ASGCT 2026: Development, Technology Transfer, and Qualification of Product-Specific Methods for AFTX-201, an Investigational New Medicine for the Treatment of BAG3-Associated Dilated Cardiomyopathy

Development, Optimization, and Qualification of a Comprehensive Analytical Panel for AFTX-201, an Investigational AAV Gene Therapy for BAG3- Associated Dilated Cardiomyopathy Shahrzad Parker, Matt Bennett, Hanna Czeladko, Lauren Sargent, Jordan Shufro, Matt Edwards, Rob May Fig 5 – Functional Potency AFTX-201 is an investigational rAAV gene therapy designed to restore BAG3 function in patients with BAG3-associated dilated cardiomyopathy. The therapy utilizes an engineered cardiotropic capsid (ATC-0187) to enable efficient myocardial transduction at reduced doses and deliver a fully human, full-length BAG3 transgene. To support GMP manufacturing and clinical evaluation in the UPBEAT trial, a robust, product-specific analytical strategy was developed to ensure consistent product quality and phase-appropriate regulatory readiness. Introduction Conclusions • This analytical panel provides a high-resolution assessment of AAV product quality, strengthening lot-to-lot comparability and enabling informed decision-making across process development and clinical product manufacturing. • This approach supports the advancement of AFTX-201 while demonstrating a scalable framework for analytical characterization of next-generation AAV gene therapies. • The framework and assays can be readily leveraged for developing additional product candidates in cardiac or skeletal muscle indications using the ATC-0187 capsid. Functional Potency Assays Relative Potency & Infectious Titer (TCID₅₀) A cell-based relative potency assay was developed early in the product life cycle to quantify BAG3 protein expression in support of process development. For the relative potency assay, transducibility across multiple cell lines, transduction duration, seeding density, and the use of different cell bank lots were evaluated to identify conditions that produced a consistent dose–response relationship. These optimizations resulted in improved assay sensitivity and inter-assay consistency. Fig 1 – Relative Potency Assay Fig 3 – ddPCR determination for ATC-0187 Capsid Packaging Quality For ddPCR-based vector genome titer determination of the engineered cardiotropic AAV capsid ATC-0187, a proteinase K (ProK) treatment was evaluated and compared to heat-based capsid disruption. ProK treatment resulted in improved assay precision, dilution linearity, and run-to-run reproducibility, with increased genome recovery observed across inputs. These improvements are consistent with enhanced capsid disruption and the high full-capsid content enabled by the optimized plasmid design. Collectively, these optimizations supported successful qualification of the ddPCR assay with high precision and accuracy for GMP lot testing. Capsid packaging quality during development was assessed using mass photometry, allowing direct differentiation and quantification of empty, full, and partially packaged AAV capsids generating comparable results to AUC. Fig 4 – Mass Photometry Analysis 2215 Droplet Digital PCR (ddPCR) References: Portions of the data presented here were generated in collaboration with Forge Biologics and Ncardia. Duong, Tam, et al., 2023, https://doi.org/10.1089/hum.2023.014 BDS Method Empty Partial Full Mass Photometry 1.9% 2.0% 96.1% AUC 1.0% 4.0% 95.0% Mass photometry demonstrated consistent profiles across multiple lots. ATC-0187 Capsid (Engineered AAV9) + Muscle-Specific Promoter + BAG3 cDNA Infectious titer is typically determined by a TCID₅₀ assay using qPCR with 10-fold dilutions. Alignment of ddPCR-based genome titration with a two-fold dilution scheme for the TCID₅₀ assay generated reliable measurements while yielding comparable infectivity estimates to the log dilution (4.00E+13 TCID₅₀/mL for 2-fold dilution vs 3.83E+13 TCID₅₀/mL for log dilution with 3%CV). A functional potency assay is under development. Cardiomyocyte contractility was evaluated as an exploratory functional readout. Impedance measurements demonstrate stable rhythmic activity in control iPSC-derived cardiomyocytes and reduced contractile function following BAG3 knockdown (day 5 post-transfection). These data highlight a biologically relevant in vitro phenotype associated with BAG3 loss-of-function. 1:2 dilution scheme 1:10 dilution scheme AFTX-201 Qualification Preliminary Runs vg/mL Average %CV 1.03E+14 1.04E+14 6.7%1.11E+14 9.72E+13 Accuracy vg/mL %RE Intra-assay precision (%CV) 0.1-fold nominal 1.07E+13 2.7 5.3 0.2-fold nominal 2.10E+13 1.5 3.6 Nominal 1.09E+14 4.8 3.5 Nominal 1.20E+14 15.9 2 5-fold nominal 5.08E+14 2 3.1 10-fold nominal 1.09E+15 5.3 3.6 Inter-assay precision 1.09E+14 5.80% R2 (Linearity) 1.00 Control BAG3 siRNA at day 5 post-transfection Fig 2 – TCID₅₀ Assay Optimization Baseline traces (red) overlayed with siRNA traces (Black). 1e25e21e35e31e45e41e55e51e65e6 0 20 40 60 80 100 Cell Line Selection MOI %GFP Cell line A AC16 HEK293 Sol8 Hskmc 1e25e21e35e31e45e41e55e51e65e6 0 20 40 60 80 100 Cell Densities MOI %GFP CD: 1E4 CD: 1E5 1e2 5e2 1e3 5e3 1e4 5e4 1e5 5e5 1e6 5e6 1e7 0 20 40 60 80 100 Transduction Enhancers MOI %GFP No Enhancer + Enhancer 104 105 106 107 0 2000 4000 6000 8000 Log MOI [BAG3] (pg/mL) Untransduced BAG3 Protein Expression

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