Slides
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CircVec: A DNA Vector Platform Harnessing Intracellular Circular RNA Biogenesis for Next-Generation T-Cell Therapies Dr Thomas Hansen, CTO ESGCT, 2026
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2 Conflict of Interest & Employment Disclosure The speaker is a full-time employee and corporate executive at Circio and holds material equity, including stock options and shares, in the company. The scientific research, data, and viewpoints presented during this session are intrinsically linked to the speaker’s role within the organization. Disclaimer
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Circio´s unique and proprietary circRNA- based gene expression platform technology 3 mVec - standard mRNA approach circVec - proprietary circRNA approach Viral or DNA vector RNA Protein ??? . circVec 75x prolonged half-life vs. mRNA Up to 60x enhanced protein expression An .
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circVec protein quantification, Western blot Optimizing circular RNA-based gene expression: circVec construct design and evolution from Gen 1 → 4 The circVec evolution 4 ~36x ~3x ~2x ~4x ~1.5x ~40x increase for circVec 4.x vs. 1.1 State-of-the art in literature
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Bioinformatic simulation demonstrating circRNA accumulation over time - driving increased expression 5 Accumulation phase circVec Peak mVec Peak mRNA circRNA
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In vivo validation of bioinformatic simulation: 75x prolonged RNA half-life drives enhanced and durable gene expression Absolute luminescence, IVIS quantification over time 6 135h 9h 8h 610h Inferred RNA half-life, MCMC1 Inferred peak expression, MCMC1 38d 2.3d Intramuscular DNA injection, Longitudinal IVIS analysis 15x mVec circVec 1Markov Chain Monte Carlo 75x
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7 Genetic cardiomyopathy In vitro PoC In vivo technical PoC In vivo disease model IND enabling Technical concept Ophthalmology B-cell malignancies + autoimmunity Circio is deploying the circVec platform for development of next generation gene and cell therapies Heart, eye and CNS Enhanced, safer and lower cost AAV gene therapy Research collaboration with global pharma Cancer, autoimmune disease LNP: DNA format, redosable CNS
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8 40-fold enhanced expression in heart for circVec-AAV vs. conventional mRNA-based AAV D10 17 28 51 71 D10 17 28 51 71 AAV-mVec AAV-circVec 3.2 Gene expression quantification, f-luc IVIS signal 40x increased signal vs mVec +40x gene expression Systemic delivery, AAV9 vector, 5e12 vg/kg dose D10 17 28 51 71 AAV-circVec 2.1 Heart region
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circVec increases heart penetrance and reduces cellular stress response vs. conventional AAV Increased RNA penetrance in heart, RNAscope analysis AAV9-cTnT-circVec3.2 AAV9-cTnT-mVec Nuclei/Firefly transcript 80% of heart cells positive for RNA expression Reduced stress response, RNAseq analysis Toxicity from mVecToxicity from circVec3.2 Impact on UPR pathway p=0.003 n.s. p=2.5e-4p=3.3e-6 Significantly stronger toxicity signal from mVec
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10 circVec optimization for heart increases expression advantage to ~200-fold with 95% on-target specificity Enhancement of translation in heart, In vivo heart luminescence Enhancement of biogenesis in heart, In vivo heart luminescence mVec circVec Translation screen ~190x 5x Biogenesis screen mVec circVec ~70x 3x Reduced off-target liver expression, Ex vivo luminescence 95% on-target heart expression <1% off target liver expression circVec5.1 circVec5.2
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Visit CIRCIO posters for more information on the AAV programs 11 Poster: P0604 Thursday 2-4pm Poster: P0089 Wednesday 1:30-3:30pm
