Eligo Bioscience Next-gen biotherapeutics for bacteria-associated diseases

Company presentation – January 2017 SectionHIGHLIGHTS Eligo Bioscience: a promising young biotech company

Spin-out from MIT & - currently incubated at Institut Pasteur in Paris

Cofounded in 2014 by world-renowned experts in CRISPR-Cas systems, phage engineering, microbiome editing, and synthetic biology

$2.6M seed round from Seventure Partners $1M from highly-selective Research Grants and SBIR-like grants

A fast-growing team of talented scientists (Stanford, MIT, Inserm, Institut Pasteur…) Coupled with experienced executives (Sanofi, EMA. and Medincell)

The technology platform has been published in peer-reviewed journals (Nature Biotech, NAR…) and covered by international media (Wired, BBC, Forbes, Bloomberg, Fierce. Biotech...)

Eligo Bioscience -Company presentation 01/2017 2 TEAM

Who is behind Eligo?

• A strong founding team of renowned scientists • A highly skilled, international and interdisciplinary operational team

Eligo Bioscience -Company presentation 01/2017 SectionTEAM A strong founding team of renowned scientists In total, have published more than 20 peer-reviewed articles in Nature, Science, Nature , and Cell

Timothy Lu, MD-PhD Luciano Marraffini, PhD • Associate Professor, MIT • Associate Professor, Rockefeller University • World leader in phage engineering • Seminal inventor of CRISPR technology • Founder, Synlogic • Founder, • Founder, Sample6

David Bikard, PhD Xavier Duportet, PhD • Director, Synthetic Biology Lab, • Former PhD at MIT & INRIA Institut Pasteur • Expert in genome engineering • World leader in CRISPR research • MIT TR35 French Innovator of the Year • Former post-doc at Rockefeller Univ. • 10+ science & entrepreneurship prizes

Eligo Bioscience -Company presentation 01/2017 4 SectionTEAM A highly skilled operational team

Board Board Board & CEO Board & CSO CBO

Jean-Pierre Lehner, PhD Sebastien Groyer, PhD Xavier Duportet, PhD David Bikard, PhD Sebastien Enault, M.Eng. Former Chief Medical Former PhD Director SynBio Lab, Former Chief Bus. Partner, Seventure Officer, Sanofi MIT & INRIA Pasteur Institute Officer, Medincell

R&D - Animal Models R&D – Delivery vectors R&D – Automation & Robot R&D – DNA circuits R&D –

Matthieu Galtier, PhD Antoine Decrulle, PhD Erica Lieberman, BS Jesus Fernandez, PhD Laure Saujet, PhD Former PhD Former PhD Former BS Former Post-doc Former Post-doc Institut Pasteur INSERM Stanford MIT INSERM-CEA

Advisors Thomas Lonngren, PhD Former Director, EMA Jason Clark, PhD Former CTO, Novolytics

Eligo Bioscience -Company presentation 01/2017 5 BACKGROUND

Background

• Microbiome-associated diseases: a tale of bacterial strains • Eligo’s platform: a modular approach to target the cause of diseases • The potential of DNA delivery to the microbiome • Eligo’s core value: novel, optimized and proprietary DNA delivery vectors

Eligo Bioscience -Company presentation SECTIONSectionBACKGROUND TITLE Microbiome-associated diseases: a tale of strains

A growing number of serious unmet medical needs are linked with the presence of specific bacterial strains within our microbiome. These strains can be discriminated from harmless bacteria by targeting specific genetic determinants they carry in their chromosomes or their plasmids.

Examples of determinants Examples of outcomes Virulence factors Infections (invasion factors, toxins)

Inflammation Metabolic enzymes (toxic compounds synthesis, inactivation of drugs) Organ failure

Antibiotic resistance Treatment failure

Eligo Bioscience -Company presentation 01/2017 7 SectionAPPROACH A modular platform to target the cause of the diseases

At Eligo, we are developing a new generation of biotherapeutics that enable the expression of therapeutic RNAs, proteins or small molecules within target microbial populations. They can be programmed either to diagnose, kill or control the metabolism of the causative bacterial strains while sparing the rest of the microbiome.

LSI Live Strain Identification SSAMs Rapid and modular system for the Sequence-Specific detection of specific live bacterial AntiMicrobials strains in complex microbiome samples. Nuclease-based antimicrobials that selectively kill bacteria based on their genetic signature. GEMs Gene Expression Modulators Expression of DNA-binding proteins to regulate target genes expression levels.

