Towards liver-directed gene therapy: Retrovirus-mediated gene transfer into human hepatocytes

Liver-directed gene therapy is being considered in the treatment of inherited metabolic diseases. One approach we are considering is the transplantation of autologous hepatocytes that have been genetically modified with recombinant retroviruses ex vivo. We describe, in this report, techniques for isolating human hepatocytes and efficiently transducing recombinant genes into primary cultures. Hepatocytes were isolated from tissue of four different donors, plated in primary culture, and exposed to recombinant retroviruses expressing either the LacZ reporter gene or the cDNA for rabbit LDL receptor. The efficiency of gene transfer under optimal conditions, as determined by Southern blot analysis, varied from a maximum of one proviral copy per cell to a minimum of 0.1 proviral copy per cell. Cytochemical assays were used to detect expression of the recombinant derived proteins, E. coli β-galactosidase and rabbit LDL receptor. Hepatocytes transduced with the LDL receptor gene expressed levels of receptor protein that exceeded the normal endogenous levels. The ability to isolate and genetically modify human hepatocytes, as described in this report, is an important step towards the development of liver-directed gene therapies in humans.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45540/1/11188_2005_Article_BF01233625.pd

Induction of cyclin mRNA and cyclin-associated histone H1 kinase during liver regeneration

International audienceCyclins and cyclin-associated cdc kinases are key regulators of oocyte maturation (Maller, J. L. (1990) in The Biology and Medicine of Signal Transduction (Nishizuka, Y., Endo, M., and Tanaka, C., eds) pp. 323-328, Raven Press, New York), yeast cell cycles (Nurse, P. (1990) Nature 344, 503-508), DNA replication in cell-free systems (D'Urso, F., Marraccino, R. L., Marshak, R. R., and Roberts, J. M. (1990) Science 250, 786-791), and amphibian cell proliferative transitions (Hunt, T. (1991) Nature 350, 462-463). The extent to which these regulatory molecules participate in the growth control of differentiated epithelial cells like hepatocytes is unknown. Therefore, we investigated the expression of “G1” (E, C, and D) and “G2/M” (A, B1, and B2) cyclin mRNAs, the relative levels of cyclin A- and B1-associated histone H1-kinase activity, and the appearance of cyclin-associated kinases (p32/p33cdk2 and p33/p34cdc2) in regenerating rat liver and in control tissues from sham hepatectomized rats. To do this, we exploited a battery of human cyclin cDNAs and cyclin antisera that recognize rat molecules. The results suggest an apparent sequence of regeneration-specific changes: 1) elevated and induced expression of cyclins E (2.1 kilobases (kb)) and C (4 kb), and D mRNAs (4 kb), within 12 h, respectively; 2) induction of cyclins A (3.4 and 1.8 kb), B1 (2.5 and 1.8 kb), and B2 (1.9 kb) mRNAs at 24 h; 3) induction of cyclin A- and B1-associated nuclear histone H1 kinase at 24 h; and 4) enhanced levels of PSTAIRE-containing proteins of Mr approximately 32-33 and 33-34 kDa in nuclear extracts from 24-h regenerating liver that co-immunoprecipitate with cyclin A and B1 antisera, respectively. These observations provide an intellectual framework that unifies the biology of hepatocyte mitogenesis, proto-oncogene expression, and the machinery of the cell cycle