Slide 1: Disruptive circRNA technology for genetic medicine – R&D webinar
circio LF Disruptive circRNA ° technology for genetic medicine R&D webinar 29 November 2023
Slide 2: Forward-looking statements
This report contains certain forward-looking statements based on uncertainty, since they relate to events and depend on circumstances that will occur in the future and which, by their nature, will have an impact on the results of operations and the financial condition of Circio ASA and the Circio Gr
Slide 3: Presenters
Co-discoverer of circRNA, Deeply experienced tumor World-leading pioneer Circular RNA pioneer and Pharma consultant at immunology scientist from and co-discoverer of founder of ORNA McKinsey & Co and various academia and industry, incl circular RNA; 10 years as Therapeutics commercial and R&D roles
Slide 4: Agenda
1 Introduction Dr Erik Digman Wiklund - CEO 15 min 2 Circular RNA Technology - Advances and Challenges Dr R Alexander Wesselhoeft – Dir of RNA Therapeutics, Mass General Brigham 30 min 3 circVec technology overview Dr Thomas B Hansen – VP & Head of Research 20 min 4 Development Strategy Dr Victor Le
Slide 5: Dr. Erik Digman Wiklund, CEO
Dr. Erik Digman Wiklund, CEO 5
Slide 6: circRNA will disrupt gene therapy and vaccines
circRNA will disrupt gene therapy and vaccines by improving potency and adding novel functionality Extended RNA aS Enhanced protein durability > ay expression % Y circRNA is resistant to leading to significantly prolonged half-life vs. \. MRNA within cells a My ly microRNA ; Regulatory sponging func
Slide 7: Circular RNA deal and financing activity
Licensing Series A Series A Series A+B $150M Upfront $490M $3,5B Total deal value $270M $300M May/Aug 2021 Aug 2022 April 2023 May 2023 Feb 2021 Jul 2022 Aug 2022 Oct 2022 Aug 2023 $80M $42M $221M Undisclosed $30M Series A Series A Series B M&A Series A 7
Slide 8: Citations of circRNA founding publications
6,373 citations 30 September 2011 922 citations 2,291 citations 8
Slide 9: circVec DNA-format circRNA
` circVec DNA or viral vector DNA Inject circRNA biogenesis circRNA Intra-cellular protein expression 9
Slide 10: Genetic code and DNA vs RNA expression
TRANSCRIPTION TRANSLATION AAAA Genetic Code Instruction to Performing specific Recipe for Proteins generate protein function Durable (“eternal”) Transient: 6-24h in Can be structural or in the cell nucleus cell cytoplasm chemical 10
Slide 11: Circular RNA
With new technology, mRNA can be made circular tone 11 «Circle
Slide 12: Circular RNA vs mRNA
Circular RNA mRNA IRES-mediated initiation 5’cap-dependent initiation of (Internal Ribosome Entry Site) translation – More efficient than 5’ cap- – Rate-limiting step dependent initiation Short half-life Extended half-life – Hours to days – Days to weeks 5`cap & 3’poly-A tail – accessible No 3’ or 5
Slide 13: Dr. Alexander Wesselhoeft
Dr. Alexander Wesselhoeft 13
Slide 14: Circular RNA Technology: Advances and Challenges
Circular RNA Technology: Advances and Challenges R. Alexander Wesselhoeft Director of RNA Therapeutics, Gene and Cell Therapy Institute, MGB
Slide 15: Why circularize RNA?
