Abstract
Cardiac endothelial cells (ECs) are important targets for cardiovascular gene therapy. However, the approach of stably transducing ECs in vivo using different vectors, including adeno-associated virus (AAV), remains unexamined. Regarding this unmet need, two AAV libraries from DNA shuffling and random peptide display were simultaneously screened in a transgenic mouse model. Cardiac ECs were isolated by cell sorting for salvage of EC-targeting AAV. Two AAV variants, i.e., EC71 and EC73, enriched in cardiac EC, were further characterized for their tissue tropism. Both of them demonstrated remarkably enhanced transduction of cardiac ECs and reduced infection of liver ECs in comparison to natural AAVs after intravenous injection. Significantly, persistent transgene expression was maintained in mouse cardiac ECs in vivo for at least 4 months. The EC71 vector was selected for delivery of the endothelial nitric oxide synthase (eNOS) gene into cardiac ECs in a mouse model of myocardial infarction. Enhanced eNOS activity was observed in the mouse heart and lung, which was correlated with partially improved cardiac function. Taken together, two AAV capsids were evolved with more efficient transduction in cardiovascular endothelium in vivo, but their endothelial tropism might need to be further optimized for practical application to cardiac gene therapy.
Highlights
The vascular endothelium plays a pivotal role in the regulation of vascular function and homeostasis and is an attractive therapeutic target for cardiovascular diseases.[1]
associated virus (AAV) successfully evolved in a transgenic mouse model in which AAV variants enriched in mouse retina photoreceptors were isolated by fluorescence-activated cell sorting (FACS) followed by polymerase chain reaction (PCR) amplification.[11]
Selection of AAV variants enriched in cardiovascular endothelial cells (ECs) in vivo AAV libraries from DNA shuffling and random peptide display have both been successfully applied in the evolution of AAV with novel tissue tropism.[8,9,11,12]
Summary
The vascular endothelium plays a pivotal role in the regulation of vascular function and homeostasis and is an attractive therapeutic target for cardiovascular diseases.[1]. 148 Molecular Therapy: Methods & Clinical Development Vol 22 September 2021 a 2021 The Author(s) Genetic engineering of the capsid gene has been verified to be very efficient in enhancing AAV transduction in various tissues, including ECs. Earlier work on rational design of AAV capsids was exemplified by insertion of specific peptides from phage display on the surface of the capsid structure, usually at site 587. DNA shuffling among AAV serotypes followed by biopanning in a mouse model was successfully performed to engineer AAV to efficiently target the myocardium with remarkably reduced liver infection.[8] random sevenamino-acid peptides were inserted after arginine 588 of the AAV2 capsid with the resulting libraries selected in in vitro cell or in vivo mouse models in the context of the capsid to enhance AAV transduction to human coronary artery ECs9 or mouse brain microvasculature ECs.[10]
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