Comparative analysis of adeno-associated virus serotypes for gene transfer in organotypic heart slices
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Journal of Translational Medicine Open Access
METHODOLOGY
Comparative analysis of adeno‑associated virus serotypes for gene transfer in organotypic heart slices Zihou Liu1, Kristin Klose1 , Sebastian Neuber1,2,3, Meng Jiang1, Manfred Gossen4,5 and Christof Stamm1,2,3,5*
Abstract Background: Vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo and have gained increasing interest as shuttle systems to deliver therapeutic genes to the heart. However, there is little information on their tissue penetration and cytotoxicity, as well as the optimal AAV serotype for transferring genes to diseased hearts. Therefore, we aimed to establish an organotypic heart slice culture system for mouse left ventricular (LV) myocardium and use this platform to analyze gene transfer efficiency, cell tropism, and toxicity of different AAV serotypes. Methods: LV tissue slices, 300 µm thick, were prepared from 15- to 17-day-old transgenic alpha-myosin heavy-chainmCherry mice using a vibrating microtome. Tissue slice viability in air-liquid culture was evaluated by calcein-acetoxymethyl ester staining, mCherry fluorescence intensity, and the tetrazolium assay. Four recombinant AAV serotypes (1, 2, 6, 8) expressing green fluorescent protein (GFP) under the CAG promoter were added to the slice surface. Gene transfer efficiency was quantified as the number of GFP-positive cells per slice. AAV cell tropism was examined by comparing the number of GFP-positive cardiomyocytes (CMs) and fibroblasts within heart slices. Results: Slices retained viability in in vitro culture for at least 5 days. After adding AAV particles, AAV6-infected slices showed the highest number of GFP-expressing cells, almost exclusively CMs. Slice incubation with AAV1, 2, and 8 resulted in fewer GFP-positive cells, with AAV2 having the lowest gene transfer efficiency. None of the AAV serotypes tested caused significant cytotoxicity when compared to non-infected control slices. Conclusions: We have established a readily available mouse organotypic heart slice culture model and provided evidence that AAV6 may be a promising gene therapy vector for heart failure and other cardiac diseases. Keywords: Myocardial slice, Organotypic heart slice culture, Adeno-associated virus, Gene transfer, Gene therapy Background Heart failure (HF) is one of the most important causes of morbidity and mortality worldwide. More than 26 million people suffer from HF globally, and its prevalence is still growing [1]. Despite improvements in previous decades of available medical treatments, the *Correspondence: [email protected] 1 Berlin Institute of Health Center for Regenerative Therapies, Charité – Universitätsmedizin Berlin, Berlin, Germany Full list of author information is available at the end of the article
prognosis of HF remains poor, with an average 1-year mortality rate of 33% [2]. Although heart transplantation is the gold standard therapy for patients with endstage HF, routine treatment is still challenging due to the increas
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