Immediate DNA Counteragent SystemPI: Dr. Stephen Hughes Institution: Applied DNA Sciences LineaRx Team

Teaming Overview and Objectives• Confirmed Team: LineaRx (subsidiary of Applied DNA Sciences); Hudson Robotics

• Relevant experience: LineaRx commercial large gram-scale PCR production and gene therapy design and key personnel holding relevant Ph.Ds, DARPA/DoD award PI experience, commercial transitions, enterprise IT implementations; Hudson Robotics offers commercial data collection software, robotics, and integration of advanced instrument clusters.

• Institutional assets: LineaRx proprietary continuous PCR-based DNA production; Hudson advanced AI software / database applications for remote robotic integration of laboratory equipment platforms and on-demand sample collection and sequencing.

• Collaborators sought for technical challenges: Analytical systems with remote continuous datalinks to DNA therapeutic production; photolithographic production of oligonucleotides; M1135 vehicle communications knowledge; and design of M53 JSGPM drug hydrodynamic delivery systems.

Impact• Technique and Capability: Roadmap to deliver ultrapersonal DNA therapeutics at point

of diagnosis. Primary focus on nucleic acid therapeutic countermeasures in hundreds of doses to soldiers in the field, ultimately within 24 hours.

• Metrics and milestones: 1) Demonstrate miniaturized continuous GMP production unit for synthesis of oligonucleotides and utilization of these oligos in continuous PCR to produce durable long-lasting targeted terminally-mediated DNA therapeutic for hydrodynamic delivery into soldiers. 2) Demonstrate analytical detection equipment in miniaturized mobile unit for use on the M1135 vehicle with remote electronic transmission of sample analyses straight from and back to the soldier;

• Transition partnering discussions are in progress with such organizations as US Army lab in Natick, Aberdeen proving grounds (partnered in previous DoD CRADA), CDC and CBRN for defense and commercialized medicines.

Project Overview• The proposed system will be an integrated platform to detect, capture and identify biological threats on the battlefield with a mobile analytical component of the M1135 armed

forces detection and surveillance vehicle; data communications to remote facility such as CBRN/CDC using AI-enabled algorithms to identify biothreats and countermeasure sequences which are transmitted to the point of care for local oligo synthesis, DNA production, purification, formulation, testing, finish and fill into vials and eventually to soldiers.

• A rapid instrumentation cluster already developed will be obtained (modeled after DHS TSA airport systems), including miniaturized triple gas chromatograph, high performance liquid chromatograph, Raman spectroscope to detect, capture and sequence DNA for transmission to CDC or other facility where known libraries of biothreats and countermeasures exist or can be readily formed for transmission electronically back to a second local unit for production and delivery, demonstrated on 2 DNA targets.

• Linear DNA-based countermeasures using nucleic acid sequences derived from previous step will have added to them novel aptamer sequence elements at their ends, which upon production, will improve uptake expression in the cell. A mobile miniaturized continuous oligonucleotide synthesis and polymerase chain reaction (PCR) manufacture with GMP processes will target 12 - 24 hour hours completion from sample collection.

• The proposed project will consist of 3 phases: 1) Manufacture doses in < 7 days: an alpha-prototype of a miniaturized on-site detection, capture and screening of biothreats with air-gapped end-to-end system to synthesize, purify, analyze, formulate, fill and finish for 1 known DNA target. Engagement with FDA for GMP and quality process reviews.2) Manufacture doses in 1-2 days: Integrate modules from Phase 1 through fill-finish vialing, expand to second DNA target and demonstrate remote communications to confirm target, and perform GMP production of sequences for DNA countermeasures in animal models; engagement with FDA.3) Manufacture doses in 24 hours: Phase I human clinical study targeting a DoD-relevant disease. Next-gen enhancements in delivery through M53 JSGPM drug delivery system.