Eligo Bioscience -Company presentation 01/2017 8 SectionDELIVERY TECHNOLOGY Novel vectors to deliver genetic programs to the microbiome

Eligo has developed a strategy to package genetic programs into highly efficient delivery vectors derived from bacteriophages. These « biocarriers » do not contain any of the original phage DNA anymore

These vectors are non-replicative, stable at 4°C and can be used to deliver genetic payloads in the gut, skin or lung microbiomes.

Eligo has developed a process to extend the host range of a biocarrier from ~5% to ~90% of the strains of a species. Data on the left from an experiment on 159 clinical strains of E. coli

Eligo Bioscience -Company presentation 01/2017 9 SectionAPPROACH From DNA to in-situ biotherapeutics production

A wide variety of therapeutic proteins can be expressed in the bacterial populations of interest as soon as the DNA payload has been injected.

Eligo Bioscience -Company presentation 01/2017 10 VALIDATION

Proofs of concept

• Eligobiotics as Sequence-Specific AntiMicrobials (SSAMs) • Eligobiotics as Gene Expression Modulators (GEMs) • Eligobiotics as a Live Strains Identification tool (LSI)

Eligo Bioscience -Company presentation SectionPROOFSPROOF OF OF CONCEPT: CONCEPT SSAMs Sequence-Specific AntiMicrobials: delivery of programmable nuclease

① SSAMs are based on the delivery of a nuclease (CRISPR/Cas, Zinc Fingers, etc) that can be programmed to create a double-strand break in a genetic sequence of choice (virulence factor, antibiotic resistance gene, etc).

② The target sequence can be modularly specified by the single-guide RNA from the delivered genetic payload.

③ Once the complex (Cas9 nuclease + tracrRNA + sgRNA) is assembled, it scans the genome for the target sequence.

④ If the targeted genetic sequence is found, the nuclease create a double-strand break within the target sequence, which leads to the death of the bacteria due to the lack of DNA reparation systems in most bacteria.

Eligo Bioscience -Company presentation 01/2017 12 SectionPROOFSPROOF OF OF CONCEPT: CONCEPT SSAMs SSAMs – Sequence-Specific AntiMicrobials

Since SSAMs kill bacterial strains based on their genetic signature, SSAMs are a new generation of biotherapeutics that can be used to remove specific pathogenic strains while leaving the rest of the microbiome intact.

Strain A Strain B Eligobiotic Dead cell

Eligo Bioscience -Company presentation 01/2017 13 SectionPROOFSPROOF OF OF CONCEPT: CONCEPT SSAMs SSAMs – In-vitro proof of concept / Efficient & specific killing

In our 2 Nature Biotechnology publications*, we demonstrated that we could selectively eradicate bacterial strains from a mixed population. These results were obtained by delivering a CRISPR- Cas system targeting a genetic sequence only carried by the targeted strain.

Selective killing of Staphylococcus aureus Selective killing of Escherichia coli (Gram positive) carrying the KanR gene (Gram negative) carrying the aph gene

108 108

7 10 7 S. aureus strain 10 E. coli strain 6 not carrying 10 the aph gene 6 not carrying 10 the KanR gene 5 S. aureus strain 10 5 E. coli strain carrying 10 carrying

Viable cells (CFU/mL) Viable cells the aph gene 4 (CFU/mL) Viable cells the KanR gene 10 104

3 10 103

gene gene Non-targeting Cas9 targetingaph Non-targeting Cas9 KanRtargeting Cas9 nuclease the Cas9 nuclease the

* Bikard et al,. 2014 – Citorik et al., 2014 14 SectionPROOF OF CONCEPT Selective decolonisation in an complex microbiome environment

We recently demonstrated a similar efficacy in a mouse model of gut decolonization.

In-vivo experiment protocol Gavage without Eligobiotics Gavage with Eligobiotics (Control group) (Test group) Day=0 9 Stable gut colonisation of 20 mice with 2 109 10 different E.coli strains (ratio 1:100) 8 Non-targeted strain: 10^9 bacteria/g of faeces 108 10 ^7 Targeted strain: 10 bacteria/g of faeces 7 107 10

6 Day=2 106 10

Control group: gavage of 10 mice without 5 105 10 or Eligobiotics (PBS only) Test group: gavage of 10 mice with 4 4 10 10 Bacterial count (CFU/g faeces) (CFU/g Bacterial count eligobiotics programmed to eradicate the faeces) (CFU/g Bacterial count

3 targeted strain 10 103 D=0 D=3 D=0 D=3 Day=3 After gavage Before gavage Before gavage After gavage Control group: no change in colonisation model Test group: targeted strain eradicated in 7 out of 10 mice; reduced by 3+ logs in the 3 others

Eligo Bioscience -Company presentation 01/2017 Non-targeted strain Targeted strain 15 SectionPROOF OF CONCEPT: GEMs Delivery of DNA binding proteins to modulate gene expression

Concept: GEMs (Gene Expression Modulators) are based on the delivery of a DNA binding proteins that can be addressed to promoter regions of specific genes from microbial populations.

