODULATOR DEVELOPMENT

TA2 is focused on the design and development of PGMs that can be used preemptively or post-exposure to alter gene expression levels of target alleles to confer protection against (or directly neutralize) selected threat(s) in vivo in a manner that leaves primary DNA sequence intact and avoids life-long alteration of transcripts or gene expression patterns. As such, PGMs may include, but are not limited to: targeted DNA-binding protein scaffolds linked to transcriptional activators, repressors, and epigenetic modifiers; targeted RNA-nucleases; and RNA base editors.

PGMs may also transiently alter the abundance or half-life of gene products or transcripts or the primary RNA sequence of a transcript itself for the desired effect.

Proposers must establish candidate PGM libraries of component parts and perform high-throughput, combinatorial screens to test PGMs in in vitro cell, organoid, and/or in vivo systems to determine optimal designs for programmable and controllable modulation of target genes across a broad dynamic range of expression (e.g., 2-fold to 10,000-fold) and duration of effect (e.g., hours to months). Proposers must validate PGM performance in vivo. Given that there are multiple ways in which optimal level and duration of gene modulation may be achieved (e.g., epigenetically, at the transcript level, through delivery formulation (see TA3 below), or combinations therein), proposers must clearly indicate how their PGM testing approach will enable the selection of the best and most robust approach among them to address both gene modulation level and duration challenges. Proposers are expected to leverage bioinformatic tools and models to expedite PGM design, development, and performance prediction. Proposers must clearly indicate how their selected PGM methodologies will address: gene target specificity;

mitigation of risk for off-target effects; tunable control of gene modulation level and duration;

ease of manufacture and scale-up; adaptation to new gene targets; and multiplexability and generalizability. Proposers must also assess and mitigate risk for intended downstream in vivo applications, including potential for pre-existing immunities and/or de novo immune response.

Successful completion of TA2 should yield a component capability that can be tested to determine the most appropriate molecular tools to modulate protective gene targets. By the end of month 12, proposers are expected to use the TA2 component capability to generate a prioritized list of top candidate PGMs (sequence, format, etc.); an understanding of the mechanism of gene modulation (e.g., epigenetic, transcriptomic, etc.); and key PGM features including range and duration of gene modulation to enable effective, safe, transient, specific, and multiplexable modulation of target genes to confer protection against the selected threats to be tested in CD1. These key TA2 deliverables define the PGM specifications that inform the draft TPP and integration with innovations from TA1 and TA3 in a synergistic manner and help refine performance metrics for PGM-MCMs in vivo beginning in Month 13.

TA3: CLINICAL FORMULATIONS

TA3 is focused on developing novel clinical formulations to enable cell-, tissue-, and/or organ-specific delivery of PGMs to access gene targets that are protective against the selected threats.

Proposers may pursue any delivery formulation including, but not limited to, viral vectors, lipid nanoparticles, polymer-based systems, mRNA, or completely novel formulations.

Proposers should generate high-throughput, combinatorial screens of delivery formulations for PGMs. Proposers are encouraged to either explore delivery in organoid, microtissue, 3D cell culture, or similar systems that may be more predictive of in vivo success while maintaining sufficient throughput for iterative optimization of a chosen clinical formulation or initiate studies directly in vivo. It is important to note that the cell and tissue context within which intrinsic gene-encoded host protections are activated may be as critical to the protective response as the genes themselves. Similarly, the “normal” spatial expression pattern of an endogenous threat-protective gene may not always correspond to the most optimal target for induction of preemptive or post-exposure protection. Therefore, proposers must describe the methods that will be employed to determine the optimal cell/tissue/organ targets for robust protective response. Proposers must validate delivery formulation performance in vivo, and are expected to leverage bioinformatic tools and models to expedite delivery formulation design and testing.

Proposers must clearly indicate how their selected delivery formulation approach will address, and mitigate risk for potential failure modes, such as immunogenicity (adaptive and pre-existing) that would interfere with re-administration/multi-dosing treatment regimens, endosomal escape, toxicity, lack of cell tropism diversity, suboptimal ADME kinetics, cargo size limitations, off-tissue effects, and ease of manufacture and scale-up.

