Targeting transgene to the heart and liver with AAV9 by different promoters

Abstract

Adeno-associated virus (AAV) has become one of the most promising gene transfer tools for gene therapy. This work aims to evaluate tropism, gene transfer efficiency and safety of AAV9 vectors produced with recombinant baculovirus (rBac)-based system. AAV9-CMV-GFP and AAV9-CBA-GFP were produced using a rBac system, 1 × 10(11) particles of each vectors were administered intravenously (i.v.) into mice and animals were killed at 1, 2, 3, 4, 5 and 8 weeks after administration. The GFP expression in different organs was analyzed by fluorescence imaging and Western blot. Viral genomic quantities were measured using qPCR. In vitro transduction efficiency of AAV9 vectors in primary cardiomyocytes and hepatocytes was determined by flow cytometry. Toxicity of AAV9 vectors was evaluated by determining certain cardiac and liver injury biomarkers and renal function test in vivo and TUNEL analysis in vitro. The data showed that AAV9 viral particles packaged by the rBac system were fully functional in vivo and in vitro. The CMV promoter predominantly induced higher cardiac GFP transgene expression and DNA copy numbers while the CBA promoter resulted in robust GFP expression and high vector DNA copy numbers in mouse liver, both in a time-dependent increased manner. Such distinct preferential effects were also observed in the heart and liver as early as 3 and 5 days after co-infection. Both the AAV9-CMV and AAV9-CBA viral packages did not induce heart, liver and renal damage and cell apoptosis. These results indicated that AAV9-CMV can efficiently and safely direct cardiac gene transfer, whereas AAV9-CBA is preferential for liver gene delivery.

Recombinant adeno-associated virus serotype 9 in a mouse model of atherosclerosis: Determination of the optimal expression time in vivo

“…Immunofluorescence staining and western blotting for GFP indicated peak expression at 35 days, indicating a time-dependent process. These findings are in agreement with our previous study, which determined that rAAV9-CMV-GFP may efficiently transfect the heart for a long time, with peak expression occurring at the same time point of 35 days (21). This time delay in peak expression may occur as in order for rAAV9 to mediate transgene expression, single-stranded DNA should be converted into double-stranded DNA (25,30).…”

“…Tissue-specific promoters have also been used to reduce unintended gene intervention; however, their efficiency is lower compared with that of non-specific promoters, such as the cytomegalovirus (CMV) promoter (20). Previous studies have focused on identifying effective AAV serotypes for cardiac or hepatic gene therapy (16,18,20,21); however, to the best of our knowledge, there has been no investigation on AAV9 gene transfer driven by CMV in apolipoprotein E −/− (ApoE −/− ) mice that are prone to form plaques at different time points.…”

Spacial and Temporal Patterns of Gene Expression After Cardiac MEK1 Gene Transfer Improve Post‐Infarction Remodeling Without Inducing Global Hypertrophy

“…Rather than introduce numerous confounding variables with a change to a larger species compared to our foundational transgenic studies, utilization of simple left ventricular cavity injection together with a vector that has a strong tissue tropism for cardiac myocytes provided an attractive initial translational in vivo gene transfer model. AAV9's natural cardiac tropism also makes the vector an attractive and potentially safe human clinical tool [Zincarelli et al, 2008;Chen et al, 2015]. Although a cardiac-myocyte-specific promoter system might have conferred greater tissue-specificity in gene delivery-and may be important for eventual human vector design-we were most concerned in this study with the limitation of in vivo transgene delivery and expression efficiency.…”

“…This constitutively active MEK1 has basal activity up to 400 times greater than that of the unphosphorylated wild-type kinase [Mansour et al, 1994]. Expression of both aMEK1 and LacZ was driven by the CMV promoter previously documented to yield measurably greater cardiac than hepatic transgene expression after systemic AAV9 delivery [Chen et al, 2015]. The aMEK1 gene also contained a hemagglutinin (HA) tag in order to track AAV-mediated expression and differentiate transgene expression from native MEK1 expression both in vivo and in vitro.…”

MicroRNAs: pleiotropic players in congenital heart disease and regeneration

“…Due to the natural tropism of AAVs to cell types, receptors or organs, a cardiac-specific expression of the miR-mimicking constructs could also be realized (86). In addition to serotype that are used to enhance cell type specificity, the choice of a strong or tissue-specific promoter within the viral vector system could furthermore increase the efficiency of miR-mimic expression (e.g., CMV or TnnT promoter) (87,88). Several AAV approaches expressing miR-mimics have already undergone clinical trials.…”