Lightning Round / Poster

Dr. Stephen Hughes – Stephen.Hughes@adnas.com – 1-309-863-8096

Erich Izdepski, CTO of BTS

Teaming Overview and Objectives

Project Overview

• BTS wants to join a team that believes team collaboration will improve the project outcome

• Our team offers our BIFROST cloud platform, developed with DARPA funds, as an asset for data sharing and cloud analytics.

• No cost for BIFROST other than web hosting and software engineering operations and maintenance

• Our platform and capabilities can help on any TA• Currently providing cloud-based data search, sharing and

analysis platform to DARPA/BARDA

• Our cloud-based system, BIFROST, helps organizations share data and analytics making your team more efficient

• BIFROST features:• Share experiments, datasets, and reports/papers/forms• Index all content and provides advanced search• Securely share data between teams, with auditing• Run automated processes, like QA/QC• Provide cloud servers for heavy computing loads• Data visualization• Backed by expert cloud engineers and architects

Impact

• Track project data and documents from all teams in a single, secure repository with search

• Easy for PM/PI to review progress• Simpler sharing/communication with DARPA on all

projects aspects since in BIFROST• Improve collaboration for a geographically dispersed

team• Leverage cloud storage and compute

Come see the poster session demo!

Distribution Statement 1

Project OverviewWe have previously developed Rolling Circle Amplification (RCA) to rapidly produce gram quantities of DNA that can be utilized as is, or converted to RNA for therapy needs. We have experience in industrializing manual workflows and fabricating functionally closed automated systems to perform multi-step scaleup of biologics along with in-line QC, compatible with GMP requirements. This paired with rapid milligram- to gram-scale biosynthetic production of DNA and RNA constructs from initial templates enables the capability to rapidly produce nucleic acid-based therapeutics on-demand at the point of need.

Contact Information – John Nelson- john.nelson@ge.com – (518) 387-7272

FOR OFFICIAL USE ONLY – NOT FOR PUBLIC RELEASE

GE Research

Synthesize DNA

construct

Amplify DNA by RCA, purification

Optional- RNA Formulation Purify/Aliquot

Partner need Partner need

Human Testing

Partner need

Closed system engineering/Disposables

Teaming Overview and Objectives•GE Research is one of the most diverse industrial research facility in the world•Current team has expertise in molecular biology, cell biology, systems integration, automation (platforms for single use processing), complex fluid architecture

Partner needs:•DNA creation automation•RNA therapy formulations•Human subject testing•GMP manufacturing

Impact•Our goal is to assemble a team capable of implementing a realistic plan for accomplishing the DARPA goals towards rapid generation of nucleic acid therapies.

•These therapies revolve around DNA vaccines, DNA expression constructs, mRNA vaccines and mRNA expression constructs.

•Unique metrics:•Nucleic acid product <7 day workflow•Design for GMP•FDA-approved safety profile

Supercoiled DNA

RCA DNA

1.4 grams made in 18 hours

0

200,000

400,000

600,000

800,000

1,000,000

1,200,000

1,400,000

1,600,000

1,800,000

2,000,000

Supercoiledplasmid

RCA

GFP IntensityDay 2Day 4Day 7Day 10

g

Infectious Disease Research Institute Team IDRI

Teaming Overview and Objectives

Project Overview

• IDRI: co-PIs Amit Khandhar, PhD (materials scientist) and Jesse Erasmus, PhD (RNA molecular virologist)

• Aldevron: RNA manufacturing and enzyme supply partner• Gennova: Southeast Asia manufacturing partner• Major accomplishments:

• Erasmus, Khandhar et al, Molecular Therapy, 2018• US Patent Application PCT/US2018/037783• RNA manufacturing scale: 200 mg; cGMP compatible

process• NLC manufacturing scale: 1,000 ml

• Institutional assets: cGMP facility capable of manufacturing NLC up to 200 L scale

• We are seeking to team with engineers in the automotive and energy sector to transfer our established technology to a fully mobile system for DARPA NOW