Successful completion of TA3 should yield a component capability that can be tested to determine the most appropriate delivery formulations to access target cells and tissues for a given threat. By the end of month 12, proposers are expected to use the TA3 component capability to generate a prioritized list of top candidate delivery formulations to enable highly-specific PGM cargo delivery in vivo and optimal cell types, tissues, and organs for maximal threat protection through target gene modulation to be tested in CD1. These key TA3 deliverables define the delivery formulation specifications that inform the draft TPP and integration with innovations from TA1 and TA2 in a synergistic manner and help refine performance metrics for PGM- MCMs in vivo.

PLATFORM INTEGRATION AND CAPABILITY DEMONSTRATIONS

Beginning in Month 13, proposers are expected to address the topics below (in order of occurrence):

1. Target Product Profile: submit to DARPA a draft TPP for PGM-MCMs in development.

2. Platform Integration: integrate innovations from TA1, TA2, and TA3 components into a platform capability.

3. Capability Demonstrations: test integrated PGM-MCMs to protect against selected threats in 3 increasingly difficult CDs.

4. PGM-MCM Characterization: comprehensively evaluate and characterize PGM-MCM activity in vivo after each CD.

Target Product Profile:

PGM-MCM use cases should be specified in a draft TPP that states the overall intent, development process, and end specifications of the PGM-MCM. The draft TPP should include foundational knowledge established in Year 1 including gene target(s) specifications (TA1), PGM specifications (TA2), and delivery formulation specifications (TA3). Advantages of using a TPP early in the development process can facilitate the efficiency of FDA interactions and communications. A TPP helps focus the development of a product and can help address issues early on in the development process thereby preventing late-stage development failures and decreasing the total time involved with development. Early TPPs can be brief depending on the status of the development process.

Platform Integration:

Proposals must address how platform integration and optimization are expected to proceed from Months 13 through 48, including optimization steps, expected risks, and risk mitigation strategies. The component capabilities from TAs 1-3 should work as an integrated whole to generate PGM-MCMs. However, it is anticipated that various component development strategies may be re-visited at varying times throughout the program (e.g., following each Capability Demonstration) to optimize PGM-MCM performance and refine platform functionality. For example, following a given in vivo test, if a given PGM-MCM provides encouraging results, but does not reach performance targets for level of gene expression, proposers may revisit the TA2 programmable gene modulator component to identify PGM tools with an improved dynamic range of expression control and/or TA1 gene target identification component to identify improved target sequences (e.g., regulatory elements) that are supportive of higher levels of expression.

Capability Demonstrations:

PGM-MCM safety, efficacy, specificity, and transience must be tested in vivo in CDs. While proposers are encouraged to test PGM-MCM multiplexability (e.g., multiple targets) and generalizability (e.g., multiple threats) in vivo prior to the end of Phase I, in vitro proof-of-concept for these two criteria will fulfill program requirements during this period.

A series of three sequential CDs of increasing difficulty should be designed to test the ability of integrated PGM-MCMs to protect against selected threats prophylactically (pre-exposure or immediate post-exposure administration) and/or therapeutically (post-exposure administration) in healthy threat-relevant model organisms (Figure 3). It is intended that CDs follow design-test-learn-iterate principles where experimental outcomes and regulatory feedback from CD1 should inform design and implementation of CD2, and so on, to facilitate eventual clinical translation of

PGM-MCMs. Specific investigations of the mechanisms of protection (MoPs), failure modes, and a thorough in vivo characterization of PGM-MCMs and outcomes should be included at each CD in order to inform subsequent designs. CDs can begin at any time but must be completed by Month 22 (CD1), Month 32 (CD2), and Month 40 (CD3).

Figure 3 PREPARE Capability Demonstrations

For each CD, proposers must compare the performance of PGM-MCMs head-to-head against SoA MCMs for a given threat and must demonstrate improved performance compared to SoA MCMs (≥2× SoA for CD1; ≥10× SoA for CD2; ≥100× SoA for CD3) where improvement over SoA should focus on improvements in host functionality and survival, including, but not limited to, improved level of protection (e.g., survival rate), time to protection, breadth of threats covered (e.g., broad activity against multiple strains/universality), and/or duration of protection (e.g., months). For additional details regarding metrics, please see Section 1.4 PROGRAM

METRICS.

