Journal
GENE THERAPY
Volume 19, Issue 4, Pages 425-434Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/gt.2011.117
Keywords
hepatocytes; lentiviral vector; transduction; EGF; ex vivo gene therapy
Categories
Funding
- Deutsche Forschungsgemeinschaft [SFB 738, SPP 1230]
- Excellence Cluster REBIRTH
- Improvement of conventional and innovative transplants [SFB 738]
- Bill and Melinda Gates Foundation
Ask authors/readers for more resources
Partial resistance of primary mouse hepatocytes to lentiviral (LV) vector transduction poses a challenge for ex vivo gene therapy protocols in models of monogenetic liver disease. We thus sought to optimize ex vivo LV gene transfer while preserving the hepatocyte integrity for subsequent transplantation into recipient animals. We found that culture media supplemented with epidermal growth factor (EGF) and, to a lesser extent, hepatocyte growth factor (HGF) markedly improved transduction efficacy at various multiplicities of infection. Up to 87% of primary hepatocytes were transduced in the presence of 10 ng EGF, compared with similar to 30% in standard culture medium (SCMs). The increased number of transgene-expressing cells correlated with increased nuclear import and more integrated pro-viral copies per cell. Higher LV transduction efficacy was not associated with proliferation, as transduction capacity of gammaretroviral vectors remained low (<1%). Finally, we developed an LV transduction protocol for short-term (maximum 24 h) adherent hepatocyte cultures. LV-transduced hepatocytes showed liver repopulation capacities similar to freshly isolated hepatocytes in alb-uPA mouse recipients. Our findings highlight the importance of EGF for efficient LV transduction of primary hepatocytes in culture and should facilitate studies of LV gene transfer in mouse models of monogenetic liver disease. Gene Therapy (2012) 19, 425-434; doi: 10.1038/gt.2011.117; published online 18 August 2011
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available