ASGCT 2025: A Novel Investigational AAV Gene Therapy for Treatment of BAG3 Dilated Cardiomyopathy

AFTX-201: A Novel Investigational AAV Gene Therapy for Treatment of BAG3 Dilated Cardiomyopathy Matt Edwards1, Lisa Stanek1, Bryan Mastis1, Charles Gualtieri1, Tyler Ironside1, Cynthia Pryce1, Elizabeth Scott1, Nick Robinson1, Emily Grandell1, Mayara Ribeiro1, Giridhar Murlidharan1, John Reece-Hoyes1, Sherry Cao1, Roberto Calcedo1, Charles F. Albright1, and Laura K. Richman1 Fig 4 – Further Packaging Improvements ATC-0187 next generation cardiotropic capsid 96-well assay; 50K cells/well; 72h incubation. Avg GFP per cell was determined by analyzing fluorescence images. Cell boundaries were defined, and pixel counts within each cell were recorded. The avg pixel intensity was calculated across 25k cells/well AAV9 ATC-187 0 50 100 % GFP +ve (NHP heart) 10 8 10 9 10 10 vg per well Avg GFP per cell 5000 8000 1000 2500 AAV9 ATC-187 IHC staining of NHP heart sections showing GFP-positive staining in the myocardium ATC-0187 performance is superior relative to AAV9 in iPSC-derived human cardiomyocytes and NHP heart % Cardiomyocytes GFP positive quantified in heart left ventricle from IV delivered AAV9 and ATC-0187 at 3e13 vg/kg in NHPs (28 days in life) AAV-BAG3 restores normal cardiac function following myocardial infarction WT Control MI Untreated MI+AAV 0 20 40 60 80 Ejection Fraction % ns ✱✱✱✱ ✱✱✱✱ AAV-BAG3 improves cardiac function in MI mice 3 weeks post injection Experimental protocol and BAG3 construct design AAV expressing human BAG3 under the control of a muscle promoter Ejection fraction (EF%) in WT and MI mice (9 weeks post MI surgery and 3 weeks post AAV injection). ****p <0.0001; one-way ANOVA with Tukey’s multiple comparisons test. Data are shown as means ± SEM 5 6 7 8 9 10 11 12 13 14 15 16 17 18 35 40 45 50 55 60 Weeks old EF% WT BAG3 cKO +/- 5 6 7 8 9 10 11 12 13 14 15 16 17 18 15 20 25 30 Weeks old FS% WT BAG3 cKO +/- 5 6 7 8 9 10 11 12 13 14 15 16 17 18 2.5 3.0 3.5 Weeks old LVIDs (mm) WT BAG3 cKO +/- 5 6 7 8 9 10 11 12 13 14 15 16 17 18 3.5 4.0 4.5 Weeks old LVIDd (mm) WT BAG3 cKO +/- WT BAG3 cKO +/- 0 5×10 3 1×10 4 1.5×10 4 BAG3 Protein (Area under the curve) ✱✱ Conditional BAG3 KO mouse model with cardiac-specific haploinsufficiency (BAG3 cKO+/-) Reduced BAG3 protein expression in BAG3 cKO +/- mouse heart BAG3 Haplo-insufficient conditional KO generation paradigm Time course of cardiac dysfunction by ultrasound in BAG cKO +/- mice vs. WT Representative M-Mode ultrasound images from WT and BAG cKO +/- mice at 10 Weeks of age Ejection fraction (EF), fractional shortening (FS), Left ventricular internal dimension during systole (LVIDs), left ventricular internal dimension during diastole (LVIDd) Total BAG3 protein measured in 10-week-old mouse heart lysate by JESS automated Western blot WT cKO Adeno-associated virus (AAV)-mediated gene therapy is a promising modality for cardiovascular disease, but efficient and safe myocardial gene delivery remains a major hurdle to clinical translation. Through a machine learning-guided rational design approach, we identified novel AAV capsids with enhanced cardiac tropism, enabling robust cardiomyocyte transduction at lower doses than wild-type AAV9. Dilated cardiomyopathy (DCM), characterized by left ventricular dilation and systolic dysfunction, is a leading cause of heart failure and the most common indication for heart transplantation in young patients. Over half of DCM cases have a genetic etiology, including pathogenic variants in BAG3, a co-chaperone protein implicated in sarcomere integrity and cellular stress response. BAG3-associated DCM (BAG3-DCM) is a severe, progressive disorder with no approved disease-modifying therapies. We are developing AFTX-201, an AAV-based gene replacement therapy using a novel cardiotropic capsid to deliver full-length human BAG3. In a cardiac-specific BAG3 haploinsufficient (BAG3 cKO+/−) mouse model that recapitulates key aspects of BAG3-DCM, a single intravenous administration resulted in significant improvements in cardiac function, with no safety concerns observed. In nonhuman primates, low systemic doses of AFTX-201 achieved therapeutic cardiac BAG3 expression (>80% cardiomyocyte transduction and expression) with minimal off-target expression and no