For each successive CD of increasing difficulty, proposers are expected to demonstrate further utility of PGM-MCMs, which may include, but not be limited to:

Increasing threat exposure dose while maintaining or improving protection and/or duration of protection

Increasing number of PGM-MCM targets (e.g., multiple host genes, or host gene plus direct viral genome neutralization targets) for increased protection

Expanding from one threat category (e.g., chemical) to multiple (e.g., chemical and radiological)

Expanding from one threat to multiple threats within the same category (e.g., protection against multiple viral flu subtypes (e.g., A and B) or strains (e.g., H1N1, H3N2, H7N9, and H5N1) simultaneously)

Increasing efficacy of existing MCMs, when used in combination with PGM-MCMs

PGM-MCM Characterization:

Throughout the Integration and Capability Demonstration activities that are anticipated during Months 13-48, in addition to assessment of survivability and duration of protection outlined above, proposers should pursue a comprehensive characterization of PGM-MCM activity in vivo.

Proposers must demonstrate progress against intermediate performance and characterization goals and develop an increased understanding of the mechanisms, and potential failure modes, that underlie PGM-MCM activity in vivo. These intermediate assessments are expected to occur in parallel with the CDs and must increase in complexity and comprehensiveness with each subsequent program Phase and CD, including (but not be limited to) an understanding of the mechanisms of protection, failure modes, impact on symptomatology and pathology, measurement of ADME properties, toxicity, on- and off-target activity in host transcriptome, on-and off-tissue activity, immune response (pre-existing and adaptive). More specific examples include:

Months 13 – 24/CD1: Characterization in Months 13-24 is focused on acute responses (minutes to hours). Proposers must further refine the kinetics of PGM-MCM modulation of protective host targets and establish a quantitative representation of system kinetics to include:

o temporal requirements for modulation (ramp up and ramp down) and magnitude of activation/repression response;

o measurement of on/off-target activity or toxicities at the level of the genome, transcriptome, and epigenome as well as the cell, tissue, and organ level;

o ADME measurements over a minimum 24-hour period to understand system kinetics;

o physiological responses, including immune responses (i.e., immune cell activation, inflammatory response, and cytokine release);

o standard toxicology studies;

o molecular and histological analyses of cellular targets and tissues;

o behavioral responses to include waking, sleeping, feeding, grooming, etc.

Proposers must assess, beyond survival/death, the impact of the PGM-MCM on all of the subcategories described above and syndromes relevant to the specific threat (s) investigated.

Months 25 – 36/CD2: Characterization as above for Months 13 – 24 (acute responses), and additionally:

o persistent responses (days – weeks);

o route of administration;

o dosage;

o any requirements introduced through regulatory engagement.

Months 37 – 40/CD3: Characterization as above for Months 13 – 36, and additionally chronic responses (months) and extensive longer-term toxicology studies, including:

o genetic toxicology;

o carcinogenicity;

o reproductive and developmental toxicity;

o any requirements introduced through regulatory engagement.

Data generated during intermediate characterizations and assessments should provide the basis for optimization and improvement in subsequent iterations of PGM-MCM design and implementation. Proposers must also not only quantitatively define and describe a method to measure duration and magnitude of protective response in vivo, but also demonstrate the transient nature of PGM-MCMs either passively through basal cellular processes or actively through self-contained timed reversal to return the system to a baseline state. Proposers may consider exogenous application of gene targets (i.e., gene-encoded or as biologics) to assist with determination of dosing regimens, etc.

FURTHER RECOMMENDATIONS FOR PGM-MCM TESTING

Proposers must select a threat (influenza, opioid overdose/poisoning, organophosphate poisoning, or gamma radiation) that should remain consistent throughout their proposal to ensure the most cohesive and impactful program outcome. Additional national security or public health relevant threats may be explored in a limited manner in Phase I (e.g., in vitro demonstration only) to demonstrate the feasibility of generalizability and multiplexability of the PGM-MCM approach. If additional threats are proposed for investigation in a limited manner, appropriate technical justification, such as overlap in MoPs or simulation of real-world scenarios, must be included. Additional threat-specific recommendations are included below.