Impact• Our two-vialed approach provides an agile and target-

independent mechanism to rapidly deliver RNA-encoded countermeasures

• Stable and stockpiled formulation eliminates need for on-site formulation manufacturing, freeing capacity for streamlined RNA manufacturing

• Potential applications enabled by this technology • Mobile end-to-end cGMP manufactured RNA

countermeasure• Novel RNA synthesis processes• Tailor-made formulations for various delivery routes

• Unique metrics and milestones• Streamlined RNA synthesis process for

transferability to a mobile platform

Jesse Erasmus – Jesse.Erasmus@idri.org – 206-858-6035 1

NLCstock-pile

stable for >1 year at 25°C

“Plug-and-play” = target adaptable

• Two-vial approach enables instant RNA formulation (no encapsulation)

• Formulation and RNA synthesis raw materials readily available

• saRNA provides dose sparing

From sequence ID

to RNA in2-3 weeks

Self-amplifying RNA (saRNA)

Nanostructured Lipid Carrier (NLC)

Formulate by bedside

mixing

Self-contained mobile GMP facility

Erasmus/Khandhar

Founded: 2010 IPO: 2018 Focus: exclusively on mRNA

therapeutics and vaccines Employees: 800+; ~50% with

Ph.D., MD, JD, or Master’s degrees Pipeline: 20+ development

programs across 4 therapeutic areas: infectious disease, immuno-oncology, rare disease, and cardiovascular disease; 4 programs in or preparing for Ph. 2

Clinical data: 10 positive Ph. 1 readouts (including 6 intramuscular vaccines and 1 IV administered mRNA-encoded antibody)

CMC: 70 GMP batches manufactured in last 14 months

Publications: ~30 peer-reviewed publications in the last few years

Select partners:

Eliot Green – Eliot.Green@modernatx.com 1

Moderna headquarters, Cambridge, MAModerna’s 200,000 sq. ft. manufacturing facility, Norwood, MA

Next step: additional optimization to enable even faster cycle times and miniaturization to increase capacity and portability

Tissue SamplesTumor (biopsy) and

Normal (blood)

Next Generation Sequencing (NGS)

Mutations identified in protein neoantigen and major histocompatibility

complex Vaccine DesignUp to 34 neoantigensAutomated algorithm

integrated with workflow

Administration

ManufacturingManufacturing of mRNA

Aim for one lot per patient

70 unique PCV batches manufactured to date; ~130 additional batches planned through YE 2020

3’x4

’ foo

tprin

t

Manufacturing of our Personalized Cancer Vaccine (PCV)

Current 50-60 day turnaround time

© 2018 Precision NanoSystems Inc 1

Precision NanoSystems (PNI): The Leading Platform for Mobile Medical Countermeasure Manufacturing of Nanoparticle Enabled Nucleic Acid TherapeuticsJames Taylor, PhD, Padma Kodukula PhD. CONTACT INFORMATION: 50- 655 West Kent Ave N., Vancouver, BC, Canada. Ph: 1-888-618-0031 . Email: jtaylor@precision-nano.com.

Final Drug ProductNucleic acid nanoparticle therapeutic

NanoAssemblrTM

Continuous Flow MicrofluidicsmRNA, Replicon, DNA, siRNA

GenVoyTM Lipid NanoparticlesOr other deliver components Fully Automated & GMP Compliant

Fast setup, non-expert users, small footprintConfigurable platform : mL to 100s L

Any nucleic acid payload

Final DosesFull manufacturing

completed within hours

Impact

• A fully automated drug product manufacturing system• Rapid response and in country manufacturing• Reproducible drug product manufacturing with minimal

operator expertise• Platform for rapid response, mobile manufacturing, single to

thousands of doses• Interoperable with PNI’s commercial development systems,

for rapid deployment of novel therapeutics.