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12 Genetic cardiomyopathy In vitro PoC In vivo technical PoC In vivo disease model IND enabling Technical concept Ophthalmology B-cell malignancies + autoimmunity Circio is deploying the circVec platform for development of next generation gene and cell therapies Heart, eye and CNS genetic disease Enhanced, safer and lower cost AAVs Research collaboration with global pharma Cancer, autoimmune disease LNP: DNA format, redosable CNS
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Non-viral circVec: LNP-formulated DNA for durable and repeat-dosable in vivo cell therapy DNA formulation dsDNA (or ssDNA) in lipid nanoparticle LNP tropism Using systemic delivery ( tailvein) Gene expression kinetics in target cells Determined by production and decay -rates, and translational efficacy LNP-formulated DNA (not RNA!) Liver and spleen targeting RNA biogenesis and translation or An mRNA circRNA
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In vivo cell therapy: circVec expression duration up to 6 months vs. 2 weeks for mVec 14 LNP-mVec, luminescence Systemic I.V. delivery, single dose on Day 0 LNP-circVec luminescence Systemic I.V. delivery, single dose on Day 0 Mainly short -term liver expression Non-viral DNA circVec 2.1 LNP delivery, non-targeted No liver expression circVec in vivo spleen expression confirmed for 6 months on one dose Accumulation in spleen from week 3 -12
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Nucleofection of Jurkat T-cell line in vitro shows enhanced and prolonged expression from circVec 15 Enhanced and prolonged signal from circVec circVec expression temporal kinetics Vector-based expression of tdTomato in Jurkat Signal distribution. tdTomato fluorescence at day 7 after nucleofection of jurkat cells circVec mVec EV circVec mVec EV Density tdTomado fluorescence High dose Low dose Mid dose circVec: Right-tailed distribution
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Systemic delivery of T-cell tropic LNP:circVec recapitulate spleen targeting at 10x reduced dose 0.20mg/kg 0.10mg/kg 0.05mg/kg LNP-mVec w/ T-cell tropic LNP* Systemic delivery, three dose levels LNP-circVec w/ T-cell tropic LNP* Systemic delivery, three dose levels Spleen signal detectable at Day 7 for high dose only Spleen signal at all doses with >2 months durability 16 Non-viral DNA circVec 3.2 LNP delivery, T-cell-targeted
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Take home messages 1 Accumulation of circRNA from circVec leads to higher steady state expression 2 circVec delivers high RNA penetrance in heart at low dose 3 circVec reduces translational stress and unwanted expression in liver 4 circVec enables transient spleen signal for 3+ months
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Acknowledgements – Circio R&D team in Stockholm 18 Victor Levitsky Eoghan O’Leary Juan Zhang Summer Stefanelli Mohamed Warsame Fahima Akter Yaxin An Narmadha Subranian Jiangnan Liu Ola Melin Erik Wiklund €80 million Capital raised in 2026 to fuel the next phase of Circio R&D We’re hiring Scientists at all levels Early-career Experienced Senior
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PosterID: P0089 Presented at ESGCT 2026 O’Leary ET1, Zhang J1, Warsame M1, Stefanelli S1, Akter F1, An Y1, Liu J1, Subramanian N1, Melin O1, Wiklund ED1, Levitsky V1, Hansen TB1 1. Circio AB, Huddinge, Sweden Eoghan.oleary@circio.com SA SD IRIR IRES + ORFcircVec 1 IRES + ORFcircVec 2.0 IRES + ORFcircVec 2.1 circVec 3.x IRES + ORF A B 1. Optimized circVec architecture outperforms conventional circRNA expression constructs by ~36× Figure 1: circVec platform evolution. A) Schematic representation of circVec designs from generation 1 → 4. B) Relative protein yield from different generations of circVec in HepG2 cells (n=2). ~36x ~3x ~2x ~4x circVec 4.0 IRES ORF A B D2 Figure 2: Superior circRNA stability leads to circRNA accumulation and prolonged protein expression in vivo. A) Longitudinal IVIS analysis of luminescence from BALB/c mice receiving intramuscular (i.m.) injection of 1ug pcDNA3 vectors expressing a Firefly luciferase-encoding mRNA (mVec, left hindleg) or circular RNA (circVec, right hindleg). B) Quantification of signal from hindleg muscles presented as geometric mean ± s.e.m. (n=4). C) Inference of RNA half-life based on MCMC modelling (10000 iterations) of expression profile over time presented as violin plot with median. 