Why circularize RNA? Human exosome complex Garneau et al, 2020 Lima Lab Better More protein Dosing Circularization therapeutic per RNA advantage index
Slide 16: Two types of circles
Two types of circles DNA format Circular RNA RNA format Circular RNA Li et al., 2020
Slide 17: Key scientific milestones in the circRNA field
Key scientific milestones in the circRNA field 1991 2013 2018 First natural First functional First in vivo protein circular RNA characterization of expression from identified circRNA engineered circRNA
Slide 18: Circular RNA translation to industry
Circular RNA translation to industry Kjems & Hansen 2013 Circio DNA Format CircRNA Sponges Nigro & Vogelstein 1991 Litke & Jaffrey 2019 Chimerna Backsplicing Tornado Circularization Meganck & Asokan 2021 Torque Engineered Backsplicing Puttaraju & Been 1992 Wesselhoeft & Anderson 2018 Qiu & Zuo 2022
Slide 19: Different platforms, similar challenges
Different platforms, similar challenges DNA Format RNA Format IRES Circularization elements Purification Viral vector Codon optimization Circularization process Off-target functions Nonviral Delivery
Slide 20: Challenge 1: Circularization
Challenge 1: Circularization Permuted intron Enzymatic ligation Precursor 338nt circRNA ~50% circularization efficiency Precursor 124nt circRNA Intron bits Puttaraju and Been, 1992 Beaudry and Perreault, 1995 • RNA circularization is not a new idea • There were multiple attempts at circularizing RNA
Slide 21: Additional design strategies improve circularization
Additional design strategies improve circularization Spacers can dramatically facilitate correct intron folding Alternative introns can be more efficient MIT
Slide 22: Very large circRNAs have been successfully made
Very large circRNAs have been successfully made • Generating circular RNA encoding full-length • A cell-free dystrophin and smaller variants of dystrophin is translation assay a major challenge shows products of RNase R - the correct size for Circular RNA all three constructs • These circRNAs can al
Slide 23: Challenge 2: Translation
Challenge 2: Translation • mRNA has a cap structure that initiates ribosome scanning • Circular RNA doesn’t have a cap • How can we get cap-independent translation within big synthetic circular RNAs? Chen and Guo, 2016 Viruses have a solution: The Internal Ribosome Entry Site (IRES) And get protein!
Slide 24: What is the IRES?
What is the IRES? • There are a few different types of mRNA caps: • Like caps, there are a few types of IRESs: m6A 3’-OMe m7G A/C/G/U A/C/G/U ARCA 2’-OMe 2’-OMe Cap 1 Cap 2 • With key decisions being first nucleoside and methylation pattern • What kind of chemical and functional diversity can we exp
Slide 25: Unlocking a new world of IRES elements
Unlocking a new world of IRES elements • Orna identified hundreds of new IRES elements highly active in oRNA • Some IRESs are 10-40x stronger than the commonly used EMCV-type IRESs • There can be significant differences even between IRESs from highly similar viruses, showing the importance of empiri
Slide 26: Technology optimization leads to improved preclinical signal
Technology optimization leads to improved preclinical signal Orna
Slide 27: CircRNA applications
CircRNA applications • DNA and RNA format Genetic Disease circRNA technologies share several broad application areas, but delivery and duration of expression bias use towards specific areas Infectious • Improving translation Oncology Disease potency through circularization reduces dosing requirement
Slide 28: Perspectives on the future of circRNA
Perspectives on the future of circRNA • CircRNA has RNA format and DNA format applications, wherein the core advantage is increased potency through a combination of enhanced stability and expression • Extensive optimization is required to realize the full potential of circRNA • CircRNA already compa
Slide 29: Dr. Thomas Birkballe Hansen, VP & Head of Research
Dr. Thomas Birkballe Hansen VP & Head of Research 14
Slide 30: High natural prevalence of circular RNA in all eukaryotes