Project OutcomeEquivalent Size and PDI Achieved Across the Scales and Systems

Treatment Induced serum Epoexpression Across Scales

Treatment stimulated Erythrocyte Production Equivalently Across Scales

Rapid & Scalable Manufacture: Epo-encoded mRNA-LNP scaled with minimal process optimization

• 4 commercial instruments, reagents & tech. expertise• >350 installs; 100 pharma, >150 publications• Network of collaborators with expertise from RNA

manufacturing to drug product fill & finish

Mobile Medical Countermeasure ManufacturingIndustry Leadership

GenVoyTM Lipid Nanoparticles

NxGenTM Microfluidics for nanoparticle manufacturing

Core TechnologiesSelect Publications Using PNI Technologies

• Efficient Targeting and Activation of Antigen-Presenting Cells In Vivo after Modified mRNA Vaccine Administration in Rhesus Macaques. Liang et al. 2017 Molecular Therapy 25(12) 2635-2647.

• A lipid-encapsulated mRNA encoding a potently neutralizing human monoclonal antibody protects against chikungunya infection. Kose et al. 2019. Science Immunology 17 May 2019:Vol. 4, Issue 35

• Vaccine Mediated Protection Against Zika Virus-Induced Congenital Disease. Richner et al. 2017. Cell 170(2) 273-283.• A Modular Platform for Targeted RNAi Therapeutics Kedmi et al. Nature Nanotechnology volume 13, pages 214–

219 (2018)• Formulation and manufacturing of lymphatic targeting liposomes using microfluidics. Khadke et al. Journal of Controlled

Release. Volume 307, 2019, Pages 211-220.

Nucleic Acid Manufacturing: PartnerDelivery: PNI or Partner

Drug Product Manufacturing: PNI Fill Finish: PartnerTEAM Requirements:

Teaming Overview and Objectives Impact

Dr. Julien Vantourout – julienv@scripps.edu – (858) – 209 - 3244

FOR OFFICIAL USE ONLY – NOT FOR PUBLIC RELEASE

Elsie – Scripps Research – Baran lab A Redox Neutral Platform for Oligonucleotide Synthesis

• Ideality in phophorothioate oligonucleotide construction• Minimization of concession steps• Easy and rapid implementation• Direct modification of native sequences• Complete control of chemoselectivity and stereoselectivity• Enabling access to new and unprecedented chemical space

• Prof. Phil Baran (PI), Dr. Julien Vantourout (Staff Scientist), Dr.Natalia Muñoz Padial (Postdoc), Kyle Knouse (PhD student)

• P(V) platform already validated (4 reagents – 3 linkages)• Science paper published in 2018• Nature paper in preparation• Oligopeptide synthesizer and HPLC purification system availablein our laboratory

• We are seeking collaborators to implement our P(V) platform tomobile devices.

• We are also seeking collaborators to help with formulation andrapid analysis of the prepared oligonucleotides

• Simple protocol and stable reagents• P(V) platform is as fast as P(III)• P(V) platform is not sensitive to air or moisture• This technology will allow access to chimeric sequences

Stereorandom Stereoselective

Project Overview

Contact Information – Sarah.Milsom@Touchlight.com – (+44)7482607473

FOR OFFICIAL USE ONLY – NOT FOR PUBLIC RELEASE

Status: Multi kb blocks, 1/3000 error rate,

multi species generated from enzymatic assembly

Project objective:Improve length and fidelity of

fragment synthesis / assembly; Develop synthetic clonal selection

Touchlight’s technologySynthesise /

assemble clonal template

Automated enzymatic amplification from

single template vector

Automated, in-line DSP (enzymatic +

single step column)Formulate for

delivery

Status: Benchtop process est. (isothermal, multigram, GMP, pDNA fidelity);

machine prototype functional (100mg scale)