0 15x increased signal vs mVec C 75x prolonged half-life vs mVec 2. circRNA stability confers enhanced protein expression from equal vector dose in vivo Figure 2: 0 3. Intra-vitreal delivery of AAV2-circVec increases protein expression in eye by up to ~50x mVec circVec 4.0 D2 D7 D14 D21 D42 D63 83x 18x 5e8 VG/eye 5e9 VG/eye n.s. 5e8 VG/eye 5e9 VG/eye AAV2-circVec4.0 AAV2-mVec A B D E Figure 3: In vivo expression of CMV-driven AAV2-circVec 4.0 in the eye by intravitreal delivery. A) Longitudinal IVIS imaging of ocular luminescence at the indicated timepoints after intravitreal injection of 5e9 or 5e8 vg of AAV2-mVec (right eye) and AAV2-circVec4.0 (left eye) in BALB/c mice. B) Ocular luminescence over time for AAV2-mVec and the AAV2-circVec generations, shown as geometric mean ± s.e.m. C) RNA half-life inferred by MCMC modelling (10,000 iterations) of the expression profile over time, shown as a violin plot with median. D, E) Firefly RNA (D) and AAV genome (E) quantified in eye tissue 63 days after intravitreal injection of 5e8 or 5e9 vg/eye of AAV2-circVec4.0 or AAV2-mVec, as indicated. Significance was determined by t-test on log-transformed values; n.s., not significant. 50x 50x Graphical Abstract AA[...]AAA mVec: conventional mRNA-based gene expression Linear mRNA -> short lived circVec: novel circRNA-based gene expression Stable circRNA -> accumulates More stable RNA → higher transcript level → more protein per AAV degraded rapidly Up to 60x higher gene expression at equal dose Less toxicity, wider therapeutic window HeartEye CNSAAV (Capsid agnostic) AAV-circVec Get PDF using the QR code (do not distribute without permission) Conclusions o circVec is a platform technology to generate stable circRNA from viral and non-viral vectors: circRNA accumulates intra-cellularly, leading to higher and more durable protein expression vs. standard linear-mRNA-based vectors o AAV-circVec outperforms conventional mRNA-based AAV by up to 60× across eye, heart and CNS in vivo: the advantage is driven by increased RNA stability and transcript accumulation, with no change in AAV transduction o New heart-optimized circVec 5.2 design achieves significantly higher on target gene expression than conventional mRNA-based AAV9, with 94% heart-specific expression and less than 1% off-target liver expression. o Next steps: testing circVec in genetic heart disease models, single-cell analysis for eye & CNS, validation in NHPs Eoghan.oleary@circio.com 0 5. Systemic delivery of AAV9-circVec shows ~10–20x dose-sparing potential in heart Figure 5: Dose-response relationship for AAV9-circVec vs. AAV9-mVec targeting heart. A-B) Super-linear dose response curve on ex vivo luminescence (A) and Firefly RNA quantities (B) from heart 54 days after injection of AAV9-mVec or AAV9-circVec3.2 using 4 different dose levels: 5e12, 1e13, 5e13, and 1e14 vg/kg. Dashed line represents linear dose-response with a slope of 1. C) RNAscope images on representative hearts form mice subjected to the different doses of AAV9-mVec or AAV-circVec as denoted. D) Impact of dose level on transcript distribution based on RNAscope. Black line represents estimated mRNA distribution of a therapeutic gene, such as Pkp2. 