Adenoviral gene delivery in long-term cultured human living myocardial slices: a proof-of-concept study to advance human pre-clinical models for cardiac gene therapy
B Pamias Lopez, F G Pitoulis, N Bouhrira, K Bedi, B L Prosser, K Margulies, M E IbrahimAbstract
Background
Cardiac gene therapy holds promise for the treatment of cardiomyopathy, yet clinical translation remains limited by the lack of reliable, high-throughput human myocardial models to assess vector efficacy, tropism, and safety prior to first-in-human studies. Animal models frequently fail to recapitulate the structural, functional, and pathological complexity of the adult human heart, contributing to a persistent translational gap. Living myocardial slices (LMS), 300 µm sections of cardiac tissue obtained from explanted human hearts, serve as an in vitro model that can close this gap. While already accepted on the main stage of cardiovascular research, they are yet to be used for gene therapy studies.
Aims
To establish the feasibility and functional safety of a novel platform for viral gene delivery in long-term cultured, electromechanically active human LMS, to advance pre-clinical gene therapy research and accelerate cardiac nucleic acid drug development.
Methods
LMS were prepared from explanted failing and non-failing human hearts and cultured for ≥7 days in a biomimetic system providing electrical stimulation, mechanical loading, and humoral support. Slices were transduced via direct application with an adenoviral vector carrying a fluorescent probe (mCherry) at a range of multiplicities of infection (MOI). Contractile force was continuously monitored throughout culture. Structural and biochemical assessments included native fluorescence imaging, immunostaining, and Western blotting. Functional performance was evaluated during culture and via post-culture force–length relationships.
Results
Adenoviral transduction resulted in robust mCherry expression within cardiomyocytes, displaying a mosaic distribution across the slices (Figure 1A). mCherry protein abundance was significantly increased compared with untransduced controls (UTC), with no significant differences across the tested MOI range (Figure 1B). Importantly, viral transduction did not impair myocardial function: daily force generation during culture and post-culture force–length relationships were preserved across multiple donor hearts, indicating maintained viability and contractility (Figure 2).
Conclusion
Our proof-of-concept study demonstrates viral gene delivery in long-term cultured, beating human myocardium with continuous contractile assessment. This human organotypic platform enables integrated structural and functional evaluation of cardiac gene therapy vectors and represents a critical step toward bridging the translational gap between preclinical studies and clinical application. Our expertise in LMS methodology positions us to develop this platform as a novel pre-clinical tool for genetic therapies, with future studies focusing on optimising MOIs, expanding to other adenoviral and AAV vectors, and transducing failing human hearts with cardiac recovery-associated proteins.Figure 1For image description, please refer to the figure legend and surrounding text.Figure 2For image description, please refer to the figure legend and surrounding text.