Project objective:Scale up (e.g. 1-2L=~1-2g); speed up

(1 day process scoped); GMP align and characterise

Status:Single step, closed loop DSP

established for GMP multi-gram scale batches

Project objective:Build in column and aseptic filter

step, adapt enzymatic purification for starting material

Status: Multiple formulations and devices

approved for clinical use

Project objective:Identify best option based on target

product profile; improve dose sparing

Seeking collaboratorSeeking collaborator

Seeking collaborator – automation / machine development

•Existing team: Touchlight Genetics DNA process dev team, delivery partners (device, single step lipid – TBD)

•Experience: Process development (GMP), machine prototype building, vaccine development

•Assets: Patented and scaled DNA process, backbone-free DNA vector, GMP facilities, existing machine, research facility and delivery technology options (EP device and lipid - collaborations)

•Seeking collaborators for:•Starting template fragment/GOI synthesis•DNA drug delivery technologies•Machine development / automation partner

•Impact: With the level of existing development (DNA + delivery) and a rapid fragment synthesis partner, the project could develop an end to end solution

•Potential applications: on demand vaccines), personalized vaccines, DNA launched antibodies, gene therapy, etc.

•Unique metrics and milestones:•Fully closed loop drug-grade DNA manufacturing and purification machine

•1-day amplification of 1-2g batch•Approach to transition: Further collaboration with defense, commercialization for personalized cancer vaccines

Teaming objectives and overview Impact

PROJECT: DEVELOP 24-HOUR DNA SYNTHESIS-TO-DELIVERY SYSTEM LEVERAGING TOUCHLIGHT’S ENZYMATIC DNA MACHINE

Touchlight Genetics Ltd | Sarah Milsom

Vaccine & Immunotherapy Center

DNA vaccines and immunotherapeutics: Clinical successes and preclinical advancements

DNA plasmids are usually delivered by intramuscular injection. This plasmidDNA is taken into cells at the site of injection and transcribed and translatedintracellularly. The resulting protein is secreted from transformed cells andprocessed and presented on major histocompatibility complex-1 (MHCI)molecules for presentation to CD8+ T cells (cytotoxic T lymphocytes, orCTLs). Secreted protein antigen can activate B lymphocytes directly or bephagocytosed and presented to CD4+ T cells on MHCII to promote humoralimmunity (Fig. 1). Advanced delivery methods such as gene gun deliveryand electroporation (EP) (Fig. 1c) have enhanced the immunogenicity of theDNA platform.

DNA vaccines targeting emerging and re-emerging infectiousdiseases have had recent clinical success. These include DNA vaccinestargeting ZIKA virus (ZIKV), Ebola virus (EBOV), and Middle East respiratorysyndrome coronavirus (MERS-CoV).The ability to generate syntheticconsensus (SynCon) sequences make the platform useful for generatingimmune responses against pathogenic proteins which exhibit antigenicvariation or for which there are multiple variants or serotypes in the targetpopulation. SynCon DNA technology also allows for the generation of cellularand humoral responses against self-antigens which may be overexpressedby cancerous or pathogenic tissues. Finally, the ability to express complexbiological molecules using the DNA platform is of important clinicalsignificance. DNA encoded monoclonal antibodies (dMAbs) and bi-specificT-cell engagers (dBiTEs) has shown great promise in preclinical studies.

Ebony N. Gary1, Mansi Purwar1, Ziyang Xu1,2, Ami Patel1, Emma Reuschel1, Kar Muthumani1, Trevor Smith3, Kate Broderick3, Laurent Humeau3, and David B. Weiner1

Background and platform

Clinical Successes Preclinical Achievements

Figure 3. Seroreactivity and total IgG antibody titers against EBOV GP.(A) Seroreactive percentages following 1, 2, and 3 immunizations (up ton = 15 subjects per cohort). (B) Anti-EBOV GP antibody end point titers.Open symbols represent the reciprocal end point titer of individualsubjects 2 weeks following each immunization. Horizontal lines are atthe GMT with error bars representing the 95% confidence intervals,from Wilcoxon rank-sum test. Abbreviations: EBOV, Ebola virus; EP,electroporation; GMT, geometric mean titer; GP, glycoprotein. Adaptedfrom Tebas, Patel, and Weiner et al. JID. 2019.