High natural prevalence of circular RNA in all eukaryotes, particularly in humans 15
Slide 31: Growth-decay model: two ways to increase expression
Growth-decay model: Two ways to increase expression Expression 𝑃 = 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 1) Technical enhancement of production −𝐷𝑡 𝑃 2) Natural reduction of 𝑋 𝑡 = 𝐶𝑒 + decay 𝐷 log 𝑒 2 𝑃 𝐷 = 𝑑𝑒𝑐𝑎𝑦 𝑟𝑎𝑡𝑒 = lim 𝑋 𝑡 = 𝑇½ 𝑡→∞ 𝐷 Gene expression is determined by…
Slide 32: Temporal vector-based RNA expression dynamics; circRNA vs. mRNA
Temporal vector-based RNA expression dynamics; circRNA vs. mRNA Input assumptions for simulation: circRNA reaches Non-dividing target cells substantially higher and more durable steady-state levels mRNA production: 10 molecules / hr mRNA half-life: 9 hrs * circRNA production: 2 molecules / hr 20% of
Slide 33: Expression of human endogenous circRNA
´ Expression of human endogenous circRNA NGS analysis of 300+ RNAseq datasets mRNA AAAAAAAA Linear splicing Backsplicing Maximum biogenesis-rate circRNA of endogenous circRNA Circular RNA expression cassette Screening of most effective natural sequences promoter SA SD 5 circRNA Exon 4 expression / G
Slide 34: circRNA expression and RNase R resistance
IR-dependent signal RNAseR resistant promote SA SD circRNA: r Exon promote SA SD ∆IR: r Exon circEGFP RNA Expression 15 No production without IR mRNA circRNA GAPDH 10 element //GAPDH RNaseR: - + - + 5 Exon 0 Resistance GAPDH circRNA ∆IR towards IR 1 es exonucleolytic _Δ _D FP FP degradation G G E rc
Slide 35: circRNAs are translated in a cap-independent manner
CONFIDENTIAL circRNAs are translated in a Protein expression, cap-independent manner Western blot No IRES IRES ⍺-GFP ⍺-Loading Ctrl IRES element is required for circRNA translation 20
Slide 36: De-circularize into linear DNA cassette
De-circularize into linear DNA cassette promoter SA SD Exon D10 D1 D2 D3 D4 D5 D6 D7 D8 D9 ⍺-Payload IRES_1 ⍺-Loading ctrl ⍺-Payload IRES_2 ⍺-Loading ctrl 21
Slide 37: circVec1.0
circVec1.0 promoter SA SD IR Exon promoter SA SD IR Exon 22
Slide 38: Expression
circRNA expression, RT-qPCR eGFP expression from circRNA, western blot promoter SA SD IR Exon IR1_11 circEGFP/GAPDH IR1_10 IR1_1 IR1_2 IR1_3 IR1_4 IR1_5 IR1_6 IR1_7 IR1_8 IR1_9 80 RNA expression Expression 60 ⍺-Payload / GAPDH 40 ⍺-Loading Ctrl Relative 20 0 93 de 1 90 de ty 87 de ty 79 ide ity 7 5
Slide 39: circVec design optimization, protein expression
CONFIDENTIAL circVec design optimization, protein expression level @48h post-transfection 15 Relative expression 10 circVec v2.0 achieves up to 10x1 higher protein 5 10x yield than v1.0 design 0 VE H iE K C C P1 R lo 3 C _ f C er r PC CV _F V V R BV ru ve PV i H V_ C VB SV iHR H B H V_ v B tM 3_ EM
Slide 40: circVec vs. mRNA luciferase reporter expression
circVec vs. mRNA luciferase reporter expression; time course 50 45 40 Relative luminescence 35 mRNA “Due to its significant advantages, 30 circVec1.0 circRNA systems can be expected to 25 replace mRNA-based expression for 20 8.5x circVec2.0 DNA format therapeutics in the future 15 circVec2.1 – just
Slide 41: Monitor expression over time
mRNA-FL circVec2.1-FL Monitor expression over time Day1 Day8 Day14 Day21 Realtime monitoring ongoing Efficient circRNA expression established in mouse model demonstrating progressive accumulation 26 circRNA mRNA circRNA mRNA circRNA mRNA circRNA mRNA
Slide 42: circVec: expression of missing protein
promoter SA SD Exon circVec Expression of missing protein in Removal of protein in gain-of- loss-of-function scenarios! function scenarios? Remove-and-Replace design! 27
Slide 43: circVec-remover
promoter SA SD Exon Remover circVec-remover Expression of missing protein in Removal of protein in gain-of- loss-of-function scenarios! circRNA Remover function scenarios! Sequence-specific gain-of-function target cleavage An Loss-of-function protein expression An 28
Slide 44: circVec-remover data
promoter SA SD Exon Remover circVec-remover Expression of missing protein in Removal of protein in gain-of- loss-of-function scenarios! function scenarios! circVec-remover 2.5 2.5 Effective depletion of target gene by remover Relative Luminescence Relative Luminescence circVec 2.0 2.0 CircRNA Target