Influenza viruses:

Proposers pursuing the influenza viral threats must investigate the ability of PGM-MCMs to neutralize multiple influenza strains and sub-types to meet generalizability requirements for Phase I, but proposers may also pursue limited investigation (e.g., in vitro demonstration) of the ability to neutralize additional ssRNA viruses with clear national security relevance for additional demonstration of PGM-MCM platform capability. De novo synthesis or re-creation of eradicated viral threats or genesis of novel pathogens, or pathogens with increased transmission or pathogenicity, are not permissible under the PREPARE program. To identify improvements in the symptomatology, pathology, and understanding of mechanisms of protection (or failure), proposers should utilize assays, methods, and animal models appropriate to the investigation of influenza to quantify relevant exposure phenomena such as viremia, anti-viral cytokine profiles, host transcriptomic responses, transmission, etc.

Opioids and organophosphates:

If opioids are selected as a threat target, proposers should focus on mitigating the toxic effects of opioids (e.g., respiratory depression, increasing metabolic turnover) or developing non-opioid approaches to pain management. Development of non-addictive pain solutions in general or treatments for addiction is considered out of scope for the PREPARE program. To identify improvements in the symptomatology, pathology, and understanding of mechanisms of protection (or failure), proposers should utilize assays and methods appropriate to the threat(s) chosen to quantify relevant exposure phenomena such as serum levels of metabolites or enzymatic activity, respiratory rate, opioid receptor occupancy, etc.

Gamma radiation:

It is highly encouraged that proposers assess increasing doses of total body irradiation (e.g., 0.5 to 10 Gy) to identify gene targets that may confer radiation protection using a proposer-chosen radiation source that can recapitulate radiation exposure relevant for therapeutic radiation uses or occupational exposures. Proposers should quantitatively define and provide technical justification for the screening doses and timing of PGM-MCM intervention, and indicate how the selected approach relates to public health and national security need. To identify improvements in the symptomatology, pathology, and understanding of mechanisms of protection (or failure), proposers should utilize assays and methods appropriate to the radiological exposure chosen to quantify physiological phenomena such as, measurement of hematopoietic response, DNA damage, cell death, cognitive impairment, and other physiological changes associated with Acute Radiation Sickness (ARS) subsyndromes (gastrointestinal, hematopoietic, and neurovascular syndromes) or Delayed Effects of Acute Radiation Exposure (DEARE).

Animal models:

For each threat type, proposers must identify an animal model used in each CD, and relevance to downstream human clinical translation. Proposers must provide technical justification as to why the model is appropriate for the chosen threat type (e.g., mode and magnitude of exposure, citation of literature supporting MCM development for specified threat using the model with physiological and molecular responses relevant to human exposure). Furthermore, it is expected that animal models used in the CDs are healthy, wildtype organisms. Proposers may use transgenic organisms, such as reporter lines, but must clearly articulate how the transgenic features do not interfere or bias results or responses to the selected threat.

1.4. PROGRAM METRICS

In order for the Government to evaluate the effectiveness of a proposed solution in achieving the stated program objectives, proposers should note that the Government hereby promulgates the following program metrics that may serve as the basis for determining whether satisfactory progress is being made to warrant continued funding of the program. Although the following program metrics are specified, proposers should note that the government has identified these goals with the intention of bounding the scope of effort, while affording the maximum flexibility, creativity, and innovation in proposing solutions to the stated problem. Note that individual metrics for TA1, TA2, and TA3 must be met by Month 13, while all other metrics refer to the remainder of the program.

Quantitative performance metrics are expected to vary for each proposer-selected threat area and system. Proposers to the PREPARE program are required to define ambitious, specific, and quantitative metrics in support of program goals, including intermediate metrics (e.g., every six months or sooner) to help further evaluate progress. Some exemplary milestones and metrics are included below for proposers to consider, but proposers should adjust accordingly for their given threat and system. Final metrics are to be determined at time of award negotiation and are subject to DARPA approval. Proposers should note that program metrics may serve as the basis for determining whether satisfactory progress is being made to warrant continued funding of the program.