5e12 vg/kg mVec circVec3.2 1e13 vg/kg 5e13 vg/kg 1e14 vg/kg A B C D ~10-20x dose sparing 63x ICM (intra-cisterna magna) injection, 1e11vg/mouse ~10x ~30x ICV (intra-cerebroventricular) injection, 9e10vg/mouse IT (intrathecal) injection, 1e11vg/mouse ~60x Neck Brain Spine 4. Local delivery of AAV9-circVec increases protein expression in CNS by up to 60x 0 mVec circVec3.2 Figure 4: In vivo expression of CMV-driven AAV9-circVec 3.2 in CNS by three delivery routes. A-C) Schematics of the routes of administration and doses of AAV9-circVec3.2 or AAV9-mVec expressing Firefly luciferase from a ubiquitous promoter in BALB/c mice: (A) intra-cisterna magna (ICM), 1e11 vg/mouse; (B) intra-cerebroventricular (ICV), 9e10 vg/mouse; (C) intrathecal (IT), 1e11 vg/mouse. D–F) Longitudinal IVIS imaging of luminescence for AAV9-circVec3.2 vs AAV9-mVec after ICM (D), ICV (E) or IT (F) injection. G–I) Luminescence quantified from the site of injection after ICM (G), ICV (H) or IT (I) injection, shown as geometric mean ± s.e.m. A B C D E F G mVec circVec3.2 H I mVec circVec3.2 Genetic cardiomyopathy – systemic delivery In vitro PoC In vivo technical PoC In vivo disease model IND enabling Technical concept Ophthalmology - local delivery CNS - local & systemic Circio pipeline B-cell malignancies & autoimmunity Expression Cross- correction Stress Off-target expression tbd tbd tbd tbd tbdtbd tbd circVec: A novel circular RNA-based expression system to increase AAV potency and reduce therapeutic dose Covered in Circio oral presentation SESSION 3d: Non-viral vectors/nanotechnology and RNA therapeutics I D2 D7 D14 D21 D28 DNA RNA 0 5. Novel circVec features show potential for 5-fold further enhancement Novel circVec designs 5x Novel circVec designs 3x 94x 171x A C Figure 5: circVec optimization of circular RNA biogenesis and translation efficiency for heart shows 3-fold and 5-fold improvement over circVec3.2, respectively. A-B) Characterization of in vivo heart expression from an array of circVec cassettes after 5e12vg/kg i.v. injection of AAV9. The circVec cassettes aimed to enhance biogenesis (A) or translation (B). C) Relative luminescence from heart and liver across all organs (heart, liver, diaphragm, ovaries, hindlegs, thyroid gland, spleen, intestine, and lung) as determined from ex vivo luminescence at day 70. BEnhanced biogenesis Enhanced translation 94% signal from heart 0 5. Novel circVec5 features unlock up to 5-fold higher cardiac expression C Figure 5: Screening circVec design for enhanced biogenesis and translation in heart. A-B) Quantification of in vivo heart luminescence from an array of AAV9-circVec cassettes expressing firefly luciferase upon i.v. injection of AAV9 at dose 5e12vg/kg. The circVec cassettes aim to independently enhance biogenesis (A) or translation (B). The best performing circVecs are coined circVec5.1 and circVec5.2, respectively. C) Longitudinal IVIS imaging of luminescence for AAV9-mVec, AAV9-circVec3.2, AAV9-circVec5.1, and AAV9-circVec5.2. D) Ex vivo luminescence from heart at day 70 post transduction. E) Relative luminescence from heart and liver across all organs (heart, liver, diaphragm, ovaries, hindlegs, thyroid gland, spleen, intestine, and lung) as determined from ex vivo luminescence at day 70. 94% signal from heart circVec3.2mVec circVec5.1 circVec5.2 Enhancement of translation in heart Enhancement of biogenesis in heart Biogenesis screen mVec circVec circVec5.1 circVec5.2A B E mVec circVec Translation screen ~70x ~190x 5x 3x D2 D7 D14 D21 D28D2 D7 D14 D21 D28 D2 D7 D14 D21 D28 D2 D7 D14 D21 D28 D2 D7 D14 D21 D28 D10 D20 D28 D42 D69D10 D20 D28 D42 D69 D10 D20 D28 D42 D69D10 D20 D28 D42 D69 D2 D28 D56 D84 D170 13.5 408 30x C <1% in liver D ~163x ~1.5x
