A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes

Human immunodeficiency virus (HIV, lentivirus) type-1 based vectors have a number of attractive features for gene therapy, including the ability to tr ansduce non-dividing cells and long term transgene expression. Mie used a t hree-plasmid expression system to generate pseudotyped lentivirus-based sec tors by transient transfection of human embryonic kidney 293T cells in the presence of sodium butyrate, which is known to activate the long terminal r epeat-directed expression of HIV. Using this system we successfully generat ed versatile high titer lentivirus at titers of up to 2 x 10(8) transducing units/ml (TU/ml), and improved transduction efficiency in various cell typ es from seven to over twenty fold, We demonstrate its applicability of thes e Vectors for the efficient transduction of non-dividing cells, including p ost mitotic beating rat cardiac myocytes and well-differentiated rat L6 myo fibers, While both lentivirus-based and murine retrovirus-based vectors eff ectively transduced dividing cardiac fibroblasts and L6 muscle myoblasts in culture, lentivirus-based vectors also efficiently transduced cardiac myoc ytes and yielded titers of (6.3 +/- 1.2) x 10(5) TU/ml; however murine retr ovirus-based vectors showed low transduction efficiency with titers reachin g only (8.9 +/- 2.1) x 10(2) TU/ml. Furthermore, even 12 days after inducti on of differentiation of L6 myofibers, lentivirus-mediated transduction of beta-galactosidase (beta-Gal) at approximately 30-40% of the maximum expres sion levels achieved in replicating myoblasts, In contrast, the expression of beta-Gal following transduction of the myofibers by murine retrovirus-ba sed vectors fell to less than 1% of an already reduced level of transductio n in undifferentiated confluent myoblasts. These results demonstrate that l entivirus-based vectors can efficiently transduce both well-differentiated cardiac myocytes and differentiated myofibers. This appears to be an effici ent method and provides a new tool for research and therapy for cardiovascu lar diseases. (C) 1999 Academic Press.