Abstract
BackgroundVectors 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.MethodsLV tissue slices, 300 µm thick, were prepared from 15- to 17-day-old transgenic alpha-myosin heavy-chain-mCherry 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.ResultsSlices 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.ConclusionsWe 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.
Highlights
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
Application of AAV vectors for gene transfer into the heart was first demonstrated more than 20 years ago [12], and significant progress has been made in the clinical translation of AAV-mediated cardiac gene therapy in recent years [13]
Among the different AAV serotypes, AAV1, 6, 8, and 9 have been identified as the most cardiotropic ones [5]; data regarding their efficiency of cardiac gene transfer are inconsistent
Summary
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. Liu et al J Transl Med (2020) 18:437 appealing candidates because of their ability to mediate efficient transfer and stable expression of therapeutic genes in a wide variety of tissues, such as brain, liver, and heart [5,6,7]. As a gene therapy vector, AAV provides the ability to target a particular tissue or cell type by using a specific AAV serotype [8, 9]. Application of AAV vectors for gene transfer into the heart was first demonstrated more than 20 years ago [12], and significant progress has been made in the clinical translation of AAV-mediated cardiac gene therapy in recent years [13]. Among the different AAV serotypes, AAV1, 6, 8, and 9 have been identified as the most cardiotropic ones [5]; data regarding their efficiency of cardiac gene transfer are inconsistent. More evidence is required to determine the ideal AAV serotype for gene therapy in heart disease
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