Throughout the course of the program, proposers must compare the performance of PGM-MCMs head-to-head against SoA MCMs for a given threat and must demonstrate improved performance that increases over time with each subsequent Capability Demonstration: ≥2× SoA for CD1;

≥10× SoA for CD2; ≥100× SoA for CD3. Performance metrics should focus on improvements in host functionality and survival, including, but not limited to, the following categories:

level of protection/survivability (e.g., survival rate) improved survivability at increased threat exposures/doses time to protection from MCM administration time window for effective treatment (i.e., time window between threat exposure and administration of MCM with maximal efficacy) breadth of threats covered at maximum protection (e.g., broad activity against multiple strains/universality) duration of protection (e.g., months)

Proposers must clearly indicate their target performance metrics for each CD. These metrics must describe the SoA MCM that will be used to benchmark performance, must define, in quantitative and qualitative terms, what is considered threat “protection” in vivo, and describe how it will be measured (e.g., target serum concentration of protective gene product detected for threat-relevant duration). Successful completion of all CDs should yield a PGM-MCM with at least 100× improved performance in vivo over SoA for a given threat agent in at least one performance category.

Table 1 below is exemplary of proposed performance metrics relative to SoA MCMs that must be provided by each proposer team. Additional program metrics appear in Tables 2-5. Proposers may select more than one performance category to exceed SoA MCMs, but must meet at least one performance category at ≥2× SoA for CD1; ≥10× SoA for CD2; ≥100× SoA for CD3. Teams that propose PGM-MCM solutions that exceed 2×, 10×, and 100× SoA for more than one performance category will be viewed more favorably. The category(s) selected should remain consistent throughout the course of the program, and may include, but are not limited to:

Table 1 – Performance Metrics Examples

CD1

≥2× SoA

CD2

≥10× SoA

CD3

≥100× SoA

Threat Performance Category SoA MCM

Initial Therapeutic PGM-MCMs

Improved Therapeutic PGM-MCMs

Final Therapeutic PGM-MCMs

Neuraminidase inhibitor/antiviral (e.g., oseltamivir)

Influenza example

(Treatment)

Duration of Protection

1 – 10 hr serum half-life

20 hr serum half-life/duration of protection

100 hr serum half-life/duration of protection

1,000 hr serum half-life/duration of protection

Seasonal Flu VaccineInfluenza example (Prophylaxis)

Breadth of Threats Covered 3 – 4 strains protection

6 strains 30 strains 300 strains

Competitive opioid receptor antagonist

(e.g., naloxone)Opioid example

(Treatment)

Duration of Protection

~1 hr serum half-life

2 hr serum half-life/duration of protection

20 hr serum half-life/duration of protection

200 hr serum half-life/duration of protection

Muscarinic cholinergic receptor antagonist (e.g., atropine)

Organophosphate example

(Treatment)

Duration of Protection

3 hr serum half-life

6 hr serum half-life/duration of protection

30 hr serum half-life /duration of protection

300 hr serum half-life/duration of protection

Hematopoietic colony-stimulating factor (e.g., G-CSF)Gamma radiation example (Treatment)

Administration Efficacy Time

Window <2 hr post-exposure

<4 hr post-exposure

<20 hr post-exposure

<200 hr post-exposure

Table 2 – TA1 metrics

TA1: Gene Target Identification (Months 1-12)

Milestones and Deliverables Program Metrics

Perform high-throughput, agnostic screens to identify gene targets and regulatory elements that confer protection (or enable neutralization) against selected threat(s)

Identify gene expression range of gene modulation required for each protective allele target(s) to confer protection against selected threat(s)

Establish bioinformatics pipeline to receive and analyze omics data generated during threat screening and/or from pre-existing datasets

Identify target cells/tissues and/or organs that confer optimal protection when gene targets are modulated

Key deliverables:

o Component ready for integration o Gene target specifications and performance metrics for in vivo PGM-MCM testing and validation in Years 2-4 for in vivo PGM-MCM testing and validation, to include:

prioritized list of candidate protective gene targets and sequences and gene regulatory regions, target range of gene

Proposers must define quantitative performance metrics for:

Criteria for selection and prioritization of protective gene targets, tissues, and modulation activity

