Circio Holding Asa · OSL:CRNA
Circio Holding Asa — ESGCT Annual Meeting 2024
18 slides from Circio Holding Asa's conference presentation, Oct 24, 2024
Slide 1: In vitro and in vivo performance of circVec
In vitro and in vivo performance of circVec j a vector-based circular RNA expression platform for enhanced gene therapy Dr. Thomas Hansen, CTO ESGCT 2024, Rome
Slide 2: The unique circVec expression system
The unique circVec expression system: Turning the patient´s cells into circRNA factories circVec DNA or viral vector DNA Inject circRNA biogenesis circRNA Protein Potent and durable protein expression 2
Slide 3: Why use circular RNA?
Why use circular RNA? mRNA circRNA ? circRNA is resistant to exonucleolytic decay and consequently very stable and long-lived within cells 3
Slide 4: Bioinformatic simulation demonstrating advantage of vector-expressed circRNA vs. mRNA
Bioinformatic simulation demonstrating advantage of vector-expressed 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 mRNA production: 10 molecules / hr s
Slide 5: Circular RNA – a natural design
Circular RNA – a natural design Widespread natural prevalence of circRNA in all eukaryotes, particularly in humans 5
Slide 6: circVec is based on nature's best design
circVec is based on nature´s best design mRNA Expression of human endogenous circRNA AAAAAAAA NGS analysis of 300+ RNAseq datasets Linear splicing Backsplicing circRNA Maximum biogenesis-rate of endogenous Circular RNA expression cassette circRNA promoter SA SD Exon Screen and optimize the most effe
Slide 7: Establishing circRNA 1.0 expression
Establishing circRNA 1.0 expression DNA cassette Biogenesis RNA Protein promoter SA SD 1 – Establish circVec Exon IR-dependent signal RNAseR resistant promoter IR SA SD IR circRNA: Exon promoter SA SD ∆IR: Exon circRNA circEGFP RNA Expression 15 No production mRNA circRNA without IR RNaseR: - + - +
Slide 8: Establishing circRNA 1.0 expression
Establishing circRNA 1.0 expression DNA cassette Biogenesis RNA Protein promoter SA SD 1 – Establish circVec Exon Clean expression, RNAseq promoter SA SD Exon 8
Slide 9: Establishing circRNA 1.0 expression
Establishing circRNA 1.0 expression DNA cassette Biogenesis RNA Translation Protein promoter SA SD 1 – Establish circVec Exon circRNAs are translated in a Protein expression, cap-independent manner Western blot IRES element is required for circRNA No IRES translation IRES ⍺-GFP ⍺-Loading Ctrl 9
Slide 10: circVec 2.0: IRES optimization has resulted in >10x improvement in protein expression
CONFIDENTIAL circVec 2.0: IRES optimization has resulted in >10x improvement in protein expression vs. v1.0 DNA cassette Biogenesis RNA Translation Protein promoter SA SD 2– IRES selection Exon circVec IRES optimization, protein expression level @48h post-transfection 15 Relative expression 10 IRES
Slide 11: Correct cassette design is critical for circRNA-derived protein expression
Correct cassette design is critical for circRNA-derived protein expression DNA cassette Biogenesis RNA Translation Protein promoter SA SD 3 – Cassette design Exon Cassette design screen, western blot High protein yield only D10 D11 D12 observed with optimal D4 D2 D3 D6 D7 D1 D5 D8 D9 cassette design
Slide 12: Optimized circRNA biogenesis, circVec2.x matches mRNA expression at 48 hours
CONFIDENTIAL ... and with optimized circRNA biogenesis, circVec2.x matches mRNA expression at 48 hours post-transfection DNA cassette Biogenesis RNA Translation Protein promoter SA SD 4 – Biogenesis Exon Protein expression circVec1.0 IR SA SD IR ~16x Relative luminescence EXON circVec2.0 ~10x EXON c
Slide 13: circVec 2.1 achieves 15x prolonged circRNA half-life and increased protein expression vs. mRNA
circVec 2.1 achieves 15x prolonged circRNA half-life and increased protein expression vs. mRNA in vitro circVec vs. mRNA luciferase reporter expression; time course >8.5x expression at circVec RNA stability Day 8 for circVec 2.1 RT-qPCR, nascent vs. total RNA vs. mRNA 50 45 Relative luminescence 40
Slide 14: circRNA vs. mRNA expression in immunocompetent mouse muscle
circRNA vs. mRNA expression in immunocompetent mouse muscle mRNA-vector-Luciferase circVec 2.1-Luciferase Intramuscular injection with BALB/c Immunocompetent mice Left hindleg Right hindleg PBS; 3 different doses Monitor expression over time Low-dose mRNA circRNA 14
Slide 15: circVec in vivo advantage is enhanced at lower dose levels
circVec in vivo advantage is enhanced at lower dose levels, up to 15x higher expression than mRNA Absolute expression (luminescence) Relative expression (luminescence) circVec 2.1 vs. mRNA pDNA vector expression -fold change circVec 2.1 vs. mRNA expression 1.8x higher 3.8x higher 15x higher expressi
Slide 16: Summary - circVec has the potential to transform how proteins are expressed
Summary - circVec has the potential to transform how proteins are expressed in therapeutic settings Superior stability leads to accumulation of circRNA inside the cell, resulting in higher and prolonged protein expression vs. mRNA circVec is significantly more durable and achieves up to 15x higher e
Slide 17: Gene therapy development plan
Gene therapy development plan Modality and disease to be selected based on experimental data Therapeutic Ongoing in vivo Pipeline modality screening opportunities Muscle Limb-girdle (LGMD), Sarcopenia AAV-circVec Heart Fabry, Danon disease Payload: reporter LNP-DNA circVec gene Lung Cystic fibrosis,
Slide 18: Acknowledgements
Acknowledgements Circio RnD Dr. Eoghan O’leary Dr. Juan Zhang Dr. Daisy Hjelmqvist Ms. Summer Stefanelli Poster: Dr. Md. Victor Levitsky Thursday 14-1530 Dr. Erik D. Wiklund Mr. Ola Melin #P0947 Karolinska Institute: Prof. Michael Uhlin 18