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 transduce non-dividing cells and long term transgene expression. We used a three-plasmid expression system to generate pseudotyped lentivirus-based vectors by transient transfection of human embryonic kidney 293T cells in the presence of sodium butyrate, which is known to activate the long terminal repeat-directed expression of HIV. Using this system we successfully generated 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 types from seven to over twenty fold. We demonstrate its applicability of these vectors for the efficient transduction of non-dividing cells, including post mitotic beating rat cardiac myocytes and well-differentiated rat L6 myofibers. While both lentivirus-based and murine retrovirus-based vectors effectively transduced dividing cardiac fibroblasts and L6 muscle myoblasts in culture, lentivirus-based vectors also efficiently transduced cardiac myocytes and yielded titers of (6.3 +/- 1.2) x 10(5) TU/ml; however murine retrovirus-based vectors showed low transduction efficiency with titers reaching only (8.9 +/- 2.1) x 10(2) TU/ml. Furthermore, even 12 days after induction of differentiation of L6 myofibers, lentivirus-mediated transduction of beta-galactosidase (beta-Gal) at approximately 30-40% of the maximum expression levels achieved in replicating myoblasts. In contrast, the expression of beta-Gal following transduction of the myofibers by murine retrovirus-based vectors fell to less than 1% of an already reduced level of transduction in undifferentiated confluent myoblasts. These results demonstrate that lentivirus-based vectors can efficiently transduce both well-differentiated cardiac myocytes and differentiated myofibers. This appears to be an efficient method and provides a new tool for research and therapy for cardiovascular diseases.
Sakoda
T
T
Institute for Genetic Medicine, University of Southern California School of Medicine, Los Angeles 90033, USA.
Kasahara
N
N
Hamamori
Y
Y
Kedes
L
L
D000818:Animals; D000831:Animals, Newborn; D002087:Butyrates / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D005822:Genetic Vectors; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000187:drug effects / Q000502:physiology*; D006321:Heart / Q000502:physiology* / Q000821:virology; D006801:Humans; D007668:Kidney; D018482:Muscle, Skeletal / Q000821:virology; D009206:Myocardium / Q000166:cytology*; D051381:Rats; D012190:Retroviridae / Q000502:physiology; D014162:Transfection / Q000379:methods*; D014779:Virus Replication / Q000187:drug effects
D002087:Butyrates