Bioengineering Adeno-Associated Viral Vectors for the Targeted Expression of Therapeutic Genes

Adeno-associated virus (AAV) vectors are increasingly used viral vectors for gene delivery in human gene therapy trials. They infect both nondividing and dividing cells and show long-term gene expression with low immunological response compared to other viral vectors used for gene therapy. Different AAV serotypes exhibit a variety of tissue tropisms and transduction efficiencies. A previous study in our lab has shown that serotype AAV9 is particularly effective for cardiac applications because of its uniform gene expression throughout the myocardium as well as its short lag phase, 2-3 weeks, to maximum gene expression. Furthermore, ischemia and reperfusion to the heart creates an environment that is more conducive to AAV9 transduction than healthy myocardium. In this study, I describe the engineering and evaluation of plasmids designed for packaging into AAV9 for cardiac-targeted gene therapy. I show that a plasmid with a bicistronic expression cassette containing neuregulin-1β (Nrg-1β) and green fluorescent protein (GFP) causes dual overexpression of these genes in vitro. I also show that the Nrg-1β is a functional protein via a proliferation assay with MCF7 breast cancer cells in vitro. This bicistronic cassette has been incorporated into multiple plasmid constructs driven by the promiscuous cytomegalovirus (CMV) promoter or the tissue-specific cardiac troponin-T (cTnT) promoter and flanked by two AAV inverted terminal repeats (ITR) for packaging into the AAV9 capsid for in vivo studies of its therapeutic potential.