C.

C D

Figure 2. Safety and immunogenicity of a ZIKV DNA vaccine. Shown are geometric mean titers of binding antibody (A) and neutralizing antibody (B) atbaseline and at weeks 1, 4, 6, 12, and 14 among the study participants, according to dose group (1 mg or 2 mg). Binding antibody was measured onenzyme-linked immunosorbent assay, and neutralizing antibody on a standard 50% microneutralization assay in Vero cells. dose. Rate of survival of IFNARknockout mice infected with the ZIKV-PR209 strain after peritoneal injection of serum obtained from the study participants before or after receipt of theZIKV vaccine. (C) shows the proportion of mice surviving at 14 days after ZIKV infection, according to whether they received serum obtained from theparticipants at baseline (lanes 1 to 9) or at study week 14 (after the third dose of ZIKV vaccine) (lanes 10 to 25). Of the 112 mice that had receivedimmune serum from the participants, 103(92%) were alive at 14 days, even though five of the participants (P4, P8, P25, P37, and P39, as indicated byboldface type and an asterisk) had binding-antibody titers but no neutralizing-antibody titers at week 14. (D) shows Kaplan–Meier curves of survivalamong mice that were included in the challenge study and injected with phosphate-buffered saline as a control, with baseline serum, or with week 14serum. There were no significant differences in response between male and female mice. Adapted from Tebas, Muthumani, and Weiner et al. NEJM.2017.

Figure 4. MERS-CoV Vaccine-associated antibody responses. Antibodyresponses for each dose are shown for available specimens from the modifiedintention-to-treat population. (A) shows the geometric mean endpoint titre(95% CI error bars) and the proportion of participants who developedantibodies against Middle East respiratory syndrome (MERS) coronavirusdetermined by S1-ELISA. B shows the geometric mean endpoint titre (95% CIerror bars) and the proportion of participants who developed neutralizingantibodies against MERS coronavirus determined by EMC-2012 MERS-coronavirus infection of Vero cells. Adapted from Modjarrad, Muthumani, andWeiner et al. Lancet Infec. Dis. 2019.

1Vaccine and immunotherapy center, The Wistar Institute, Philadelphia, PA, 2Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA, and 3Inovio pharmaceuticals, Bluebell, PA

Figure 5. DNA-encoded monoclonal antibodies (dMAbs) are effective against numerous pathogens in pre-clinical setting

Figure 6. dMAb and dBiTE targeting HER2 prevent tumor growth in vivo. (A) Growth curve of OVCAR3 tumors grafted into nude mice treated withHER2dMAb or empty vector (2 independent experiments, n = 5 mice per group). (B) Growth curve of Brpkp110-hHER2 tumors grafted into C57BL/6 micetreated with HER2dMAb or empty pVax plasmid (representative of 2 independent experiments, n = 5 mice per group). Two-way ANOVA, t test, log-ranktest. ***P < 0.001. (C) In vitro cytotoxicity of sera from mice treated with HER2DBiTE after injection and electroporation of 100 μg using OVCAR3 as thetarget (triplicates). (D) Average growth curve of OVCAR3 tumors grafted into NSG mice treated with HER2DBiTE or empty vector (n = 10 mice pergroup).Adapted from Perales-Puchalt, Muthumani, and Weiner et al. JCI Insight. 2019.

Conclusions- Rapid DNA Development• DNA vaccines are safe and tolerogenic in

humans, promoting robust cellular and humoralresponses

• Ebola virus• Zika virus• MERs-CoV

• DNA –encoded biologics are easily synthesizedand are poised for use as rapid immunotherapy