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PosterID: P0604 Presented at ESGCT 2026 Circular RNA-based expression enhances AAV potency in heart, whilst minimizing dose and cellular stress O’Leary ET1, Zhang J1, Warsame M1, Stefanelli S1, Akter F1, An Y1, Liu J1, Subramanian N1, Melin O1, Wiklund ED1, Levitsky V1, Hansen TB1 1. Circio AB, Huddinge, Sweden Circular RNA (circRNA) is a novel class of endogenously expressed RNA. CircRNAs are generated by a non-linear splicing event, known as backsplicing, where an upstream splice acceptor attacks a downstream splice donor. circRNAs are resistant to exonucleolytic decay, which results in increased intra-cellular stability and persistence compared to mRNA. Circio has developed a circRNA-based gene expression platform, circVec, which utilizes this natural stability advantage to increase and prolong protein expression from viral and non-viral vectors o 40x enhanced protein expression in heart from AAV9-circVec: Prolonged circRNA stability leads to accumulation of RNA and enhanced protein expression compared to conventional mRNA-expressing AAV o Reduced stress response and off-target liver expression from circVec: Significantly lower activation of UPR pathway by as well as lower reduced off-target expression adds to the safety and specificity of circVec-based gene therapy in heart o 80% circRNA penetrance in heart using low dose (5e12vg/kg) i.v. administration: RNAscope analysis shows higher and broader expression of circRNA compared to mRNA from equivalent vector copy numbers in heart o Superior LAMP2B expression from circVec: Using two different codon-optimized hLAMP2B ORFs, circVec significantly outperforms mVec on RNA and protein levels in wild-type mice Get PDF using the QR code (do not distribute without permission) 0 Figure 2: In vivo performance of AAV9-circVec3.2 with heart-specific promoter (cTnT). A) Longitudinal IVIS analysis at different timepoints after i.v. injection of different AAV9 vectors (5e12 vg/kg) in female BALB/c mice (n=4). B) Absolute ex vivo luminescence from heart 72-days post injection shown as geometric mean + s.e.m. C) Relative luminescence from heart and liver as determined from ex vivo luminescence at day 72. circVec technology introduction Conclusions circVec increases AAV9 gene expression by over 30-fold compared to benchmark AAV thomas.hansen@circio.com AAA[...]AAAA mRNA Protein mVec circVec circRNA 0 A B Figure 4: RNAscope analysis on mouse heart subjected to intravenous injection of AAV9-circVec and AAV9- mVec (5e12vg/kg). A) Representative heart sections from AAV9-circVec3.2 or AAV9-mVec treated mice (as denoted) probed for Firefly RNA (violet) using RNAscope technology and strained with Dapi (blue). B) Using the QuPath software, number of positive cells were extracted. Ppib (peptidyl prolyl isomerase B) is used as the positive control marker (ctrl). C) Distribution of Firefly transcript copy-numbers in positive cells showing 34x-58x more RNA from circVec3.2 compared to mVec. RNAscope analysis shows 80% circRNA positive cardiomyocytes at 5e12 vg/kg dose AAV9-circVec 3.2 AAV9-mVec 0 B Figure 3: Viral genome and transcriptomic analysis on heart samples. A-B) AAV genome (A) and Firefly RNA (B) quantification on heart tissue 92 days after i.v. injection of 5e12vg/kg AAV9-circVec3.2 or AAV9-mVec, as denoted. T-test on log-transformed values is used to determine significance, n.s.: not significant. C) Volcano plot based on whole transcriptomic analysis of heart tissue (n=4/group). Only Firefly RNA shows a significant change in expression (red dot, BH-adjusted p-value (padj) = 0.01). D) Unfolded protein response (UPR) genes as a class are significantly upregulated in mVec heart tissue compared to circVec (p=0.003, Wilcoxon rank sum test). C Circular RNA accumulation drives enhanced gene expression and reduces cellular stress response A D Toxicity from mVecToxicity from circVec p=0.003 C Replicate Replicate Firefly RNA 34x 58x 4 3 n.s. 0 Novel circVec features show potential for 5-fold further enhancement 0 AAV-circVec advantage also applies to a therapeutically relevant gene (LAMP2) in vivo SA SD IRIR IRES + ORFcircVec 1 IRES + ORFcircVec 2.0 IRES + ORFcircVec 2.1 circVec 3.x IRES + ORF A B Optimized circVec architecture improves circRNA gene expression by ~36-fold Figure 1: circVec genetic construct design and platform evolution. A) Schematic representation of circVec conctruct design at RNA and DNA level. B) Relative protein yield from circVec generation 1-4 in HepG2 cells (n=2). 