We demonstrated that we could efficiently repress the expression of a reporter gene by delivering a dCas9 (deactivated Cas9) programmed to bind to the reporter gene’s promoter (forward or reverse strand).

Application: in-vivo microbiome metabolism modulation (toxic metabolites synthesis repression, drug metabolism control, etc)

Design of the experiment 1.2 Quantification of 1 the repression dCas9 target sequence A dCas9 Target 0.8 sequence B ~100x repression 0.6 of the reporter gene -35 -10 0.4 GFP relative fluorescence relative 0.2

0 Promoter region Reporter gene

No targeting Eligo Bioscience -Company presentation 01/2017 TargetingTargeting sequence sequence A B 16 SectionPROOFSPROOF OF OF CONCEPT: CONCEPT LSI New diagnostic tool: Live Strain Identification circuit (LSI)

We have created a proprietary genetic circuit which, once injected, enables the identification of live bacterial strains in a microbiome sample.

The detection of a strain-specific genetic signature triggers the expression of a reporter gene in the strain. Quantification of the Visualisation of the LSI concept reporter gene expression

8000

7000

6000 >200x difference in fluorescence signal intensity between target strain 5000 to identify and control strain

3000 Control strain 2000 GFP fluorescence (au) fluorescence GFP Strain to identify 1000

0 t=0 t=1,5h t=3h Time after injection of the LSI circuit into the microbial population Eligo Bioscience -Company presentation 01/2017 17 DEVELOPMENT

Eligo’s development plan

• Potential markets • Lead candidates clinical development plan • Strategic collaborations strategy • Series A: financing and use of proceeds • IP portfolio

Eligo Bioscience -Company presentation SectionPROOFSPOTENTIAL OF MARKETS CONCEPT Eligo’s platform can address a growing number of unmet needs

Eligo Bioscience -Company presentation 01/2017 19 SectionPROOFSCLINICAL OF DEVELOPMENT CONCEPT

Our two first lead candidates are addressing two different orphan diseases with no cure on the market or being developed. Both have a market size of approx ~$300M (peak sales). We are filing IP for these indications – can be disclosed them under CDA. Eligo Bioscience -Company presentation 01/2017 20 SectionPROOFSPARTNERSHIP OF CONCEPT OPPORTUNITIES A large number of opportunities are being discussed with partners

Eligo Bioscience -Company presentation 01/2017 21 SectionPROOFSSERIES A OF CONCEPT An ambitious Series A to lead the microbiome engineering space

Targeting $15m Series-A to advance our two lead orphan-disease indications into Phase 2

Hiring Chief Medical Officer, Chief Financial Officer, Head of Manufacturing

Expand scientific discovery and development team

IP estate protection, conversion and expansion

Support activities to expand the arsenal of biocarriers to become the one-stop shop for DNA delivery in a wide range of bacterial species

Extand laboratory facilities

Eligo Bioscience -Company presentation 01/2017 22 SectionPROOFSIP PORTFOLIO OF CONCEPT Building a strong IP portfolio to support our development

Eligo Bioscience -Company presentation 01/2017 23 SectionPROOFSFEEDBACKs OF CONCEPT

Financing

• Don’t start too early • Go fast and thrive or go slow and survive • France is awesome but don’t count grant money as « real » money • Always raise a bit more than you need • You should pick your investor, not the opposite! • Engage in a transparent relationship with your investor • High valuation is great but founders-friendly terms are much more important! • Start raising money when you don’t need it!

Eligo Bioscience -Company presentation 01/2017 24 SectionPROOFSFEEDBACKs OF CONCEPT

Development • Be ambitious but not arrogant • Don’t give away too much to the scientific founders with no executive role! • Secure IP and build IP portfolio asap, try to free yourself from your institution • In biotech, it’s not « cool » to be young, surround yourself with experienced people or you will get your head cut • Go meet and talk to R&D and BD Directors in large pharmas asap • Communicate and do some PR! • Learn how to let go from operations as soon as possible, you are here to lead • Hire international scientists! • Look for international investors • Being a CEO is one of the loneliest job ever, so meet meet meet other CEOs! • Build a product, not a technology nor a therapy

Eligo Bioscience -Company presentation 01/2017 25 Eligo Bioscience

Pasteur Institute 28 rue du Docteur Roux, 75015 Paris - France

[email protected]

+33 6 48 77 66 10

Company presentation – January 2017