histopathological findings. AFTX-201 is proposed as an investigational gene therapy for BAG3-DCM. A first-in-human, multicenter, open-label Phase 1/2 study will evaluate the safety, tolerability, and pharmacodynamics of a single intravenous dose in adults with symptomatic BAG3 mutation-associated DCM. Introduction ATC-0187-BAG3 restores cardiac function in BAG3 cKO+/- mouse model Contact Process Science: Matt Edwards – [email protected] Low systemic dose of ATC-0187-BAG3 shows robust BAG3 expression throughout NHP cardiac tissue Proof of concept pharmacology, distribution and safety in NHP cardiac tissue after ATC-0187-BAG3 treatment at 1e13 vg/kg LV RV IVIS Atria 0 20 40 60 80 100 % Cardiomyocytes positive by IHC • AFTX-201 is an investigational AAV-based gene therapy designed for BAG3 mutation– associated dilated cardiomyopathy (DCM) • Our novel ATC-0187 capsid enables efficient cardiac transduction at significantly lower doses than wild-type AAV9 (~100% GFP transduction in NHP myocardium at 3e13 vg/kg) • Proof of concept restoration of cardiac function in myocardial infarction (induced) and BAG3-haploinsufficient (genetic) mouse models. • In nonhuman primates, systemic, low-dose ATC-0187-BAG3 administration achieved: ~60- 90% cardiomyocyte transduction, robust BAG3 expression across heart and no histological abnormalities • Data support a favorable efficacy and safety profile of AFTX-201 in murine and NHP models • A Phase 1/2 first-in-human clinical trial is planned to evaluate safety, tolerability and pharmacodynamics in adults with symptomatic BAG3-DCM Summary and conclusions References: 1. Domínguez F, et al. Dilated Cardiomyopathy Due to BLC2-Associated Athanogene 3 (BAG3) Mutations. J Am Coll Cardiol. 2018 Nov 13;72(20):2471-2481 2. Knezevic T, Myers VD, Gordon J, Tilley DG, Sharp TE, 3rd, Wang J, et al. BAG3: a new player in the heart failure paradigm. Heart Fail Rev. 2015;20(4):423-34 3. Myers VD, Gerhard GS, McNamara DM, Tomar D, Madesh M, Kaniper S, et al. Association of Variants in BAG3 With Cardiomyopathy Outcomes in African American Individuals. JAMA Cardiol. 2018;3(10):929-38 WT FB 61.8% EF; 0% IHC cKO FB 43.2% EF; 0% IHC cKO 6E11 Construct 1 54.5% EF; 24% IHC cKO 2E12 Construct 1 51.4% EF; 91% IHC cKO 2E12 Construct 2 59.5% EF; 77% IHC A) BAG3 protein level quantified as area under the curve by Jess-quantitative western blot in the mouse heart; B) Cardiac function as measured by % ejection fraction using echocardiogram; C) Vector genome biodistribution quantified using digital droplet polymerase chain reaction (ddPCR) normalized to housekeeping gene; D) mRNA Expression quantified using reverse transcription-ddPCR (rt-ddPCR) normalized to %rpp30 housekeeping gene; (A-D) Colors representing various treatment groups captured in the legend; E) Representative M-mode echocardiogram images at 11-12 weeks post treatment; F) Representative images showing HA-positive immunostaining (brown) in cardiomyocytes from each treatment group. A B C D E F LV RV IVIS Atria 0 1 2 3 4 5 Vector Genomes per Diploid Genome LV RV IVIS Atria 0 200 400 600 800 1000 mRNA expression Normalized to %RPP30 LV RV IVIS Atria 0 1 2 3 4 BAG3 protein expression (Fold Change vs naive NHP) A) Vector genome biodistribution quantified using digital droplet polymerase chain reaction (ddPCR) normalized to housekeeping gene; B) mRNA expression quantified using reverse transcription-ddPCR (rt-ddPCR) normalized to %rpp30 housekeeping gene; C) BAG3 protein level quantified as area under the curve by Jess-quantitative western blot in the ATC-0187-BAG3 treated vs naïve NHP heart; D) Representative images showing HA-positive immunostaining (brown) in cardiomyocytes from two ATC-0187-BAG3 treated NHPs. No significant histopathological findings were observed in the NHP hearts (data not shown). E) Histological quantification of % cardiomyocytes positive for protein expression. A B C D E 1 Affinia Therapeutics 103 104 105 106 107 BAG3 protein expression (AUC) ~3.2X ~62.3X ~12.5X ~0.29X 1X 30 40 50 60 70 Cardiac function (% ejection Fraction) ✱ ✱ WT-FB BAG3 cKO-FB 6e11 vg/kg (Construct 1) 2e12 vg/kg (Construct 1) 2e12 vg/kg (Construct 2) 1 10 100 1000 10000 Vector mRNA %RPP30 0.001 0.01 0.1 1 Vector Genomes per Diploid Genome 1024

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