1 ~36x ~3x ~2x ~4x circVec 4.0 IRES ORF ~1.5x C Novel circVec designs 5x Novel circVec designs 3x 94x 171x A C Figure 5: circVec optimization of circular RNA biogenesis and translation efficiency for heart shows 3-fold and 5-fold improvement over circVec3.2, respectively. A-B) Characterization of in vivo heart expression from an array of circVec cassettes after 5e12vg/kg i.v. injection of AAV9. The circVec cassettes aimed to enhance biogenesis (A) or translation (B). C) Relative luminescence from heart and liver across all organs (heart, liver, diaphragm, ovaries, hindlegs, thyroid gland, spleen, intestine, and lung) as determined from ex vivo luminescence at day 70. B Figure 6: Comparison of hLAMP2B expression in heart from AAV9-circVec3.2 and AAV9-mVec using 5e13vg/kg. A) RNA quantification by RT-qPCR on heart samples 42 days after injection of CIRCIO and industry benchmark codon-optimized hLAMP2B expression vectors (AAV9-circVec3.2 and AAV9-mVec) with a heart specific promoter. B-C) Representative western blots (each mVec/circVec pair cropped from same blot, B) and quantification of relative hLAMP2B protein expression in heart (C). D) IHC analysis of hLAMP2B protein expression in mouse heart 42 days post AAV injection. B C D 5 6 CIRCIO codon- optimization CIRCIO codon-optimization In vitro PoC In vivo technical PoC In vivo disease model IND enabling Technical concept Circio pipeline Expression Cross- correction Stress Off-target expression 7 Benchmark codon- optimization CIRCIO codon- optimization A Enhanced biogenesis Enhanced translation 11x 8x 4x 4x Benchmark codon- optimization CIRCIO codon- optimization 500µm hLAMP2B IHC PBSmVeccircVec3.2 circVec3.2 mVec circVec3.2 mVec hLAMP2B GAPDH conventional mRNA- based gene expression novel circRNA-based gene expression Genetic cardiomyopathy - systemic delivery Ophthalmology - local delivery CNS - local & systemic tbd tbd tbd tbd tbdtbd tbd B-cell malignancies & autoimmunity Oral presentation: Wednesday 8.30-10.30 - Non-viral vectors/nanotechnology/RNA therapeutics I o circVec is a platform technology to generate stable circRNA from viral and non-viral vectors: circRNA accumulates intra-cellularly, leading to higher and more durable protein expression vs. standard linear-mRNA- based vectors o AAV-circVec outperforms conventional mRNA-based AAV by up to 60× across eye, heart and CNS in vivo: the advantage is driven by increased RNA stability and transcript accumulation, with no change in AAV transduction o circVec shows 10-20× dose-sparing effect for systemic heart-targeted AAV gene therapy in vivo: same therapeutic index at significantly reduced dose for safer and lower cost AAV gene therapy o Next steps: testing circVec in genetic heart disease models, single-cell analysis for eye & CNS, validation in NHPs o Optimized circVec cassettes further enhances biogenesis and translation in heart by 3-fold and 5-fold respectively AAV9-mVec D10 17 28 51 71 AAV9-circVec 3.2 D10 17 28 51 71 A B 2 35x increased signal vs mVec 94% signal from heart 0 Systemic delivery of AAV9-circVec shows ~10–20x dose-sparing potential in heart Figure 5: Dose-response relationship for AAV9-circVec and AAV9-mVec in heart. A-B) Super-linear dose response curve on ex vivo luminescence (A) and Firefly RNA quantities (B) from heart 54 days after injection of AAV9-mVec or AAV9-circVec3.2 using 4 different dose levels: 5e12, 1e13, 5e13, and 1e14 vg/kg. Dashed line represents linear dose-response with a slope of 1. C) RNAscope images on representative hearts form mice subjected to the different doses of AAV9-mVec or AAV-circVec as denoted. 5e12 vg/kg mVec circVec3.2 1e13 vg/kg 5e13 vg/kg 1e14 vg/kg ~10-20x dose sparing 63x A B C 5 96x