Efficacy: e.g., level of protection and increased survival

Safety: e.g., non-toxic, non-carcinogenic when modulated

Specificity: e.g., no collateral off-target or off-tissue effects

Table 3 – TA2 metrics

TA2: Programmable Gene Modulator Development (Months 1-12)

Milestones and Deliverables Program Metrics

Establish candidate PGM libraries of component parts that are modular, adaptable, multiplexable, and easily manufactured

Perform high-throughput, combinatorial screens to test PGMs in in vitro cell, organoid, and/or in vivo systems

Establish bioinformatics pipeline to receive and analyze omics data generated during PGM screening and development

Plan describing the PGM component designs aligned with simple and rapid production and scale-up (e.g., tens to millions of doses in <1 week)

Key deliverables:

o Component ready for integration o PGM specifications and performance metrics for in vivo PGM-MCM testing and validation in Years 2-4, to include: prioritized PGM designs (sequence, format, etc.); mechanism of gene modulation, range of gene modulation control, duration of gene modulation control for targets

Proposers must define quantitative performance metrics for:

Efficacy: e.g., dynamic range of gene modulation control (e.g., 2-fold to 10,000-fold) and duration (e.g., 30 days)

Safety: e.g., no toxicity or immunogenicity (pre-existing or adaptive)

Specificity: e.g., modulates only selected gene targets, active only in desired tissue type

Multiplexability & generalizability: e.g., modular design enables modulation new or multiple targets

Table 4 – TA3 metrics

TA3: Clinical Formulations (Months 1-12)

Milestones and Deliverables Program Metrics

Perform high-throughput, combinatorial screens with delivery formulations in vivo or in an appropriate cell, organoid, or tissue system in vitro

Establish bioinformatics pipeline to receive and analyze omics data generated during clinical formulation screening

Demonstrate delivery formulations that deliver PGM cargo and target intended cell, tissue, organs in vivo

Plan describing the clinical formulation designs aligned with simple and rapid

Proposers must define quantitative performance metrics for:

Efficacy: e.g., delivers cargo at sufficient quantities and duration to facilitate modulation and confer protection

Capacity: e.g. capable of packaging PGMs (e.g., >10 kb, >200 kDa)

Safety: e.g., no toxicity or immunogenicity either pre-existing or induced by the clinical formulation; no effects on overall animal health

Specificity: e.g., no off-tissue delivery expression that confers protection, and optimal cells and tissues for target modulation.

production and scale-up (e.g., tens to millions of doses in <1 week)

Key deliverables:

o Component ready for integration o Delivery formulation specifications and performance metrics for in vivo PGM-MCM testing and validation in Years 2-4 for in vivo PGM-MCM testing and validation, to include:

prioritized delivery formulations that efficiently, safely, and specifically deliver PGMs to target cells, tissues, and organs

Table 5 – Demonstration metrics

Platform Integration & Capability Demonstrations (Months 13 - 48)

Milestones and Deliverables Program Metrics

Generate draft Target Product Profile (Mo 13) Integrate TA1, TA2, and TA3 innovations

(Mo 24) Demonstrate multiplexability (>1 target modulated) and generalizability (>1 threat protected against) of PGM-MCMs in vitro and/or in vivo (Mo 24)

Engage regularly in informal pre-pre- IND/EUA (Mo 24) and formal pre-IND/EUA (Mo 36) discussion with the FDA in preparation for IND/EUA submission

Demonstrate efficacy, safety, specificity, and transience of PGM-MCMs in CDs 1, 2, and 3

Characterization of the mechanisms of protection and failure modes for each PGM- MCM in vivo for each CD

Complete all pre-IND/EUA submission requirements and submit MCM IND/EUA to FDA (Mo 48)

Final program deliverables:

o PGM-MCM with 100× improvement over SoA MCM o Defined Target Product Profile and use case for an IND/EUA submission o IND/EUA or equivalent submission for corresponding PGM-MCM

Proposers must define quantitative performance metrics for each metric category below for each Capability Demonstration (CD):

Efficacy: e.g., protection (i.e. survival) against increasingly more difficult threats delivered prophylactically (pre- or…