Slide 45: Technical development status
Technical development status v Substantially optimized circRNA biogenesis and protein expression Multifunctional Remove-and-Replace concept established In-vivo validation ongoing in multiple settings XNA Now testing circVec 2.1 in therapeutically relevant applications 30 circia
Slide 46: R&D Strategy – Dr. Victor Levitsky, CSO
ZA R&D Strategy Dr. Victor Levitsky - CSO 31 eircio
Slide 47: Current focus areas
Current focus areas Designed for intra-cellular circRNA supply, durable protein expression and targeted regulatory functionality
Slide 48: Rare disease indication prioritization
Rare Disease Initial Screening n Ipsum Lorem = 363 • Therapeutic Focus: Rare genetic disorders • Development Stage: Clinical or Market validation achieved 1st Prioritization Criteria n= Lorem 127 Ipsum • Etiology: monogenic disorders only (exclude polygenic disorders) • In vivo validation: availabil
Slide 49: Rare disease indications
Rare Disease Indication Second priority Lead Alpha-1 Antitrypsin Ornithine Citrullinemia Argininosuccinate Deficiency Transcarbamylase Type I Synthetase Lyase (AATD) Deficiency (OTCD) (CTLN1) Deficiency (ASLD) Third priority Commercial and regulatory assessment Pompe Wilson Glycogen Storage Tyrosine
Slide 50: AATD is a major unmet medical need
Rare Disease Current treatment options AATD is a major unmet medical need manifested in liver and lung Lung-associated AATD Replacement therapy with an alpha-1 proteinase inhibitors Weekly IV infusions Bronchodilators and inhaled steroids used for mild symptoms Lack of functional AAT protein Accumul
Slide 51: Depleting mutant form and replenishing functional protein by circVec
Rare Disease circVec v1.0 AAT expression in liver cells Depleting mutant form and replenishing functional HepG2 AAT1 Protein Expression protein by circVec 250 > durability - reverses toxic protein accumulation in liver and restores normal function in lung 24 hr w/ circRNA 200 48 hr 72 hr 150 ng/mL 9
Slide 52: Safety issues of AAV gene therapy
Rare Disease Safety issues Liver toxicity, innate immunity High dose = high immunogenicity No repeat dosing Manufacturing cost 1014 – 1015 VPs per dose 37
Slide 53: circVec potential to boost AAV-based gene therapy potency
Rare Disease vector 15x durability Shorter durability RNA 5x translation rate Lower steady-state Protein >25x maximum protein levels protein levels circVec has the potential to substantially boost the potency of AAV-based gene therapy 38
Slide 54: Temporal AAV-based RNA expression dynamics
Rare Disease Temporal AAV-based RNA expression dynamics; circRNA vs. mRNA Input assumptions for simulation: Non-dividing target cells AAV half-life: 365 days mRNA production: 10 molecules / hr >5x steady-state RNA levels → mRNA half-life: 9 hrs * >25x peak protein expression circRNA production: 5 mo
Slide 55: Non-replicating AdV advantages and upcoming milestones
Vaccines Non-replicating AdV advantages Replication-deficient AdV vector 35kb genome Large cargo capacity 7+ kb, 50% more than AAV vectors Opportunity to express multiple circRNAs from one vector circVec inserts Potent and well-described for vaccine >7kb size feasible applications Established delive
Slide 56: circVac v1.0 COVID Spike expression
Vaccines circVac v1.0 COVID Spike expression, RNA and protein level RNA expression Protein expression circRNA > mRNA SPIKE/GAPDH mRNA circRNA SPIKE protein 0.8 mRNA circRNA expression @ 96hr 48hr 48hr 96hr 96hr / GAPDH 0.6 /GAPDH 0.4 ⍺-ORF SPIKE-encoding 0.2 circRNA accumu- lates over time 0.0 ⍺-Hex
Slide 57: Circio is the only significant player in DNA-format circRNA
Circio is the only significant player in the DNA-format circRNA space Enhanced durability and protein expression from circRNA is expected to translate into lower dosing of DNA-format applications, which may solve both potency, toxicity and cost challenges facing current ”gold-standard” gene therapy